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The regulation and characterization of surfing motility in Pseudomonas aeruginosa Sun, Evelyn 2019

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     THE REGULATION AND CHARACTERIZATION OF SURFING MOTILITY IN PSEUDOMONAS AERUGINOSA    by  EVELYN SUN    B.Sc., The University of British Columbia, 2014   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (MICROBIOLOGY AND IMMUNOLOGY)   THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)    June 2019   © Evelyn Sun, 2019    ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:  The regulation and characterization of surfing motility in Pseudomonas aeruginosa  submitted by Evelyn Sun  in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology  Examining Committee: Robert E.W. Hancock, Microbiology and Immunology Supervisor  Michael Murphy, Microbiology and Immunology Supervisory Committee Member   Supervisory Committee Member Charles Thompson, Microbiology and Immunology University Examiner Stuart Turvey, Experimental Medicine University Examiner   Additional Supervisory Committee Members: Lindsay Eltis, Microbiology and Immunology Supervisory Committee Member Erin Gaynor, Microbiology and Immunology Supervisory Committee Member    iii Abstract  Pseudomonas aeruginosa is an opportunistic pathogen associated with a high incidence of infections in hospitalized and cystic fibrosis (CF) patients. P. aeruginosa is highly adaptable and exhibits diverse lifestyle adaptations depending on its surrounding environment. Here I studied a complex motility lifestyle termed surfing that occurs in the presence of mucin, a glycoprotein that is found in large abundance in the CF lung, and showed that surfing was associated with broad-spectrum antibiotic resistance, conserved in several bacterial species, and regulated by a complex network of regulators. RNA-Seq revealed ~1,024 genes dysregulated in P. aeruginosa under surfing conditions, while a screen of the PA14 transposon mutant library revealed 192 mutants that exhibited surfing deficiency, 40 of which were regulatory genes, including the putative chemotaxis regulator, PA1463, and two-component regulator, pfeR. Both PA1463 and pfeR were found to be master regulators of P. aeruginosa surfing and mutants in these genes demonstrated dysregulation of the majority of other regulators influencing surfing. Using disk diffusion assays, I investigated the adaptive antibiotic resistance associated with surfing motility. P. aeruginosa surfing cells were significantly more resistant to several antibiotics including all tested aminoglycosides, carbapenems, polymyxins, fluoroquinolones, and trimethoprim, tetracycline, and chloramphenicol. To identify the genes mediating surfing-dependent antibiotic resistance, transposon mutants in antibiotic susceptibility genes that were dysregulated under surfing conditions were screened for altered susceptibility under surfing conditions. This revealed 65 mutants, including mutants in armR, recG, atpB, clpS, nuoB, that exhibited changes in susceptibility to one or more antibiotics, consistent with a contribution to the observed adaptive resistance. It was further demonstrated that other motile bacterial species, including Escherichia coli, Salmonella enterica, Vibrio harveyi, Enterobacter cloacae, Proteus mirabilis, and Bacillus subtilis, exhibited similar characteristics of surfing as observed for P. aeruginosa in the presence of mucin, including rapid surface growth, dependence on flagella, and broad-spectrum adaptive resistance. Therefore, surfing is a conserved motile lifestyle regulated by complex networks of regulators and leads to broad spectrum adaptive antibiotic resistance.       iv Lay summary  Pseudomonas aeruginosa is highly adaptable and exhibits diverse motile lifestyles. Here I studied a novel form of bacterial motility termed surfing that occurs in the presence of mucin as a wetting agent, and showed that surfing was associated with antibiotic resistance, conserved in several bacterial species, and regulated by a complex network of regulators. Using disk diffusion assays, P. aeruginosa surfing cells were found to be significantly more resistant to several antibiotics. RNASeq revealed over 1,000 dysregulated genes under surfing. I performed a comprehensive mutant library screen to identify 44 surfing-essential regulators, of which pfeS and PA1463 were identified as master regulators involved in surfing. It was further demonstrated that other motile bacterial species also characteristics of surfing including rapid surface spread, dependence on flagella, and broad-spectrum antibiotic resistance. Therefore, surfing is a conserved motile lifestyle regulated by complex networks of regulators and leads to broad spectrum adaptive antibiotic resistance.     v Preface   A portion of the research presented in this thesis was drawn from published literature. I was the lead investigator, responsible for all major areas of concept formation, data collection and analysis, as well as manuscript composition. Below is a description of the contributions made by fellow scientists and collaborators. Dr. Robert Hancock was involved in the research presented in all chapters with regards to original conception, research planning, and extensive editing of all written work. The use of all bacterial strains presented in this thesis was appoved by UBC Risk Management Services under the UBC Biosafety Permit Number B14-0207. The American Society of Microbiology (ASM) and associated journals’ disclaimer on the republication of published work in a dissertation or thesis states: “Authors in ASM journals retain the right to republish discrete portions of his/her article in any other publication (including print, CD-ROM, and other electronic formats) of which he or she is author or editor, provided that proper credit is given to the original ASM publication. ASM authors also retain the right to reuse the full article in his/her dissertation or thesis.” Chapter 1:  Twitching motility assays and associated plate imaging (Figure 1-1) were performed by Morgan Alford.  Chapter 2:  Sections of Chapter 2 have been derived from the following published papers and copyright permissions were granted: Sun, E., Gill, E. E., Falsafi, R., Yeung, A., Liu, S., & Hancock, R. E. W. (2018). Broad-spectrum adaptive antibiotic resistance associated with Pseudomonas aeruginosa mucin-dependent surfing motility. Antimicrobial Agents and Chemotherapy 62:pii:e00848-18 (Sun et al 2018a). Sun, E., Liu, S., & Hancock, R. E. W. (2018). Surfing motility: a conserved yet diverse adaptation among motile bacteria. Journal of Bacteriology 200(23):pii:e00394-18. (Sun et al 2018b) RNA preparation and RNA-Seq was carried out by Reza Falsafi with bioinformatic generation of read-count Tables performed by Dr. Erin Gill who assisted me in writing the methods section. Dr. Amy Yeung was involved in the original conception of the surfing and swimming antibiotic screens as well as the surfing assay including providing guidance on how to perform the screens and analyze the data.   vi Chapter 3: Swarming assays and RNA extraction were performed by Shannon Coleman and biofilm assays and RNA extraction by Dr. Daniel Pletzer from our lab. RNA-Seq library preparation and quality control (QC) were performed by Reza Falsafi. Generation of RNA-Seq read count tables and assistance with bioinformatics analysis of data for surfing and swimming cells was done by Dr. Erin Gill. Dr. Amy Lee was responsible for uploading the raw data into GEO. Dr. Daniel Pletzer generated the pyoverdine and pyochelin mutant in PA14. Mutants of pfe genes in PAO1 as well as PAO6609 were generously provided by Dr. Keith Poole’s lab.  Chapter 4:  RNA-Seq library preparation and QC were performed by Reza Falsafi. Dr. Maren Smith generated RNA-Seq read count tables and assisted with bioinformatics analysis for the pfeR and PA1463 mutants and their complemented derivatives. Chapter 5: A version of Chapter 5 has been published in Sun et al 2018a, and copyright permission was granted. RNA-Seq library preparation and QC were performed by Reza Falsafi. Generation of RNA-Seq read count tables and assistance with bioinformatics analysis was done by Dr. Erin Gill. Dr. Amy Lee uploaded data onto GEO. A summer student Nicole Liu, directed by me, contributed to the generation of complemented strains and performed liquid MICs. Chapter 6: A version of Chapter 6 has been published in Sun et al 2018b, and copyright permission was granted: Nicole Liu assisted in conducting motility assays, antibiotic susceptibility screens, and motility zone growth assays. Mutants in other bacterial species besides Pseudomonas aeruginosa were generously provide by the labs of Dr. Bonnie Bassler, Dr. Avigdor Eldar, and Dr. John Gunn, Dr. Rasika Harshey, Dr. Paul Orndorff, Dr. Fitnat Yildiz, and Dr. Paula Watnick. Chapter 7: All stringent response mutants, constructs and complemented strains were made by Dr. Daniel Pletzer. He was also responsible for performing all swarming assays and RT-qPCR under swarming conditions, as well as designing the original concepts, and curating experimental results. Research presented is this chapter is currently being drafted into a manuscript with Dr. Pletzer and I as joint first authors.    vii Chapter 8: Sections of Chapter 8 have been published in Sun et al 2018a and copyright permission was granted.   viii Table of contents Abstract ......................................................................................................................................... iii Lay summary ................................................................................................................................ iv Preface ............................................................................................................................................ v Table of contents ........................................................................................................................ viii List of tables.................................................................................................................................. xi List of figures ............................................................................................................................... xii List of abbreviations .................................................................................................................. xiii Acknowledgments ...................................................................................................................... xiv Chapter 1: Introduction ............................................................................................................... 1 1.1 Pseudomonas aeruginosa: a serious threat ........................................................................... 1 1.1.1 Pseudomonas aeruginosa pathogenicity ........................................................................ 1 1.1.2 Pseudomonas aeruginosa virulence .............................................................................. 3 1.2 The role of P. aeruginosa in cystic fibrosis .......................................................................... 5 1.2.1 Cystic fibrosis lung environment ................................................................................... 6 1.2.2 Acute to chronic infections ............................................................................................ 7 1.2.3 Pseudomonas diversity in CF isolates ........................................................................... 8 1.3 Bacterial motility .................................................................................................................. 9 1.3.1 Swimming ...................................................................................................................... 9 1.3.2 Twitching ..................................................................................................................... 10 1.3.3 Swarming ..................................................................................................................... 11 1.3.4 Sliding .......................................................................................................................... 12 1.3.5 Surfing.......................................................................................................................... 12 1.4 Pseudomonas antimicrobial resistance ............................................................................... 13 1.4.1 Intrinsic resistance ....................................................................................................... 14 1.4.2 Acquired resistance ...................................................................................................... 15 1.4.3 Adaptive resistance ...................................................................................................... 15 1.4.4 Biofilm-associated resistance ....................................................................................... 16 1.5 Pseudomonas pangenome organization and gene regulation ............................................. 17 1.5.1 Gac/Rsm cascade ......................................................................................................... 18 1.5.2 Quorum sensing system ............................................................................................... 19 1.5.3 Stringent response ........................................................................................................ 22 1.5.4 Chemotaxis .................................................................................................................. 23 1.6 Hypotheses and objectives .................................................................................................. 24  ix Chapter 2. Materials and methods ............................................................................................ 26 2.1 Bacterial strains ................................................................................................................... 26 2.2 Motility assays .................................................................................................................... 27 2.3 RNA-Seq ............................................................................................................................. 27 2.4 Library Screen ..................................................................................................................... 28 2.5 RT-qPCR ............................................................................................................................ 29 2.6 Growth curves ..................................................................................................................... 29 2.7 Generating knock-out mutants and complements ............................................................... 29 2.8 Disk diffusion assay ............................................................................................................ 30 2.9 Antibiotic incorporation assay ............................................................................................ 30 2.10 Liquid minimal inhibitory concentration (MIC) ............................................................... 30 2.11 Motility zone growth assay ............................................................................................... 31 2.12 Iron titration assay ............................................................................................................. 31 Chapter 3. The transcriptomic profile and genes essential to surfing ................................... 32 3.1 Introduction ......................................................................................................................... 32 3.2 The P. aeruginosa surfing adaptation involved a transcriptionally diverse population of cells ........................................................................................................................................... 33 3.3 Regulatory genes expressed in surfing compared to swarming and biofilms ..................... 35 3.4 Mutant screens revealed more than 100 surfing-essential genes ........................................ 38 3.5 Surfing was mediated by interactions between 44 regulators ............................................. 41 3.6 Discussion ........................................................................................................................... 44 Chapter 4. PA1463 and PfeR as major regulators of surfing motility ................................... 48 4.1 Introduction ......................................................................................................................... 48 4.2 Surfing is highly dependent on the pfeRS and PA1463 operons ........................................ 49 4.3 PfeR and PA1463 as master regulators in surfing .............................................................. 50 4.4 PfeR and PA1463 regulated a large subset of Pseudomonas genes ................................... 52 4.5 Surfing dependence on iron and PfeRS regulation ............................................................. 55 4.6 Discussion ........................................................................................................................... 56 Chapter 5. Broad-spectrum adaptive antibiotic resistance associated with Pseudomonas aeruginosa mucin-dependent surfing motility .......................................................................... 59 5.1 Introduction ......................................................................................................................... 59 5.2 Surfing cells exhibited broad-spectrum antibiotic resistance ............................................. 59 5.3 Antibiotic incorporation assays to confirm adaptive resistance ......................................... 60 5.4 Adaptive antibiotic resistance was not due to the presence of mucin alone ....................... 62 5.5 Surfing-mediated antibiotic resistance is associated with multiple resistome genes .......... 63  x 5.6 Discussion ........................................................................................................................... 67 Chapter 6. Surfing motility: A conserved yet diverse adaptation among motile bacteria ... 71 6.1 Introduction ......................................................................................................................... 71 6.2 Physical characteristics of surfing motility exhibited by multiple motile bacterial species 71 6.3 Surfing motility demonstrated adaptability to various medium viscosities ........................ 73 6.4 Consistent surfing-like motility was not observed in alternative wetting agents ............... 74 6.5 Surfing cells exhibited distinct multiple antibiotic resistance ............................................ 75 6.6 Surfing dependence on flagella was conserved .................................................................. 77 6.7 Dependence on quorum sensing of P. aeruginosa surfing motility was not conserved ..... 78 6.8 Discussion ........................................................................................................................... 80 Chapter 7. Role of the stringent stress response in Pseudomonas surfing motility .............. 84 7.1 Introduction ......................................................................................................................... 84 7.2 The stringent stress response regulated swarming and surfing, but not swimming ............ 85 7.3 Differential gene expression in the stringent response mutant during motility .................. 86 7.4 Cross-complementation with the copper-transport regulator cueR or the quinolone synthase pqsH restored surfing motility in a stringent response mutant .................................. 87 7.5 Discussion ........................................................................................................................... 88 Chapter 8. Conclusions ............................................................................................................... 91 8.1 Surfing in comparison to other motile lifestyles ................................................................. 91 8.2 Regulation of surfing motility ............................................................................................. 94 8.3 Surfing as a conserved complex adaptation ........................................................................ 95 8.4 Concluding remarks ............................................................................................................ 98 8.5 Clinical implications ........................................................................................................... 98 References .................................................................................................................................... 99 Appendix .................................................................................................................................... 120     xi List of tables Table 1-1. P. aeruginosa quorum sensing systems ....................................................................... 21 Table 2-1. List of bacterial strains used in this study ................................................................... 26 Table 3-1. Regulatory genes dysregulated in surfing as well as swarming and/or biofilm cells .. 36 Table 3-2. Regulators whose transposon mutant variants exhibited surfing deficiency ............... 39 Table 4-1. Expression of surfing-essential regulators in the pfeR and PA1463 operon mutants and complements ................................................................................................................................. 51 Table 4-2. Surfing-essential effectors dysregulated in the pfeR and PA1463o mutants ............... 53 Table 5-1. Mucin addition, and its effect on diffusion, had little impact on antibiotic susceptibility ................................................................................................................................. 63 Table 5-2. Resistome genes and their corresponding changes in antibiotic susceptibility relative to wild-type when mutated ............................................................................................................ 64 Table 5-3. Complementation of selected resistome mutants that showed broad spectrum changes in susceptibility led to restoration of antibiotic susceptibility ...................................................... 66 Table 6-1. Surfing mediated diverse adaptive multi-drug resistance in different bacterial     species ........................................................................................................................................... 76 Table 7-1. Differential gene expression levels of key surfing- and swarming-essential regulators and effectors in LESB58 ............................................................................................................... 87 Table 8-1. Differences between motile adaptations in P. aeruginosa .......................................... 91 Table 8-2. Comparison of motility in diverse species .................................................................. 95 Table A-1. Concentrations of the antibiotics in the disk diffusion assay including their      solvents ....................................................................................................................................... 124 Table A-2. Effectors genes whose transposon mutant variants exhibited surfing deficiency .... 124 Table A-3. Genes dysregulated in PAO6609 wild-type surfing relative to swimming .............. 129 Table A-4. Genes uniquely dysregulated in the DpfeR mutant but not dysregulated under wild-type surfing conditions ................................................................................................................ 142 Table A-5. Dysregulated genes in the PA1463o mutant not dysregulated in PA14 wild-type surfing relative to swimming ...................................................................................................... 186 Table A-6. Influence of mucin on MIC ...................................................................................... 204 Table A-7. Resistome mutant susceptibility under surfing conditions - raw data ...................... 205 Table A-8. RT-qPCR results confirmed the dysregulation of resistome genes shown in          RNA-Seq ..................................................................................................................................... 206             xii List of figures Figure 1-1. Types of Pseudomonas aeruginosa motility in vitro ................................................. 10 Figure 1-2. Pseudomonas quorum sensing systems ..................................................................... 20 Figure 1-3. Pseudomonas chemotaxis regulation of swimming and twitching motility .............. 24 Figure 3-1. Transcriptomic analysis of surfing cells exhibited distinct expression patterns in cells collected from the edge and centre ............................................................................................... 34 Figure 3-2. Process of screening the PA14 transposon mutant library revealed over 40 surfing-essential regulators ........................................................................................................................ 39 Figure 3-3. Gene expression profiles of each individual surfing-essential regulator in mutants of other regulators found to be essential for surfing through the mutant library screen ................... 42 Figure 3-4. Putative surfing regulatory network based on regulators that had the same expression patterns in centre and edge cells ................................................................................................... 47 Figure 4-1. Surfing is inhibited when pfeR or the PA1463 operon is knocked-out ...................... 50 Figure 4-2. Surfing motility was dependent on iron and surfing persisted longer under iron-limiting conditions when pfeRS was overexpressed ..................................................................... 56 Figure 5-1. Multi-drug adaptive resistance of surfing colonies .................................................... 60 Figure 5-2. Antibiotic concentration dependent inhibition of surfing motility ............................ 61 Figure 6-1. Mucin triggered rapid surface motility in a range of bacterial species ...................... 72 Figure 6-2. Effect of medium viscosity on surfing motility ......................................................... 74 Figure 6-3. Effect of alternative wetting agents on surfing motility ............................................. 75 Figure 6-4. Surfing motility was dependent on flagella but not pili/fimbriae .............................. 78 Figure 6-5. P. aeruginosa surfing was dependent on quorum sensing ......................................... 79 Figure 6-6. Surfing-dependence on quorum sensing did not extend to bacterial species other than P. aeruginosa ................................................................................................................................ 80 Figure 7-1. Stringent response mutants exhibited surfing inhibition but wild-type swimming ... 85 Figure 7-2. Overexpression of pqsH and cueR in the stringent response mutant restored surfing in the LESB58 strain ......................................................................................................................... 88 Figure A-1. Disk diffusion assay plate set-up ............................................................................. 120 Figure A-2. Effect of viscosity in rich medium on surfing motility ........................................... 120 Figure A-3. Effect of medium viscosity on surfing images ........................................................ 121 Figure A-4. Surfing motility of PA14 in rhamnolipid deficient mutants ................................... 121 Figure A-5. Bacillus subtilis exhibits rapid surface growth under high mucin conditions and surfing-mediated broad-spectrum antibiotic resistance ............................................................. .122 Figure A-6. Effect of stringent response knock-out on swarming in LESB58 and PAO1 ......... 123 Figure A-7. Surfing and swarming dependence on pqsH and cueR in PA14 ............................. 123      xiii List of abbreviations  3OC12-HSL  N-3-oxododecanoyl-homoserine lactone Acyl-HSL   N-acylated homoserine lactone AIDS    Acquired immunodeficiency syndrome C4-HSL   Butanoyl-homoserine lactone CDC    Centre for Disease Control c-di-GMP   Cyclic di-GMP CMC    Carboxymethyl cellulose CF     Cystic fibrosis CFTR    Cystic fibrosis transmembrane regulator DGC    Diguanylate cyclase ECF    Extracytoplasmic function eDNA    Extracellular DNA EPS    Extracellular polymeric substance HAA    3-(hydroxyalkanoyloxy)alkanoic acid HHQ    4-hydroxy-2-heptylquinoline LPS    Lipopolysaccharide MDR    Multidrug resistance PDE    Phosphodiesterase ppGpp    Guanosine 3’5’-bispyrophosphoate pppGpp   3’-diphosphate-5’-triphosphate PQS    2-heptyl-3,4-dihydroxyquinoline QS     Quorum sensing RND    Resistance nodulation division ROS    Reactive oxygen species SCFM    Synthetic cystic fibrosis media T1SS    Type I secretion system T2SS    Type II secretion system T3SS    Type III secretion system T5SS    Type V secretion system T6SS    Type VI secretion system TCA    Tricarboxylic acid Usp   Universal stress protein    xiv Acknowledgments  Funding for work presented in this thesis was provided by the John Richard Turner Scholarship as well as the Canadian Institute of Health Research (CIHR) and Cystic Fibrosis Canada grants to Dr. REW (Bob) Hancock.   I wish to thank Bob for the opportunity to work under him as his graduate student and make meaningful contributions to research in this area. I am very grateful for his endless patience and support throughout the years. He is an amazing mentor and researcher who continues to be a pioneer in his field. I wish to also thank my committee members, Dr. Erin Gaynor, Dr. Lindsay Eltis, and Dr. Michael Murphy, for their amazing support and guidance throughout the course of my degree.   Much of the work presented in this thesis could not have been done without the continuous support and guidance from current and past members of the Hancock lab. I am especially grateful for their energy and familial ambience. I would especially like to thank the bioinformatics team for their help compiling and managing all the RNA-Seq data, Reza for helping prepare all the libraries for RNA-Seq, and the microbiology group for their input and collaborations. The Hancock lab is such an amazing bunch that have made my experience rich and memorable, and I wish to thank all of them for their positive attitudes.   In addition to all the amazing people who have directly impacted my research and experience as a graduate student, I would also like to thank my family for their patience in letting me pursue this degree and their endless support.   1 Chapter 1: Introduction  1.1 Pseudomonas aeruginosa: a serious threat Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium found ubiquitously in the environment (Azam and Khan, 2018; Opperman and Shachar-Hill, 2016). P. aeruginosa can be isolated from both biotic and abiotic environments, from soil and water to organic tissue (Azam and Khan, 2018). P. aeruginosa was first discovered in France in 1882 by Carle Gessard who first identified the species by its production of colored pigments which appeared blue-green (Gessard, 1984). P. aeruginosa is primarily an opportunistic pathogen, meaning that it is a commensal organism that normally does not cause infections unless the host is compromised. It is a leading cause of death from hospital-acquired or nosocomial infections, primarily involved in pneumonia, urinary tract infections, device-related infections and sepsis (Inweregbu et al., 2005). For example, P. aeruginosa is the primary agent and cause of death associated with ventilator-associated pneumonia (Sawa, 2014). P. aeruginosa is also known to be involved in several other diseases including: skin infections, bacteremia, chronic respiratory infections associated with cystic fibrosis (CF), joint infections, and infections in burn victims and immunosuppressed patients suffering from cancer or AIDS (Azam and Khan, 2018; Opperman and Shachar-Hill, 2016). Pseudomonas carries a large array of genes and broad genetic potential that contributes to its relatively high adaptability and prevalence (Silby et al., 2011). Therefore, P. aeruginosa can colonize diverse niches and is involved in several types of infections.  In 2017, the World Health Organization (WHO) released a list of 12 bacterial species classified as high threats to human health and this included P. aeruginosa (WHO, 2017). The Centre for Disease Control (CDC) also recognizes P. aeruginosa as a “serious threat” (CDC, 2018) accounting for approximately 10% of all hospital acquired infections and chronic infections in CF patients (Bennett, 1974; van Ewijk et al., 2006). In addition, approximately 13% of all Pseudomonas infections are associated with multidrug resistance, exhibiting resistance to at least three groups of antibiotics (Azam and Khan, 2018). Its high intrinsic resistance, high adaptability and quick acquisition of resistance has made P. aeruginosa one of the world’s greatest threats to human health.  1.1.1 Pseudomonas aeruginosa pathogenicity P. aeruginosa pathogenesis involves a combination of multiple factors that together  2 contribute to the threat that P. aeruginosa poses on human health. Moreover, P. aeruginosa is also involved in both acute and chronic infections. During acute infections, P. aeruginosa tends to retain a motile lifestyle, but after a series of binary signals, P. aeruginosa can undertake a sessile lifestyle growing as a biofilm which contributes to chronic infections (Valentini et al., 2018). Acute infections are characterized by tissue damage and dissemination (Turner et al., 2014). Chronic infections, on the other hand, involve more localized, persistent infections. During chronic infections, P. aeruginosa experiences loss of flagella which are immunogenic, formation of biofilms, loss of lipopolysaccharide O-antigenic side chains, and production of copious alginate exopolysaccharide, and causes persistent inflammation due in part to the production of virulence factors (Hancock et al., 1983; Kipnis et al., 2006). Quorum sensing plays an important role in maintaining ongoing inflammation and enhancing biofilm formation (De Kievit et al., 2001; Smith et al., 2002). However, one key binary signal that promotes the switch between lifestyles is cyclic di-GMP (c-di-GMP). C-di-GMP is a secondary messenger that triggers the switch between motile to sessile lifestyle by regulating key aspects of biofilm formation such as the production of the extracellular polymeric substance (EPS) (Kuchma et al., 2007). Many other factors and regulators also reciprocally regulate the planktonic and chronic lifestyles (Gooderham and Hancock, 2009; Yeung et al., 2009). Biofilm formation plays key a role in the transition between acute to chronic infections.  Biofilms are colonial aggregations of bacterial cells encased in an extracellular matrix comprising primarily polysaccharides, proteins, and extracellular DNA (Azam and Khan, 2018). The centres of biofilms exhibit low growth rates due the limited availability of oxygen and nutrients, and the up-regulation of sigma factor S, which contributes to antimicrobial resistance (Borriello et al., 2004; Brown et al., 1988; Yang et al., 2008). Biofilm formation is considered a stress adaptation and follows a defined development program involving complex regulation. It normally begins with reversible attachment to a surface, which in Pseudomonas involves pili and flagella (Rasamiravaka et al., 2015). Subsequently there is tighter, almost irreversible, attachment accompanying the production of the exopolysacharride matrix involving in P. aeruginosa the expression of exopolysaccharides Pel and Psl, and (more controversially) alginate. Microcolony development leads into the maturation of the biofilm structures and biofilms demonstrate a high level of adaptive multidrug resistance (Taylor et al., 2014). During chronic infections, P. aeruginosa can also switch into a mucoidal variant, producing large amounts of  3 alginate which is a polymer of mannuronic and glucuronic acid (Pedersen et al., 1992). Alginate protects Pseudomonas from immune responses including phagocytosis and antimicrobials (Leid et al., 2005). Although it is found to be overexpressed in biofilms, it is not actually necessary for biofilm formation (Schurr, 2013).  1.1.2 Pseudomonas aeruginosa virulence  P. aeruginosa produces a wide range of virulence factors, both cell-associated, such as flagella, pili, and lipopolysaccharides, and extracellular, including proteases, exotoxins, lipases, phospholipases, phenazines, pyocyanin, and hydrogen cyanide (Gonçalves-de-Albuquerque et al., 2016; Kipnis et al., 2006; Son et al., 2007; Strateva and Mitov, 2011). Cell-associated virulence factors, adhesins, aid in binding to host cells to initiate colonization and promote growth on surfaces (Kipnis et al., 2006). Flagella and pili can adhere and tether to epithelial cells (Conrad et al., 2011). Once bound, bacteria can produce enzymes that damage the local tissue to promote spreading. Proteases including elastases (LasA, LasB), staphylolysins, phospholipase C, alkaline proteases promote host tissue damage (Gonçalves-de-Albuquerque et al., 2016; Sawa, 2014). Exotoxins can be injected into host cells using the type III secretion system (T3SS) or secreted into the medium by type II secretion system (T2SS), and these in turn promote necrosis and apoptosis in host cells (Kipnis et al., 2006). T3SS injects cytotoxins (ExoSTUY) into host cells, while the T2SS secretes elastases, alkaline proteases, exotoxin A, and phospholipase C, which can be cytotoxic to, or damage, epithelial cells (Kipnis et al., 2006).  P. aeruginosa also expresses pigmented signaling molecules that contribute to virulence (Jimenez et al., 2012). Pyoverdine is a siderophore that in conjunction with limited iron autoregulates the pvd biosynthesis and uptake genes. Once bound to Fe3+, it is taken up across the outer membrane by the ferripyoverdine receptor FpvA in conjunction with TonB1 and other pvd genes are involved in reduction to release Fe2+ and active transport across the cytoplasmic membrane. In the inner membrane ferripyoverdine interacts with FpvR, an anti-s factor (Adams et al., 2006) which then promotes the cytoplasmic expression of PvdS and FpvI (Rédly and Poole, 2005). PvdS up-regulates toxA, prpL, and pyoverdine production. FpvI up-regulates fpvA in a positive feedback loop resulting in increased pyoverdine production (Rédly and Poole, 2003).  Other pigmented signaling molecules include the phenazines. Phenazines are redox-active compounds such as pyocyanin produced in both acute and chronic infections. Pyocyanin can suppress lymphocyte proliferation, damage the epithelium, inactivate protease inhibitors, and  4 target specific host cell functions (Jimenez et al., 2012). Pyocyanin biosynthesis genes, pyo, are regulated by the Pqs quorum sensing (QS) system (Jimenez et al., 2012).  1.1.2.1 Secretion systems  P. aeruginosa has six known secretion systems (Filloux, 2011). There are two type I secretion systems (T1SS) (Strateva and Mitov, 2011). The Apr system involves the ABC transporter, AprD, outer membrane protein, AprF, and adaptor, AprE, that work together to secrete proteases such as AprA (Guzzo et al., 1991). A second T1SS is involved in iron acquisition involving HasD, HasE, and HasF. This system is used to secrete the hemophore, HasAp (Létoffé et al., 1998). The major type II secretion system (T2SS) is comprised of 11 genes in two operons (xcpPQ, xcpRSTUVWXYZ). It makes use of a pseudopilin to excrete several virulence factors such elastases (LasB), staphylolysins (LasA), protease IV, phospholipases (PlcH, PlcN) and exotoxin A (Braun et al., 1998; Lu et al., 1993; Voulhoux et al., 2001). Two other T2SSs identified in P. aeruginosa include the Hxc system which is homologous to the Xcp system and secretes the alkaline phosphatase, LapA, independent of the Xcp system, and the Txc system which secretes the chitin-biding protein, CpbE (Ball et al., 2002; Cadoret et al., 2014).The T3SS is the most complex system and is responsible for the secretion of four known effectors (not all are present in all P. aeruginosa strains), ExoS, ExoU, ExoT, and ExoY (Yahr et al., 1997). These effectors contribute to the initial stages of infection including tissue damage used to promote dissemination and induction of inflammation (Strateva and Mitov, 2011). ExoS is a cytotoxin with two active domains: C-terminus ADP-ribosyltransferase and N-terminus Rho GTPase-activating domain. Both domains act to disrupt host cytoskeletal organization and activate Toll-like receptors (TLR2, TLR4) to promote inflammation (Epelman et al., 2004). ExoT is similar to ExoS as it also has two active domains that can disrupt the host cytoskeleton; however, it is also involved in preventing phagocytosis and wound repair (Garrity-Ryan et al., 2000). ExoY is an adenylate cyclase that induces the production of the secondary messenger, cAMP (Yahr et al., 1998), resulting in increased cell gap junction permeability allowing bacterial virulence factors to spread and induce inflammation (Castellano and Eugenin, 2014; Hritonenko et al., 2011). ExoU is a phospholipase that disrupts host membrane integrity during acute infections (Kurahashi et al., 1999). The type V secretion system (T5SS) involves a two-step process: effectors leave the inner membrane through the Sec secretion system and are transported through the outer membrane through an integral autotransporter protein. The T5SS is involved in  5 secreting the protease, LepA, which induces inflammation, and the chaperone protein, CupB5 (Kida et al., 2008; Ruer et al., 2008). The type VI secretion system (T6SS) involves a phage-like injection mechanism into target cells and is primarily involved in interstrain and interspecies competition (2 of the Pseudomonas Type V1 systems), although one system attacks host cells (Hood et al., 2017; Lien and Lai, 2017; Logan et al., 2018).  1.2 The role of P. aeruginosa in cystic fibrosis  Cystic fibrosis is an autosomal recessive genetic disease resulting from mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, which in turn results in irregular electrolyte secretion at mucosal surfaces (Folkesson et al., 2012; Oliver et al., 2000). There are more than 1,500 possible mutations that can result in CF, and 1 out of every 2,500 live births exhibit the disease phenotype (Folkesson et al., 2012). CF affects >70,000 people worldwide (Opperman and Shachar-Hill, 2016). It can result in pulmonary infections, bronchiectasis, pancreatic insufficiency, and diabetes mellitis (Opperman and Shachar-Hill, 2016). Not only are CF patients highly susceptible to infections by P. aeruginosa whereby >93% of CF patients between the ages of 18-24 acquire a P. aeruginosa infection (Son et al., 2007), but P. aeruginosa infections are also the primary cause of death in CF patients (Fothergill et al., 2012; Oliver et al., 2000).   The CFTR is a cAMP-regulated chloride ion channel which regulates electrolyte levels at epithelial surfaces such as the lung epithelium (Folkesson et al., 2012; Gellatly and Hancock, 2013). Irregular chloride levels result in the reduction of fluids within the lungs and increased dehydration leading to the impairment of mucociliary function and accumulation of thickened mucus (Folkesson et al., 2012; Gellatly and Hancock, 2013). This results in reduced microbial clearance and reduced lung immunity. The accumulation of rich mucus also promotes the growth of opportunistic pathogens such as P. aeruginosa. It also inhibits oxygen exchange and results in difficulties in breathing (Folkesson et al., 2012). Overall lung function decreases throughout the patient’s life. Although the lung flora is a mixture of several organisms, P. aeruginosa, Staphylococcus aureus, and Haemophilus influenzae being the three major players in the CF lungs, CF patients more frequently suffer from infections by P. aeruginosa or S. aureus (Folkesson et al., 2012). More than half of CF patients develop a P. aeruginosa infection by the age of 20. During early life, CF patients tend to have a higher level of S. aureus colonization but as changes in physiology of the CF sputum occur, in addition to the use of antibiotics, there is  6 selective pressure that favours P. aeruginosa (Folkesson et al., 2012). 1.2.1 Cystic fibrosis lung environment  The CF sputum environment is relatively rich in amino acids, nicotinamide adenine dinucleotide (NAD), and glutathione which contribute to the growth of P. aeruginosa; however, it is relatively poor in cofactors including biotin, pantothenate, and riboflavin (Turner et al., 2015). Synthetic CF sputum medium (SCFM) was found to closely resemble the composition of in vivo CF sputum (Palmer et al., 2007; Turner et al., 2015). However, the original SCFM lacked major molecules such as mucin and extracellular DNA which were later introduced in a modified version of the SCFM (Yeung et al., 2012). Mucin is a major glycoprotein, produced by mucosal and submucosal glands, found in the CF lung in large abundance (Li et al., 1997). Mucin is a highly glycosylated polypeptide that acts as a surfactant in the lung sputum, regulating the viscosity of mucus (Gellatly and Hancock, 2013; Yeung et al., 2012). The number of carbohydrate chains and the amount of cross-linking between chains that occurs varies depending on the hydration level, iron concentration, and pH (Celli et al., 2007; Gellatly and Hancock, 2013). Mucin helps bind and trap bacteria, which are swept away by cilia out of the lungs. In the CF lungs, however, due to dehydration, the carbohydrate chains of mucin cannot fold properly, which disrupts its bacterial trapping properties. Instead, it binds tightly to the epithelium through MUC1 and MUC4, and impedes the cilia thus inhibiting their mucocilliary function (Gellatly & Hancock, 2013). P. aeruginosa lipopolysaccharides (LPS) also promote overproduction of mucin in the CF lungs by inducing the expression of the MUC2 gene in epithelial cells (Li et al., 1997).  A study by Son et al (2007) revealed that a total of 437 genes are involved in P. aeruginosa pathogenesis in vivo in the CF lungs, and 323 are constitutively expressed. These genes appear to be mainly involved in the metabolism of fatty acids, choline, and glycerol, and virulence through phospholipase and lipase production (Son et al., 2007). Several strains of P. aeruginosa isolated from the lungs of CF patients were also found to be prone to developing auxotrophic phenotypes to certain amino acids, which in turn also had an effect on Pseudomonas metabolism (Oliver et al., 2000; Turner et al., 2015).   The CF lung is a complex environment that P. aeruginosa must adapt to with regards to nutritional changes, physiochemical changes, and challenges by the immune system and antimicrobial treatments (Folkesson et al., 2012). One key stress factor faced by P. aeruginosa in  7 the CF lung is the production of reactive oxygen species (ROS) released by host cells, resulting in oxidative stress (Folkesson et al., 2012). Oxidative stress can induce DNA, lipid, and protein damage, which can in turn induce mutations that select for stronger variants. As CF patients suffer from initial S. aureus infections, antibiotics are prescribed and enter the lung environment. This is a major factor that promotes the rise of and selection for drug-resistant P. aeruginosa (Folkesson et al., 2012).  1.2.2 Acute to chronic infections  During the early stages of CF infection, P. aeruginosa adopts a non-mucoidal, motile lifestyle and initiates infection by binding to the mucosal surface via adhesins such as flagella and type IV pili which also promotes inflammation in the surrounding tissues (Opperman and Shachar-Hill, 2016; Valentini et al., 2018). Therefore, during these early stages of CF infection, flagella and type IV pili play key roles in acute infectivity (Penesyan et al., 2015). Flagellar components such as the flagellar cap, FliD, bind to mucin (Arora et al., 1998) as well as airway epithelial cells (Bucior et al., 2012; Feldman et al., 1998). Adhesins at the end of pili also mediate attachment to epithelial cells. After attaching to the surface, bacteria can cause tissue damage leading to inflammation and produce virulence factors that are released into the extracellular space using the T1SS and T2SS, or directly into host cells using the T3SS (Kipnis et al., 2006). Multiple T2SS effectors, as well as T3SS effectors such as phospholipase ExoU and exotoxin ExoY, inhibit phagocytosis by host immune cells and promote significant tissue damage allowing the bacteria to mobilize further (Gonçalves-de-Albuquerque et al., 2016). In rare cases, bacteria can reach the bloodstream and result in septicemia (Turner et al., 2014). As the infection becomes chronic, P. aeruginosa tend to experience loss of flagella/motility, reduced expression of virulence factors and the T3SS, adopt an LPS-rough phenotype, and begin to form small colony variants (Valentini et al., 2018). Reducing the expression of motile appendages such as flagella reduces immunogenic recognition through TLR5 (Murray et al., 2007). Loss of LPS O-antigen prevents recognition by serotype-specific antibodies (Hancock et al., 1983). The production of an EPS layer also contributes to immune evasion by preventing the binding of antibodies (Tseng et al., 2013). P. aeruginosa can also develop into highly resistant, multicellular structures known as biofilms that especially resist phagocytosis. While acute infections are predominantly aggressive, chronic infections are adapted for long-term persistence and resistance to clearance (Valentini et al., 2018). Bacteria isolated from chronic cases have been  8 shown to be less virulent than those isolated from acute infections due to their loss of flagella and the down-regulation of virulence factors (Bragonzi et al., 2009).   As P. aeruginosa adapt to the CF lung and begin to transition into a more a persistent lifestyle, they produce the polysaccharide alginate (Folkesson et al., 2012). Alginate is produced in response to cell envelope stress as a means of increasing envelope integrity (Wood and Ohman, 2012). It also prevents complement activation and phagocytosis by host immune cells (Leid et al., 2005). A common mutation found in CF variants includes a mutation in mucA, an anti-s-factor, which normally regulates alginate production through the algD operon (Pulcrano et al., 2012). The mutated mucA variant results in the overproduction of alginate, which in turn promotes increased P. aeruginosa survival in the CF lungs.  1.2.3 Pseudomonas diversity in CF isolates P. aeruginosa isolated from CF patients exhibits extensive diversity. In a study done in Spain in 2013-2014, 79 isolates were recovered from 75 patients (López-Causapé et al., 2017). Of the 79 isolates, more than half exhibited multi-drug resistance to at least three different antibiotics and 16% exhibited extensive resistance (i.e. resistance to all tested antibiotics). Mutations in mutS and mutL were observed in about 15% of the isolates (López-Causapé et al., 2017). Mutations in these two genes promote a mutator phenotype (Oliver et al., 2002), increasing the rate at which mutations appear. Oliver et al (2000) identified the mutational rate of 128 P. aeruginosa isolates from 30 CF patients and determined that 36% of those patients were colonized with hypermutable or mutator strains. These hypermutable strains were not found in acutely infected patients, only in chronic cases (Oliver et al., 2000). Hypermutable strains arise due to loss of ability to repair mistakes accumulated during DNA synthesis (e.g. mutations in mutL and mutS), which result in an increased number of genetic alterations in the chromosome (Oliver et al., 2000). The high frequency of hypermutable strains in the CF lungs, which can be selected for by aggressive antibiotic therapy (Wiegand et al., 2008), is consistent with an advantage conferred by rapid adaptation, which is accomplished by such variants (Oliver et al., 2000). Common mutations were found in mucA, lasR, and rpoN which contribute to increased survivability in the CF lungs (Folkesson et al., 2012). As previously discussed, mutations in mucA promote the overproduction of alginate. Pseudomonas also tend to exhibit common mutations in lasR that promote modification of the lipopolysaccharide (LPS) specifically on Lipid A and loss of the O-antigen in order to avoid host and antibiotic recognition (Feltner et al.,  9 2016; Yang et al., 2000).  P. aeruginosa in the CF lungs can also exhibit diversity in metabolic states (Jørgensen et al., 2015; Opperman and Shachar-Hill, 2016). Opperman and Shachar-Hill (2016) identified two metabolic phenotypes of P. aeruginosa isolated from CF sputum. One phenotype includes increased flux through the tricaboxylic acid (TCA) cycle and Entner-Doudoroff Pathway (EDP) with low flux through the oxidative pentose phosphate pathway (OPPP) and another phenotype with high flux through OPPP and low flux through the TCA cycle (Opperman and Shachar-Hill, 2016). Metabolic diversity is likely a consequence of oxidative stress and nutrient-limiting conditions encountered in the CF lung environment (Opperman and Shachar-Hill, 2016). Therefore, several factors contribute to the rise of mutator variants and metabolically diverse cells in the CF lung that promote Pseudomonas survival and colonization. 1.3 Bacterial motility  Bacterial motility is used for finding a new environment through processes such as chemotaxis, contributes to virulence, and can promote social behaviours (Mitchell and Kogure, 2006). Bacteria can have several motile appendages including flagella, and pili or fimbriae. Flagella are long helical structures made of flagellin, locked in the right-hand helical conformation (Harshey, 2003; Mitchell and Kogure, 2006). Bacteria can have one polar flagellum or multiple flagella that promote movement through low viscosity media (Mitchell and Kogure, 2006). Pili, on the other hand, are helical structures that, in Pseudomonas, extend out from the poles, have tip-associated adhesins for attachment and promote movement on solid surfaces (Harshey, 2003). Bacteria also rely on motility to promote collective behaviours, including swarming (Harshey, 2003). Collective behaviours help optimize growth and survival by triggering specialized functions, promoting access to nutrients, and influencing defense against the host and desiccation.  1.3.1 Swimming  Figure 1-1 illustrates the different forms of motility as they appear in vitro. Swimming occurs under highly aqueous or low agar (0.2-0.35%) conditions and is dependent on flagella (Harshey, 2003; Yeung et al., 2012). P. aeruginosa swimming in vitro under low agar conditions occurs embedded within the agar, and can be observed to form a translucent halo. Increased viscosity or agar concentration inhibits swimming, which is dependent on high moisture content. The direction of swimming is influenced by chemotaxis, which is the ability of cells to sense  10 changes in the environment, including concentration gradients of specific nutrients, chemicals and/or oxygen. In the presence of a chemoattractant, flagella will be locked in a counter-clockwise rotation promoting a forward movement towards the attractant (Watari and Larson, 2010). Conversely, in the presence of repellant, flagella rotation will switch to a clockwise rotation allowing the bacteria to “tumble”, or in the case of Pseudomonas twitch by Brownian motion, and change directions away from the repellant (Watari and Larson, 2010).   Figure 1-1. Types of Pseudomonas aeruginosa motility in vitro. Swimming motility was grown in 0.3% agar SCFM with ammonia. Swarming was grown on 0.5% agar SCFM without ammonia. Surfing was grown on 0.3% agar and 0.4% mucin SCFM. Twitching was grown on 1.5% agar BM2. All plates were inoculated with mid-log phase culture and incubated at 37°C for 15 hours.  1.3.2 Twitching Twitching is pilus-dependent and involves movement on solid surfaces or the interstitial space between the plate and the agar (Yeung et al., 2012). Type IV pili uniquely function independently of flagella and work to help cells adhere and aggregate, as reviewed in (Burrows, 2012). Twitching relies on the retraction of pili at the cell poles (Harshey, 2003). Twitching begins after an assembled pilus attaches to the surface via adhesins at the tip. Movement occurs when the pilus retracts into the body of the cell, dragging the bacterial cell towards the point of attachment (Burrows, 2012). Twitching is also involved in Pseudomonas biofilm formation (O’Toole and Kolter, 1998) and plays a role in initial attachment and dispersal (Morgan et al., 2006; O’Toole and Kolter, 1998). Thus mutants deficient in pilus production and twitching exhibit poor biofilm formation (Conrad et al., 2011). Pilus deficient mutants also exhibit an inability to develop into mushroom-shaped structures, forming only the stalk but not the cap structure (Klausen et al., 2003). Therefore, twitching appears to be a mechanism by which cells stack on top of each other to form an aggregate (Klausen et al., 2003).   11 Twitching motility is affected by external factors such as nutrient levels, viscosity, and surface hydrophobicity as well as internal factors such as the rate of pilin and surfactant production (Burrows, 2012), and can be triggered by specific molecules such as phosphatidylethanolamine (Kearns et al., 2001). Twitching normally occurs in environments with intermediate viscosity or about 1% agar wt/vol (Burrows, 2012) In addition to environmental cues and presence of specific molecules, twitching is also highly dependent on pilus biosynthesis. Assembly of pilus is regulated by PilT which has an ATPase at the base to provide rotary power (Harshey, 2003). Type IV pilus production is regulated by PilA, and controlled by sigma N and the two-component system PilRS (Burrows, 2012). Twitching is also regulated by c-di-GMP, a secondary messenger involved in regulating biofilm formation, through PilZ which is involved in regulating pilin assembly and FimX which senses environmental cues needed to trigger twitching motility (Amikam and Galperin, 2006; Navarro et al., 2009). 1.3.3 Swarming  Swarming is a complex, community-based motile adaptation that involves both flagella and pili (Köhler et al., 2000). Swarming normally occurs on semi-viscous surfaces with a poor nitrogen source (e.g. amino acids). It is dependent on rhamnolipids and 3-(hydroxyalkanoyloxy)alkanoic acid (HAA), a precursor to rhamnolipids (Caiazza et al., 2005). Swarming is associated with increased production of virulence factors and adaptive resistance (Overhage et al., 2008). Depending on the strain, P. aeruginosa swarming can appear dendritic (as shown in Figure 1-1 for strain PA14) or solar flare patterned (PAO1 strain). In P. aeruginosa, swarmer cells exhibit polar flagella but also express an alternative motor that facilitates their movement on surfaces (Toutain et al., 2005). When transitioning from swimming and swarming, P. aeruginosa can express more than one polar flagella (Kearns, 2010; Köhler et al., 2000). Cells at the swarming front (tips of the tendrils) appear to be significantly longer and more flagellated than cells in the swarm centre which exhibit a non-vegetative morphology (Harshey, 2003). Bacteria at the swarming front appear relatively inactive, but just behind the front cells are vigorously active (Harshey, 2003). Swarming cells group together as they align along their axes (Harshey, 2003). Swarming is dependent on QS in P. aeruginosa, in part because the Rhl and Las systems regulate the expression of rhamnolipids required for swarming motility (Köhler et al., 2000). QS regulation of swarming is highly nutrient dependent (Shrout et al., 2006;  12 Verstraeten et al., 2008). Shrout et al. (2006) demonstrated that depending on the carbon source, quorum sensing would differentially regulate the vigor of swarming motility which in turn affects the structure of a biofilm. Highly active swarmer cells tend to form less structured biofilms than poor swarmers. Therefore, swarming, which has also been shown to have an inverse regulation of certain genes in relation to biofilms (Caiazza et al., 2007), is also an influencer of other adaptations.   Through a comprehensive screen of a PA14 transposon mutant library, Yeung et al. (2009) identified approximately 233 mutants with altered swarming motility. Among the 233 mutants, 12% belong to regulatory genes. In addition to flagella and pili biosynthesis regulators, swarming was found to be dependent on QS (Rhl and Pqs) and other global regulatory systems such as CbrAB, NtrBC, and Arn (Yeung et al., 2009). Interestingly, the mutant in gacA, a global regulator involved in regulating the transition between motile to sessile lifestyle, exhibited hyperswarming phenotypes (Yeung et al., 2009). A mutant in gacA has previously been shown to exhibit a 10-fold decrease in its ability to form biofilms (Parkins et al., 2001). Conversely, several mutants including ntrB, pilH, and arn that exhibited swarming deficiencies also exhibited a biofilm overproduction phenotype (Yeung et al., 2009), which correlates to the previously reported inverse relationship observed between swarming and biofilm formation (Caiazza et al., 2007).  1.3.4 Sliding  Sliding motility, such as that exhibited by P. aeruginosa, was first reported in other bacteria such as Bacillus subtilis (Fall et al., 2006). Sliding is a passive form of motility, independent of either pili or flagella, and instead Pseudomonas relies on surfactants to propel itself across semi-solid surfaces (Murray and Kazmierczak, 2008). The production of surfactants acts as an expansion force needed to grow the motility zone (Harshey, 2003; Murray and Kazmierczak, 2008). Sliding motility can occur at agar concentrations of 0.3-0.7% wt/vol in both rich and minimal media (Harshey, 2003). Like swarming motility, sliding motility is regulated by the GacA/S system and the hybrid sensor-response regulator RetS as well as the secondary messenger, c-di-GMP (Murray and Kazmierczak, 2008).  1.3.5 Surfing Swarming had been proposed to reflect the main form of bacterial motility exhibited by P. aeruginosa in the CF lungs, due to the conditional requirements for swarming (poor N source  13 and moderate viscosity) that were similar to those found in the CF lung; however, swarming models lacked a major component in the CF lung that is produced in large amounts, namely mucin (Yeung et al., 2012). The addition of mucin into swarm plates surprisingly induced a novel form of motility known as surfing. Surfing occurs on the surface and appears as a dense circular colony with a thick white outer edges and a blue-green centre (Yeung et al., 2012). The propagation of surfing, i.e how rapidly the surfing zone expands is dependent on the viscosity of the media and on the concentration of mucin (Yeung et al., 2012). It is not affected by the presence of ammonium (NH4+), which inhibits swarming motility (Yeung et al., 2012). It is also significantly faster than other forms of motility such as swimming. Mutant studies reveal that surfing is dependent on flagella but not type IV pili (Yeung et al., 2012). Electron microscopy showed that surfing cells in the centre of a surfing colony appeared to be motile and flagellated whereas edge cells were relatively immotile and atrichous (Yeung et al., 2012). Surfing was also shown to be dependent on the Las and Rhl QS systems (Yeung et al., 2012). In comparison to swarming, surfing was found to not be dependent on rhamnolipids, was less stringent with regards to growth conditions and viscosity parameters (Yeung et al., 2012). It was proposed that mucin acts as a wetting agent since replacing mucin with other wetting agents such as Tween-20 or carboxymethylcellulose (CMC) promoted surfing-like motility, albeit not as well as mucin. Yeung et al. (2012) found that the T3SS was down-regulated in surfing cells whereas the T2SS was up-regulated in both the centre and edge cells. There was an overall down-regulation of phenazines and up-regulation of pyoverdine and pyochelin at the edge and down-regulation in the centre relative to swimming. An up-regulation of genes involved in polymyxin resistance was also observed (Yeung et al., 2012). Therefore, surfing appears to be a novel motile lifestyle that involves complex regulation and adaptive phenotypes. Due to the very recent discovery of surfing, not much has been studied regarding this motility and, therefore, this thesis focussed on surfing motility.  1.4 Pseudomonas antimicrobial resistance  Antimicrobial agents, including antibiotics, are compounds/molecules that inhibit the growth of or kill microorganisms. Broad-spectrum antimicrobials work against several different species of microbes while a narrow-range antimicrobial targets specific species. Antimicrobials can work in several different ways, by inhibiting cell wall synthesis (e.g. penicillins), cell membrane function (e.g. polymyxins), protein synthesis (e.g. aminoglycosides), nucleic acid synthesis (e.g.  14 flouroquinolones), or as antimetabolites (e.g. nitrofurans) (Kapoor et al., 2017). Microorganisms can retaliate by developing mechanisms of resistance such as altering the target of the antimicrobial agents, altering membrane permeability or promoting efflux, or producing enzymes that degrade or modify an antibiotic. P. aeruginosa is notorious for its high intrinsic resistance and ability to adapt, mutate or acquire genetic elements that increase antimicrobial resistance. Resistance can be genetic (e.g. mutations, acquisition of genetic material) or adaptive (e.g. due to alterations in lifestyle that influence the expression of resisatnce genes). One of the greatest concerns concerning resistance is the evolution of superbugs, which are microorganisms that exhibit resistance to almost all drugs on the market (Breidenstein et al., 2011).  1.4.1 Intrinsic resistance Bacteria have naturally occurring features or systems that allow them to evade antimicrobial action that collectively contribute to intrinsic resistance (Azam and Khan, 2018; Zhang and Feng, 2016). P. aeruginosa is equipped with features such as low outer membrane permeability, the production of multi-drug resistance (MDR) efflux pumps, and enzymes such as Class C β-lactamase that inactive certain antibiotics, and these collectively contribute to its high intrinsic resistance (Azam and Khan, 2018; Breidenstein et al., 2011). Low outer membrane permeability occurs due to the inefficiency of so-called porin proteins that form channels enabling antibiotic uptake, leading to low outer membrane permeability to antibiotics and consequent limited (slow) entry (Fernández et al., 2012).  There are several classes of drug efflux pumps known, including the multidrug and toxin compound extrusion (MATE), major facilitator superfamily (MFS), small multidrug resistance (SMR), and resistance nodulation division (RND) families (Azam and Khan, 2018). Major P. aeruginosa efflux pumps systems including the MexAB-OprM system belong to the RND family of efflux systems (Azam & Khan, 2018; Gellatly & Hancock, 2013). RND family efflux pumps span the outer membrane, periplasm, and inner membrane due to a tripartite structure involving a gated channel, adapter protein, and transporter respectively (Azam and Khan, 2018). P. aeruginosa constitutively expresses the MexAB-OprM system which contributes to resistance to a majority of b-lactams (Masuda et al., 2000) and conditionally, or due to multidrug resistance regulatory mutations, can express many other RND assemblies. Efflux pumps in general contribute to resistance against many classes of antibiotics including β-lactams, aminoglycosides, fluoroquinolones, tetracycline, chloramphenicol etc. (Azam and Khan, 2018).   15 Bacteria also encode enzymes that recognize antibiotics and degrade or modify/inactivate them. Mechanisms include chemical modifications, hydrolyzation, and reducing the affinity of an antibiotic to its target (Azam and Khan, 2018). P. aeruginosa expresses inducible AmpC b-lactamase that hydrolyzes most variants of b-lactams, and possibly PoxB which can break down certain carbapenems (Berrazeg et al., 2015; Zincke et al., 2016).  1.4.2 Acquired resistance  Acquired resistance normally occurs through the acquisition of extracellular genetic material through horizontal gene transfer as well as through mutations (Breidenstein et al., 2011). Horizontal gene transfer includes the exchange of plasmids, transposons, integrons, prophages, and resistance islands (Breidenstein et al., 2011). Mutations, on the other hand, occur due to replication errors which can be enhanced by certain chemicals or conditional inducers. For example, there is an increased frequency of mutations when bacteria are subjected to sub-inhibitory concentrations of certain antibiotics (Breidenstein et al., 2011). Mutations resulting in resistance can occur in the antimicrobial target to prevent recognition or reduce binding affinity or in the genes/mechanisms associated with intrinsic resistance. For example, certain mutations in the DNA gyrase genes (gyrA, gyrB) and topoisomerases IV genes (parC, parE) result in resistance to fluoroquinolones (Bagel et al., 1999). Mutations can also occur to upregulate the expression of efflux pumps which then promote efflux and reduce net uptake or can occur to increase the levels of enzymes that hydrolyze or modify antibiotics to promote resistance (Azam and Khan, 2018; Breidenstein et al., 2011). Overexpression mutants in ampC, b-lactamase, and/or derepression of the mexABOprM and mexXY efflux pumps, can arise during CF infections and promote increased resistance to b-lactams such piperacillin and ceftazidime or aminoglycosides and specific cephalosporins respectively (Berrazeg et al., 2015; Cabot et al., 2011). Those genes that mediate resistance when mutated are collectively known as the resistome for specific antibiotics (Breidenstein et al., 2011). 1.4.3 Adaptive resistance  Adaptive resistance is a form of reversible resistance that is induced as a consequence of certain adaptations to environmental changes or growth conditions (Azam and Khan, 2018; Breidenstein et al., 2011). Intrinsic and acquired resistance are often irreversible, in contrast to adaptive resistance which is transient and dependent on the triggering condition (Azam and Khan,  16 2018). Therefore, when conditions are reversed, susceptibility is restored (Breidenstein et al., 2011). Adaptive resistance was first discovered when exposure to tetracycline was found to induce plasmid-mediated tetracycline resistant gene expression (Bochner et al., 1980). Analogously, biofilms were found to have several mechanism of non-mutational resistance that were dependent on the biofilm growth state (de la Fuente-Núñez et al., 2013; Minami et al., 1980).  Adaptive resistance can involve the expression of efflux pumps, cell envelope proteins, and antibiotic-modifying enzymes, to name a few mechanisms (Breidenstein et al., 2011). It can occur as a consequence of stress responses and environmental cues. For example, polymyxins and aminoglycosides, which normally self-promote their uptake through binding to outer membrane LPS lipid A, induce the expression of arn genes which are involved in the modification (addition of aminoarabinose) of the lipid A, inhibiting self-promoted uptake of polycationic antibiotics (Fernández et al., 2010). The expression of arn genes is regulated by several two-component systems, which senses the presence of divalent cations and cationic compounds including antimicrobial peptides (Fernández et al., 2012). Exposure to chemical changes in the environment such as the buildup of ROS in the CF lung can also induce stress responses that promote the up-regulation of the MexXY-OprM efflux system, which in turn promotes resistance to aminoglycosides (Fraud and Poole, 2011).  As Pseudomonas exhibits high adaptability to diverse environments, it can deploy diverse responses that include changes in lifestyle (motile to sessile), changes in motility, and stress adaptations. These adaptations including swarming and biofilm formation can lead to adaptive resistance (de la Fuente-Núñez et al., 2013; Overhage et al., 2008). 1.4.4 Biofilm-associated resistance  Biofilms are highly resistant structures, which may be substantially related to their altered physiology and transcriptional patterns, including a dysregulation of regulatory and effector genes mediating resistance (Taylor et al., 2014). For example, some cells exhibit reduced metabolism and growth rate which contributes to increased persistence (Sultana et al., 2016). Thus, antibiotics such as aminoglycosides which target growing cells are ineffective against cells in the interior of a biofilm which may also exhibit persistence (Sultana et al., 2016). Persister cells are slow growing, non-dividing cells that can withstand high concentrations of antibiotics (Breidenstein et al., 2011). Additionally, accumulated mutations and mutator variants also  17 promote the up-regulation of resistance genes. Therefore, biofilms are often saturated with b-lactamases due to overexpression mutations as an extra layer of resistance (Breidenstein et al., 2011). Consequently, several characteristics of biofilm cells contribute to its high level of resistance against a broad range of antibiotics. Other likely contributors include adaptations to stressors that has been proposed to trigger biofilm development, and quorum sensing (Hall and Mah, 2017).  1.5 Pseudomonas pangenome organization and gene regulation P. aeruginosa is known to have one of the largest bacterial genomes at 6.3 Mbp (5,567 genes) in comparison to other bacteria such as Gram-negative E. coli that has a genome size of 4.6 Mbp (4,279 genes) and the Gram-positive S. aureus with a genome size of 2.8 Mbp (2,594 genes) (Azam and Khan, 2018). Approximately 8.4-9.4% (468-521 genes) of the P. aeruginosa genome is predicted to encode regulatory genes, including two-component regulators and transcriptional regulators (Stover et al., 2000) which suggest additional complexity when compared to the estimated 5.8% regulators encoded in the E. coli genome (Azam and Khan, 2018). P. aeruginosa contains ~60 known two-component systems (Strateva and Mitov, 2011). The general pattern for these two-component systems involves a sensor kinase that in response to a signal autophosphorylates and then phosphorylates (usually activating) a response regulator. CbrAB is a global two-component system that regulates a large number of genes involved in virulence, antibiotic resistance, carbon metabolism and swarming (Yeung et al., 2011). CbrA is the sensor kinase which activates the regulator, CbrB. This system then regulates the expression of other major regulators such as phoPQ, arn, and pmrAB (Yeung et al., 2011). Recent analysis has suggested that P. aeruginosa contains more than 690 predicted regulatory genes and >1,020 predicted regulatory interactions (Galán-Vásquez et al., 2011). Regulatory hierarchies are composed of origins at the top, where activating interactions/signals occur, while the lower end of the hierarchy is normally enriched in regulators of moderate subsets of effectors (Galán-Vásquez et al., 2011). Global regulators are those that regulate a large number of genes, often regulating other transcriptional factors and sigma factors, and target the promoters of genes with more than one sigma factor regulating them (Galán-Vásquez et al., 2011). Galan-Vasquez et al. (2011) predicted that the most influential transcriptional regulators in P. aeruginosa are LasR, RhlR, both involved in quorum sensing, Fur, MexT, and Anr, although this was likely biased by the interests of researchers who performed the analyzed  18 studies. 1.5.1 Gac/Rsm cascade  GacA/S is a two-component regulatory system involving a sensor kinase (GacS) and response regulator (GacA). GacA regulates the expression of two small regulatory RNAs, RsmY and RsmZ (Brencic et al., 2009). RsmY and RsmZ sequester RNA binding protein RsmA which is a translational repressor for several virulence factors including pyocyanin, hydrogen cyanide, and elastases, but an activator of Pel, Psl, and c-di-GMP synthesis (Heurlier et al., 2004). C-di-GMP levels are regulated by diguanylate cyclases (DGC) which produce c-di-GMP and phosphodiesterases (PDE) which hydrolyze c-di-GMP (Jimenez et al., 2012). One influential DGC is encoded by the gene wspR. Under specific conditions, WspF phosphorylates WspR to activate the production of c-di-GMP (Jimenez et al., 2012). FleQ and LasR can also regulate c-di-GMP levels. LasR regulates the tyrosine phosphatase, TpbA, which dephosphorylates and inactivates TpbB, a membrane-bound DGC, which in turn decreases c-di-GMP levels (Ueda and Wood, 2009). A decrease in c-di-GMP promotes an increase in EPS production and biofilm formation (Jimenez et al., 2012; Valentini and Filloux, 2016). The inactivation of RsmA, therefore, results in an increase in EPS synthesis and reduction of virulence factors (Gellatly & Hancock, 2013; Jimenez et al., 2012). RetS is a hybrid sensor/regulator that also regulates GacA by repressing it through the production of c-di-GMP produced by WspR (Goodman et al., 2009; Valentini and Filloux, 2016).  The Gac/Rsm regulatory system is involved in the transition between motile to sessile lifestyles (Valentini et al., 2018). RsmA is a key post-transcriptional regulator that positively regulates the expression of virulence factors, more highly expressed during acute infection, and negatively regulates genes involved in biofilm formation. RsmA-regulated virulence factors include flagella, type IV pili, rhamnolipids, and type II and III secretion systems and their effectors (Valentini et al., 2018). Conversely, RsmA down-regulates the expression of genes involved in EPS production such as pel and psl genes, quorum sensing, and the T6SS (Allsopp et al., 2017; Irie et al., 2010). Mutants in rsmA exhibit reduced virulence and ability to spread during acute infections in mice but exhibit increased persistence during chronic infections (Mulcahy et al., 2006). Increased RsmY/Z levels, as regulated by RsmA, promote attachment which triggers the initiation of biofilm formation (Valentini et al., 2018). RsmZ levels are subsequently reduced by the two-component system, BfiSR and MifSR, which further promote  19 growth of a biofilm by preventing reversion of the attachment (Petrova and Sauer, 2009). Mature biofilms exhibit high levels of RsmY/Z compared to planktonic cells. Decreased levels of RsmY/Z promote dispersal (Valentini et al., 2018).   RetS is a hybrid sensor kinase-response regulator that regulates GacAS in order to regulate the expression the two small RNAs, RsmY and RsmZ (Bordi et al., 2010). Consequently, it regulates biofilm formation and the expression of several virulence factors and the T2SS and T3SS. In turn, RetS is also regulated by HptB which can also regulate the expression of rsmY (Bordi et al., 2010). HptB up-regulates RetS which inhibits GacS and the production of RsmY and RsmZ, thus inhibiting biofilm production genes and activating the expression of virulence factors and the T3SS (Bordi et al., 2010). Therefore, the Gac/Rsm system acts as a global regulator that plays an important role in the transition between acute to chronic infection. It is worth mentioning however that there are many regulators that appear to independently regulate this switch between acute/motile (swarming) and chronic/sessile (biofilm) lifestyles (Yeung et al., 2009). 1.5.2 Quorum sensing system  Quorum sensing (QS) is a system that is conceptually conserved in Gram-negative bacteria (Kipnis et al., 2006). It involves the release of small signalling molecules in response to changes in cell density (Azam and Khan, 2018). Each signalling molecule, when it reaches a given concentration in the community, acts as a cofactor that binds to a specific transcriptional regulator (Kipnis et al., 2006). Quorum sensing is a form of intercellular communication that allows bacteria to develop community-based adaptations. In P. aeruginosa, more than 300 genes are regulated by quorum sensing (Azam and Khan, 2018). P. aeruginosa QS regulates the production of rhamnolipids, pyocyanin, elastases, alkaline proteases, and hydrogen cyanide. In addition, it also regulates genes involved in biofilm development and ROS defense (Winstanley et al., 2009). There are currently four known quorum sensing systems in P. aeruginosa: the N-acylated homoserine lactone (acyl-HSL) systems Rhl and Las, and the quinolone-based systems Pqs and Iqs (Azam and Khan, 2018; Gonçalves-de-Albuquerque et al., 2016). Table 1-1 defines the respective autoinducer molecules, synthases and regulators for each of the four systems. The systems have also been shown to overlap with one another as shown in Figure 1-2. Thus LasR bound to its autoinducer, 3OC12-HSL, for example, has been shown to activate the expression of rhlR, rhlI, pqsR, and the 2-heptyl-3,4-dihydroxyquinoline (PQS) autoinducer synthase operon  20 (McGrath et al., 2004; Pesci et al., 1997). Recently, it has been proposed that RhlR can also bind to an alternative ligand to induce Rhl-dependent gene expression to compensate for a lack of LasR-induced expression of its autoinducer synthase, rhlI (Mukherjee et al., 2017). The PQS autoinducer has also been found to regulate the Las and Rhl QS systems (McKnight et al., 2000). The Iqs system is regulated by the Las system, and is triggered by phosphate starvation (Lee et al., 2013).   Figure 1-2. Pseudomonas quorum sensing systems. The Las (purple), Rhl (blue), Pqs (green) and Iqs (blue) quorum sensing systems including their respective autoinducer synthases, autoinducer molecules, and regulators. Arrows indicate the direction of binding. Dashed-line boxes list proteins of genes regulated by each regulator bound to its respective autoinducer. Modified and reprinted with permission from (Lee and Zhang, 2015).  The Pqs autoinducer, PQS, is made from anthranilate and α-keto-fatty acids that are converted into 4-hydroxy-2-heptylquinoline (HHQ) which in turn is catalyzed by PqsH to PQS which, when it reaches a certain threshold level, binds to PqsR (Jimenez et al., 2012; Kim et al., 2010). HHQ, however, can also act as an autoinducer by binding to PqsR and activating several PQS-regulated genes, all except phenazine and lectin genes (Xiao et al., 2006). PQS can also complex with iron to act a chelator. It sequesters iron closer to the cell to facilitate pyoverdine  21 and pyochelin function (Bredenbruch et al., 2006).  Table 1-1. P. aeruginosa quorum sensing systems including their respective autoinducers, synthases, and regulators (Kipnis et al., 2006; Kiratisin et al., 2002; Lee et al., 2013; Mukherjee et al., 2017; Strateva and Mitov, 2011).  Quorum sensing system Autoinducer molecule Autoinducer synthase(s) Regulator Las N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) LasI LasR Rhl N-butanoyl homoserine lactone (C4-HSL) RhlI RhlR Pqs 2-heptyl-3-hydroxy-4-quinolone (PQS) PqsABCDE, PqsH PqsR Iqs 2-(2-hydroxy-phenyl)-thiazole-4-carbaldehyde (IQS) AmbBCDE undetermined  Quorum sensing links cell density to the regulation of the production of several virulence factors (Strateva and Mitov, 2011). QS transforms environmental signals into the expression of certain genes. The Las system is known to regulate genes such as lasI, lasB, lasA, apr, and toxA which are elastases, proteases and exotoxins (Kiratisin et al., 2002; Pearson et al., 1997). The Rhl system is known to regulate rhlI as well as rhlAB, which express rhamnolipids, and rpoS, the stationary phase sigma factor involved in regulating a large number of virulence factors and T3SS effectors (Mukherjee et al., 2017; Pearson et al., 1997). The Pseudomonas Pqs system is found to regulate a number of genes including some overlapping genes with the Rhl and Las systems including elastases, rhamnolipids, and pyocyanin (Strateva and Mitov, 2011). It also regulates genes involved in biofilm formation (Guo et al., 2014). The Pqs system has been shown to be up-regulated during persistent infections where P. aeruginosa variants frequently lose LasR regulation but retain active Rhl and Pqs regulation (Feltner et al., 2016). The Las system, however, has been shown to be crucial in enabling normal biofilm architecture, since mutants in the Las system develop abnormal (flat) biofilms (Davies et al., 1998). As infections progress from acute to chronic, there is selective pressure for non-functional mutations in lasR (Feltner et al., 2016).   Quorum sensing systems are also subjected to other levels of regulation outside of the inter-regulation among the four known systems. For example, QscR can form a complex with LasR and RhlR to delay expression of QS genes (Chugani et al., 2001). RsaL is a transcriptional repressor of lasI which inhibits expression of Las-dependent genes as well as directly repressing the expression of virulence genes involved in hydrogen cyanide and pyocyanin synthesis (Rampioni et al., 2006). QteE and QslA prevent activation of Rhl and Las QS during stationary  22 phase, whereby QteE controls the post-translation levels of LasR and RhlR, while QslA complexes with LasR to prevent DNA binding (Asfahl and Schuster, 2018).  1.5.3 Stringent response  The stringent stress response is a conserved mechanism in bacteria that occurs in response to environmental cues such as amino acid and nutrient starvation leading to transcriptional changes. It is mediated by the nucleotide secondary messengers, guanosine 3’5’-bispyrophosphoate (ppGpp) and 3’-diphosphate-5’-triphosphate (pppGpp), collectively known as (p)ppGpp (Boes et al., 2008; Khakimova et al., 2013; Vogt et al., 2011). In most Gram negative bacteria (p)ppGpp is an alarmone synthesized using ATP, primarily by the enzymes RelA and SpoT, whereby SpoT has both the ability to synthesize and hydrolyze (p)ppGpp (Khakimova et al., 2013). Amino acid starvation triggers a RelA-induced response, whereas SpoT responds to several forms of stimuli including membrane perturbations, carbon-limiting conditions, and inhibited fatty acid metabolism (Boes et al., 2008). When amino acid limitation occur, the lack of amino acids loading onto tRNAs results in an abundance of unloaded tRNAs entering the ribosome, which triggers the activation of RelA bound to the ribosome (Vogt et al., 2011). (P)ppGpp is synthesized, which in turn binds to RNA polymerase to redirect transcription, resulting in a global decrease in ribosomal protein synthesis, an increase in amino acid biosynthesis and proteolysis, a decrease in DNA replication, phospholipid, murein, and carbohydrate synthesis, and increased sigma S production (Vogt et al., 2011). Sigma S up-regulates the expression of glycolysis, oxidative stress response, stasis, and osmotic stress response proteins. Thus the stringent response copes with stress by reducing macromolecular synthesis and increasing stress coping mechanisms. The SpoT-induced stringent response, but not the RelA-induced response system, was also found to be involved in regulating the expression of universal stress response (usp) genes (Boes et al., 2008). The Pseudomonas stringent response was found to play a role in the oxidative stress response, which is normally regulated by OxyR that detects hydrogen peroxide levels and activates transcription of the oxidative stress response genes, katA, katB, ahpB, and ahlpCF, encoding catalases and reductases (Khakimova et al., 2013). A stringent response mutant, DrelADspoT, was found to be more sensitive to ROS, and the stringent response was found to be involved in regulating QS, which in turn regulates the expression of oxyR (Khakimova et al., 2013). The stringent response also regulates the expression of several virulence factors since the  23 stringent response double mutant exhibits reduced production of certain secreted virulence factors (Vogt et al., 2011). The stringent response mutant was also shown to have attenuated virulence in mouse infection models; therefore, the stringent stress response appears to play a key role in Pseudomonas virulence (Pletzer et al., 2017).  The stringent response, (p)ppGpp specifically, was found to negatively regulate the production of HHQ and PQS involved in PQS quorum sensing (Schafhauser et al., 2014). More specifically, ppGpp appears to regulate the Las and Rhl systems which in turn regulate the PQS system. The Las system induces the expression of PqsH, involved in converting HHQ to PQS, and PqsR, the Pqs regulator. RhlR, however, inhibits PqsR and PqsABCD production, inhibiting the production of HHQ and Pqs-induced gene expression (Schafhauser et al., 2014). Therefore, an accumulation of HHQ in the stringent response mutants suggests that the stringent response regulates the PQS system primarily through ppGpp-induced transcription of lasR which in turn regulates the expression of pqsH involved in converting HHQ to PQS (Schafhauser et al., 2014). 1.5.4 Chemotaxis  Chemotaxis is the ability of bacteria to sense gradients of changes in the environment and results in bacterial movement away from increased concentrations, in the presence of a repellant, or towards increased concentrations of an attractant (Mitchell and Kogure, 2006). Chemotaxis involves two types of proteins, sensors and transducers, which work coordinatively to respond to specific changes in the environment (Porter et al., 2008). The chemotaxis sensor is a cluster of sensory proteins called methyl-accepting chemotaxis proteins (MCP) at the cell envelope that act to amplify and trigger response to chemotactic signals (Porter et al., 2008). The presence of a chemoattract will trigger the dephosphorylation of CheA, the sensor kinase, which dephosphorylates the response regulators, CheB and CheY, as shown in Figure 1-3 (Porter et al., 2008). CheY is bound to flagellar motor switch proteins and dephosphorylated CheY triggers a counter-clockwise rotation of the flagellar motor, allowing the bacterium to move towards the attractant or “run” (Porter et al., 2008). The presence or change in a chemical gradient can trigger a net change in the frequency of runs, or tumbles which reorient the bacterium (Mitchell and Kogure, 2006). The presence of a chemoattractant reduces the frequency of tumbles (NB. Pseudomonas does not tumble per se but instead reorients by Brownian motion), and promotes more runs, allowing the bacterium to move towards the chemoattractant (Mitchell and Kogure, 2006). CheB and CheR work together to regulate the methylation of MCP to trigger and reset the  24 system. The dephosphorylation of CheB results in the methylation of the MCP. CheR also methylates the MCP which phosphorylates CheA, triggering a clock-wise rotation or tumble (Porter et al., 2008). CheZ is another chemotaxis protein that regulates the phosphorylation status of CheY to reset it to a “stalled” state. Besides regulating swimming motility, P. aeruginosa also has a chemotaxis system that regulates pili retraction during twitching motility through a similar set of chemotaxis proteins encored by chp genes that work in a similar fashion as the Che proteins, illustrated in Figure 1-3 (Sampedro et al., 2015).  Figure 1-3. Pseudomonas chemotaxis regulation of swimming and twitching motility. Reprinted with permissions from (Sampredo et al., 2015). 1.6 Hypotheses and objectives Due to the novelty of surfing motility, first discovered in P. aeruginosa under host-like conditions (Yeung et al., 2012), there is still much to be determined about how this motility is  25 regulated and how it may be linked to other aspects of the bacterium’s physiology. I hypothesize that surfing motility is mediated by a complex network of regulators and is orchestrated by global regulatory systems such as quorum sensing and the stringent stress response. Surfing, being a social motility like swarming, is hypothesized to be similarly involved in broad-spectrum adaptive resistance as a result of the dysregulation of resistome genes. Surfing and its complex characteristics are also predicted to be conserved in other motile bacteria. Therefore, I pursued six specific aims in addressing my hypotheses. The first aim is to determine the transcriptomic profile of surfing cells collected from the edge and centre of a surfing colony in order to better characterize surfing cells within the motility zone (Chapter 3) as they have previously been shown to exhibit differential physical features (Yeung et al., 2012). This will allow me to determine if cells from the edge and centre of a surfing colony also exhibit differential transcriptomic profiles in addition to their physical differences. The second aim, also presented in Chapter 3, focuses on identifying master regulators involved in surfing motility and mapping a regulatory network. A comprehensive mutant library screen was used to determine surfing-essential genes, both effectors and regulators. Among the regulators, a network was determined by analyzing the expression profile of each surfing-essential regulator in each of the regulatory mutants. Two regulators, PfeRS and PA1463, were identified as potential master regulators involved in surfing. Therefore, the third aim of this study is to determine the roles of PfeRS and PA1463 in mediating surfing motility through the regulation of other surfing-essential regulatory genes (Chapter 4). Swarming, like surfing, is a social motility. Because swarming cells exhibit adaptive resistance (Overhage et al., 2008), Aim 4 sought to determine if surfing motility is associated with adaptive antibiotic resistance and to identify the resistome genes involved (Chapter 5). The fifth aim of this study is to determine the level of conservation of surfing motility in other motile bacterial species. As presented in Chapter 6, this included testing the conditional requirements for surfing, adaptive resistance, and dependence on key regulators in other Gram-negative and one Gram-positive bacterial species. Finally, Aim 6, presented in Chapter 7, is to determine the dependence of surfing on the stringent stress response and to identify surfing-essential regulators mediated by the stringent response that may be contributing to its regulation of surfing.     26 Chapter 2. Materials and methods 2.1 Bacterial strains  Bacterial strains used in the research presented in this thesis are listed in Table 2-1. P. aeruginosa PA14 mutants used in this study, unless otherwise stated, were derived from the PA14 Transposon Mutant Library (Liberati et al., 2006). Table 2-1. List of bacterial strains used in this study.  Bacterial Species Description Reference P. aeruginosa Wild-type strain UCBPP-PA14 (Rahme et al., 1995) P. aeruginosa UCBPP-PA14 DPA1463-5 This study P. aeruginosa UCBPP-PA14 DPA1463-5/ PA1463-5::pUCp18 This study P. aeruginosa Wild-type strain PAO1 (Hancock and Carey, 1979) P. aeruginosa PA06609, spontaneous pyoverdine mutant (Dean et al., 1996) P. aeruginosa PAO1/ pfeRS::pUCp18 This study P. aeruginosa DpfeR; parent strain PAO6609 (Dean et al., 1996) P. aeruginosa DpfeA; parent strain PAO6609 (Dean et al., 1996) P. aeruginosa DpfeR (PAO6609)/ pfeRS::pUCp18 This study P. aeruginosa PA14 Dpvd/pch, pyoverdine and pyochelin mutant (Pletzer et al., 2017) P. aeruginosa PAO1 DrelADspoT (Pletzer et al., 2017) P. aeruginosa PAO1 DrelADspoT / relA+ chromosomal complement (Pletzer et al., 2017) P. aeruginosa PAO1 DrelADspoT / spoT+ chromosomal complement (Pletzer et al., 2017) P. aeruginosa Wild-type strain LESB58 (Cheng et al., 1996) P. aeruginosa LESB58 DrelADspoT (Pletzer et al., 2017) P. aeruginosa LESB58 DrelADspoT / relA+ chromosomal complement (Pletzer et al., 2017) P. aeruginosa LESB58 DrelADspoT / spoT+ chromosomal complement (Pletzer et al., 2017) P. aeruginosa LESB58 DrelADspoT / cueR::pBBR5 This study P. aeruginosa LESB58 DrelADspoT / pqsH::pBBR5 This study Enterobacter cloacae Clinical strain FC1165 (Pollard et al., 2001) Proteus mirabilis Wild-type strain UNSW059300 (Gram et al., 1996) P. mirabilis Wild-type strain BA6163 (Mobley et al., 1996) P. mirabilis Strain BB2401 ∆flaD; parent strain BA6163 (Mobley et al., 1996) P. mirabilis Strain HI4320 ∆mrpA; parent strain BA6163 (Bahrani et al., 1994) Salmonella enterica Wild-type ATCC14028/ JSG210 (Prouty et al., 2002) S. enterica Strain KK105 fliA::Tn10d-Tet mutant derived from ATCC14028  ( Prouty et al., 2001) S. enterica Strain JSG1240 luxS::MudJ mutant derived from ATCC14028  (Prouty et al., 2002)  27 Escherichia coli Wild-type strain 0157:H7 (Nataro and Kaper, 1998) E. coli Wild- type strain MG1655 (Partridge et al., 2015) E. coli Strain RP3098 ∆flhDC; parent strain MG1655 (Partridge et al., 2015) E. coli Strain ORN172 ∆fim; deletion of entire fim region (Woodall et al., 1993) Vibrio harveyi Wild-type strain BB120 (Nackerdien et al., 2008) V. harveyi Strain KM664 ∆luxR::Tn5; parent strain BB120 (Nackerdien et al., 2008) Bacillus subtilis Wild-type strain NCIB3610 (Pollak et al., 2016) B. subtilis Strain AES1403ΔcomA::Cm; parent is NCIB 3610 (Pollak et al., 2016) B. subtilis Strain AES2135 ΔcomQXP::tet; parent is 3610 (Pollak et al., 2016) B. subtilis Δhag::kan; parent is wild-type 3610 (Pollak et al., 2016) 2.2 Motility assays Surfing, swimming and swarming assays were performed on either Luria Broth (LB; Difco), Basal Media 2 (BM2) (Yeung et al., 2012) or synthetic cystic fibrosis media (SCFM) (Palmer et al., 2007) with 0.5% glucose, containing 0.3-0.5% (wt/vol) agar with 0.4% (wt/vol) mucin for surfing, 0.5% agar without mucin for swarming, or 0.3% agar without mucin for swimming. Briefly, as described in more detail in Palmer et al., 2007, SCFM consists of each amino acid at an average concentration of 19mM, 1.3mM NaH2PO4, 1.25mM Na2HPO4, 0.35mM KNO3, 10mM MOPS, 0.05M NaCl, 0.01M KCl, 1.75mM CaCl2, 0.6mM MgCl2, and 3.6µM FeSO4.  Other wetting agents tested besides mucin included carboxymethyl cellulose (CMC) added at 1.0%, and Tween-20 added at 0.01% wt/vol into LB with 0.3% agar. Bacterial species were sub-cultured 1 in 100 and grown to an OD600 of 0.4 - 0.5 in liquid LB medium, and 1 µL was inoculated onto the plates and incubated for 13-18 hours at 37ºC unless otherwise stated. Surfing and swarming plates were air-dried for approximately 1 hour before inoculation. Inoculation involved stabbing bacteria mid-way through the agar using the pipette tip. For the agar titration assay, bacterial species were grown on SCFM and LB with and without 0.4% mucin at varying agar concentrations (0.3%, 0.5%, 0.8%, and 1.0%). Bacterial cultures were grown and inoculated as described previously. Percent plate coverage was measured using ImageJ.  2.3 RNA-Seq PA14 was grown in liquid LB medium overnight and sub-cultured to an OD600=0.4-0.5. Mid-log phase cultures were used to inoculate SCFM (Palmer et al., 2007) for surfing and swimming plates, prepared as described above. Using sterile swabs, cells from the centre and edge of a surfing colony and centre of a swimming colony were collected into RNA protect bacteria reagent (Qiagen). Swimming liquid cultures were harvested at mid-log phase  28 (OD600=0.5) for RNA extraction. RNA extraction was conducted using a RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Deoxyribonuclease treatment was performed using a TURBO DNA-free kit (Thermo Fisher) and rRNA depletion was performed using a RiboZero Bacteria Kit (Illumina). Single end cDNA libraries were constructed using a Kapa stranded Total RNA Kit (Kapa Biosystems) and libraries were sequenced on an Illumina HiSeq 2500 in rapid run mode with 100 bp reads that were base-called and de-multiplexed using built-in software on the sequencer. Fastq file quality control was performed using FastQC v0.11.5 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MulitQC v0.8.dev0 (Ewels et al., 2016). Fastq files were aligned to the UCBPP-PA14 genome (GenBank gene annotations) using bowtie-2 (Langmead and Salzberg, 2012). Bam-sam file conversion and sorting were performed with samtools (Li et al., 2009). Read count tables were generated with htseq-count v2.5 (Anders et al., 2015). Differential expression analysis was performed using DESeq2 (Love et al., 2014). Fold-changes in surfing were calculated relative to swimming. Gene annotations were taken from the Pseudomonas Genome Database (Winsor et al., 2016). RNA-Seq was performed for the following samples using artificial sputum media (SCFM): • PA14 WT surfing (SCFM + 0.3% agar + 0.4% mucin) compared to PA14 WT swimming in liquid culture (SCFM) – Results in Chapter 3 • PA14 WT surfing compared to PA14 WT swimming in low agar conditions (SCFM + 0.3% agar) – Results in Chapter 5 • PA14 DPA1463 operon (PA1463o) and DPA1463o::PA1463o/pUCp18 under surfing conditions compared to PA14 WT surfing – Results in Chapter 4 • PAO6609 WT surfing compared to PAO6609 WT swimming in low agar conditions – Results in Chapter 4  • PAO6609 DpfeR and DpfeR::pfeRS/pUCp18 under surfing conditions compared to PAO6609 WT surfing – Results in Chapter 4 2.4 Library Screen  A screen of the PA14 transposon mutant library (Liberati et al., 2006) was performed on large Corning square bioassay dishes (245 mm x 245 mm x 18 mm) using SCFM with 0.5% agar and 0.4% mucin. 96-pin stamps were used for high-throughput initial screens of approximately 5,500 mutants. Subsequent surfing screens as described in the motility assays (Section 2.2) were  29 performed on standard, circular petri dishes (100 mm x 15 mm) using SCFM with 0.3% agar and 0.4% mucin. Mutant phenotypes were divided into four different categories: hypersurfing, wild-type surfing, other forms of motility (e.g. swarming or swimming), or 1-directional motility which appeared as a single streak from the centre towards the edge of the plate. Surfing deficiency was considered: a complete lack of motility, exhibiting swimming or swarming, or 1-directional motility.  2.5 RT-qPCR RNA was collected as described for RNA-Seq. Reaction samples were prepared using the qScript one-step SYBR green RT-qPCR Kit (QuantaBio) with 5 ng of RNA per 25 µL reaction amplified in a Roche LightCycler 96. Quantification analysis was done using the comparative Ct method (Schmittgen and Livak, 2008) using rpoD as the normalizing gene.  2.6 Growth curves Bacterial strains were grown overnight (15-18 hours) in liquid LB, shaken at 750 rpm in 37°C. Overnight cultures were diluted to an OD600=0.05 in a total volume of 100 µL and grown in round-bottom 96-well plates for 16 hours shaken at 567 cpm at 37°C in a TECAN Spectrofluor Plus. OD readings were taken every hour. Three replicates were done per sample. 2.7 Generating knock-out mutants and complements Complemented mutants were generated as follows. PCR primers were used to amplify the desired genes from genomic DNA. The amplified products were cloned into a TOPO vector using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). TOPO vectors containing amplified product were digested using two different enzymes, which differed depending on the gene of interest, and ligated into a desired vector containing the lac promoter. Vectors containing the desired genes were then transformed into their respective mutant using electroporation.  The knock-out mutant of the PA1463 operon was generated as previously described (Pletzer et al., 2014). Briefly, primers were used to amplify 500 bp regions up-stream and down-stream of the PA1463 operon and combined into a 1 kb fragment using an overlapping PCR reaction involving the forward primer that binds 500 bp up-stream and reverse primer that binds 500 bp down-stream of operon. The combined fragment was subsequently cloned into a Zero Blunt TOPO vector using the accompanying kit (Invitrogen) to be sequenced for verification that the correct fragment had been cloned. This 1 kb fragment was then cloned into a pEX18Gm suicide  30 vector using BamH1/XbaI. The vector was transformed into E. coli ST18 then transferred into PA14 via conjugation (mixing 100 µL of the transformed ST18 with 200 µL of PA14). Selection on 5% sucrose media was used to identify clean knock-out mutants. The knock-out region was then sequenced to verify that a complete deletion had been made.  2.8 Disk diffusion assay Disk diffusion assays were performed on SCFM prepared as described by Palmer et al (2007) with 0.3% agar and 0.4% (wt/vol) mucin (surfing conditions), or with 0.3% agar without mucin (swimming conditions). Control disk diffusion assays were performed on SCFM 1.5% agar with and without 0.4% mucin. Bacterial strains were grown in Luria broth (LB; Difco) liquid medium overnight then sub-cultured to mid-log phase (OD600=0.4-0.5). To assay motility, mid-log cultures were spotted on agar surfaces at four points around an antibiotic disk (Appendix Figure A-1) impregnated with 10uL of antibiotic at the concentrations indicated (Appendix Table A-1). Agar plates were air-dried at 37°C for 30 min before inoculation and application of antibiotic disks. Once inoculated, plates were incubated at 37°C for 15-18 hours. The zone of inhibition surrounding the antibiotic disk was measured in millimeters using a ruler. In the case of asymmetric zones of inhibition, the average of the four sides was taken. Disk diffusion controls or growth controls were spread as lawns on plates and antibiotic disks were applied to the centre. Two-way ANOVA was used to determine if any significant difference existed between surfing and swimming conditions. All statistical analysis was done using Graphpad Prism 7.  2.9 Antibiotic incorporation assay Incorporation assays were done on SCFM (Palmer et al., 2007) using 0.3% agar with 0.4% mucin (surfing conditions) and 0.3% agar without mucin (swimming). Antibiotics were added into the agar before solidification. Once hardened, plates were air-dried for 30 minutes at 37°C before being inoculated with 1 µL of a sub-culture at an OD600=0.4-0.5. Plates were incubated at 37°C for 15-18 hours. Spot inoculation involved stabbing bacteria midway into the agar. The percentage of area growth on the plates was measured using ImageJ. Two-way ANOVA was used to determine if significant differences occurred between the two conditions (surfing and swimming) and between concentrations for surfing. 2.10 Liquid minimal inhibitory concentration (MIC) Liquid MICs were conducted as described by (Wiegand et al., 2008). This assay was  31 performed in liquid SCFM (Palmer et al., 2007) with and without 0.4% mucin. An inoculum of 2 to 7 x105 cells was used. Significant differences between MICs were taken as a 3-fold or greater change.  2.11 Motility zone growth assay Motility growth assays were done on SCFM/0.3-0.5% agar (Palmer et al., 2007) without (swimming motility within the agar) or with 0.4% mucin (surfing motility on the agar surface). Motility assays were performed as previously described in Section 2.2. Measurements of the visible growth zone at 37ºC were taken every hour for 10 hours in the incubator to prevent interruption of incubation. Notches were drawn at the ends of the motility zones at each time point to ensure that measurements were consistently taken from the same sides of the motility colony. Measurements were taken using a ruler in mm.  2.12 Iron titration assay  2,2’-Dipyridyl (Sigma) was added into SCFM with 0.3% agar and 0.4% mucin at various concentrations. Bacterial strains were spot inoculated as described for the motility assays. Iron (FeSO4) was added at various concentrations in addition to the dipyridyl at 50µM and 500µM. Plates were incubated at 37ºC for 15 hours before imaging.      32 Chapter 3. The transcriptomic profile and genes essential to surfing 3.1 Introduction  P. aeruginosa has a relatively large genome compared to other bacterial species and, therefore, high genetic potential. The P. aeruginosa transcriptional network is the third largest known bacterial regulatory network (Galán-Vásquez et al., 2011). P. aeruginosa has a genome size of 6.3 Mbp and about 5,567 predicted genes, 690 predicted regulators and more than a thousand predicted regulatory interactions (Azam & Khan, 2018; Galán-Vásquez et al., 2011).  Galan-Vasquez et al. (2011) used computational predictions to estimate the most influential transcriptional factors in P. aeruginosa (based on published literature and thus biased towards well studied regulators) and identified lasR, Fur, mexT, Vfr, algR, Anr, Ihf, ptxR, rhlR, and algW. Gene regulation is well known to be crucial in mediating virulence and adaptability. Pseudomonas adaptions such as swarming motility and biofilm formation have been shown to exhibit unique transcriptomic profiles and rely on networks of regulators that work cooperatively to induce and maintain each adaptation. Biofilms and swarming have also been shown to have many inverse regulatory mechanisms (Caiazza et al., 2007; Yeung et al., 2009).   With regards to surfing motility, in relation to virulence and resistance, Yeung et al. (2012) previously found using qRT-PCR that genes involved in the T3SS are down-regulated in both the centre and edge while the T2SS is up-regulated (Yeung et al., 2012). Both pyoverdine and pyochelin biosynthesis genes appear to be up-regulated at the edge but down-regulated in the centre. Phenazine biosynthesis, however, is down-regulated in both population of cells (Yeung et al., 2012). A significantly high up-regulation in oprH, phoP, arnB, and pmrB was also found in both the centre and edge cells (Yeung et al., 2012). All of these genes have been shown to be associated with antimicrobial resistance (Bell et al., 1991; Macfarlane et al., 2000; Olaitan et al., 2014).  In regards to motility genes, transposon mutant screens revealed a surfing-dependence on flagella, particularly fliC, fleR, fleS, fliQ, fliD, flgB, and flgC (Yeung et al., 2012). FleRS is a two-component regulatory system found to involved in motility and adherence (Gellatly et al., 2018). Despite the dependence on flagellar biosynthesis genes, a dependence on correlated motor genes, motABCD, however, was not determined (Yeung et al., 2012). It is predicted that alternative motor proteins could be involved. Surfing was also shown to be independent of pili genes (Yeung et al., 2012). Surfing is, however, dependent on the Rhl and Las quorum sensing  33 systems. Disruption mutants in rhlI and lasI were shown to be surfing deficient whereas the addition of each respective autoinducer exogenously restored surfing motility in these mutants (Yeung et al., 2012).  In this chapter, I have extended these preliminary data to show that, like swarming and biofilm formation, surfing also exhibited a unique and extensive transcriptomic profile with distinct gene expression patterns in cells collected from the edge and centre. RNA-Seq revealed >1,000 genes dysregulated in the surf centre and at the surf edge compared to swimming cells. These differential expression patterns revealed that cells in the centre and edge were metabolically distinct. Here I have shown that surfing is dependent on a set of approximately 40 regulators, involved in various regulatory systems. Transposon mutant screens for cells with altered surfing, revealed that surfing was dependent on the GacAS master regulator, three of Pseudomonas’ quorum sensing systems namely Las, Rhl, and Pqs, chemotaxis regulators, the two-component system CbrAB, and many others. Cross analysis of the expression levels of each surfing-essential regulator in each of their mutants revealed three regulatory systems that appeared to have relatively high influence on surfing regulation, specially PfeRS, PA1463, and CbrAB.  3.2 The P. aeruginosa surfing adaptation involved a transcriptionally diverse population of cells  Physically, a surfing colony exhibits differences in the centre and at the edge, with a thick white outer edge and a blue-green centre (Yeung et al., 2012). RNA-Seq data (NCBI GEO accession number GSE110044) on surfing cells collected from the centre and edge of surfing colonies revealed that surfing involves distinct cell populations with different transcriptomic profiles. There were 1,094 genes dysregulated at the edge and 1,617 genes in the centre of surf colonies (SCFM + 0.3% agar + 0.4% mucin) relative to swimming cells (SCFM + 0.3% agar) with 487 genes overlapping between the two surfing zones. Figure 3-1 shows a cohort of functionally grouped genes and their expression levels in the centre and at the edge. Interestingly, although surfing was shown to be independent of pili, certain pilus assembly genes were found to be expressed in the centre and at the edge in an inverse manner. Thus alternative pilus genes such as cupE4, cpaB, and tadD were up-regulated in the centre but down-regulated at the edge, while major Type IV pilus biosynthesis genes (pilH, pilG, and pilJ) were down-regulated in the centre but up-regulated at the edge. On the other hand, flagella biosynthesis genes were similarly  34 regulated in both the edge and centre. However, certain flagella genes such as flgN and flgM, which are negative regulators of flagellin synthesis, were down-regulated while structural genes (flgK, flgE, flgJ) as well as flhA, which is a positive regulator of flagella synthesis, were up-regulated in surfing cells. Therefore, regardless of the observed flagellation status previously reported (Yeung et al., 2012), surfing cells were actively expressing flagella assembly proteins throughout the surf colony.   Figure 3-1. Transcriptomic analysis of surfing cells revealed distinct expression patterns in cells collected from the edge and centre. Heatmaps were generated using INVEX. Blue represents down-regulation and red up-regulation relative to a swimming cell control. Three  35 biological replicates were prepared for each sample group (e.g. edge and centre).   Coupled with flagella regulation was chemotaxis, which directly influences the rotation of the flagellar motor. According to the RNA-Seq data, chemotactic transducers, pct genes, were generally down-regulated at the centre and edge, while the wbp chemotaxis genes were generally up-regulated. Core chemotaxis genes (che genes) appeared to not be expressed at the edge but relatively more highly expressed in the centre relative to swimming cells. Therefore, centre cells appeared to be more chemotactic.  Centre and edge cells appeared to have an inverse relationship with regards to virulence gene expression. Quorum sensing regulators for Rhl, Las, and Pqs were more highly up-regulated in the centre, and there was a significant up-regulation of lipases and phospholipases in the centre as well. However, pyoverdine and pyochelin genes were relatively more expressed at the edge whereas they were significantly down-regulated in the centre. Therefore, the centre and edge had relatively distinct virulence characteristics.  Metabolic genes were mostly down-regulated in the centre and generally un-expressed at the edge. This same pattern was observed for F0F1 ATPase genes involved in energy production. The most dysregulation in terms of metabolic genes in the centre were genes involved in arginine and ornithine metabolism. Cells at the edge also revealed a modest down-regulation of these genes. Cells in the centre exhibited a relatively extensive down-regulation of nitrogen metabolism genes especially genes involved in nitrite reduction. In addition to metabolic activity, cell division and protein synthesis genes were also down-regulated in the centre and up-regulated at the edge, speaking to the differential metabolic states of the centre and edge cells.  3.3 Regulatory genes expressed in surfing compared to swarming and biofilms A comparison between RNA-Seq data collected from surfing edge, swarming (courtesy of Shannon Coleman) and biofilm cells (courtesy of Dr. Daniel Pletzer) relative to swimming revealed 21 predicted and known regulators dysregulated in all three conditions (Table 3-1). In addition, surfing motility led to the dysregulation of 63 additional regulators that were also dysregulated under biofilm conditions but not during swarming motility, and 10 that were dysregulated under swarming motility conditions but not in biofilms. Thus, with regards regulation, surfing motility appeared to have more in common with biofilm formation when compared to swarming motility. Table 3-1 summarizes the regulators dysregulated under swarming and biofilm conditions that were also dysregulated in surfing.   36  Table 3-1. Regulatory genes dysregulated in surfing as well as swarming and/or biofilm cells. RNA-Seq was performed on surfing edge cells, swarming tip cells (by Shannon Coleman), and biofilm cells (by Dr. Daniel Pletzer) and analyzed relative to swimming as a control. The relative direction of dysregulation is shown as “-“ for down-regulation, “+” for up-regulation, and “0” for no change. Fold-change cut-off of ± 1.5 was used. Gene annotations and descriptions come from www.pseudomonas.com (Winsor et al., 2016).   Gene ID  Gene name  Description Direction of dysregulation (RNA-Seq) Swarming Surfing edge Biofilm PA0612 ptrB Repressor, PtrB - + + PA3719 armR Antirepressor for MexR, ArmR + - + PA3007 lexA Repressor protein LexA - + + NA tpnC TpnA repressor protein + - - PA3410 hasI RNA polymerase ECF-subfamily sigma-70 factor/HasI - + - PA2387 fpvI RNA polymerase sigma factor/FpvI + - - PA1912 femI ECF subfamily RNA polymerase sigma-70 factor/ECF sigma factor, FemI - - - PA1300   RNA polymerase ECF-subfamily sigma-70 factor/ECF sigma factor + - - PA4896   RNA polymerase sigma factor/probable sigma-70 factor, ECF subfamily - - - PA0520 nirQ Regulatory protein NirQ - - + PA3932   Probable transcriptional regulator - - + PA3391 nosR Regulatory protein NosR - - + PA2825 ospR MarR family transcriptional regulator/OspR - + + PA2383   Probable transcriptional regulator + + - PA2320 gntR Transcriptional regulator GntR + - - PA2303 ambD Regulatory protein/AmbD + + - PA1243   Probable sensor/response regulator hybrid + + + PA1179 phoP Two-component response regulator PhoP - - + NA rcsB Two-component response regulator + - + PA4659   MerR family transcriptional regulator - - + PA4843 gcbA Two-component response regulator/GcbA - - - PA4878 brlR Transcriptional regulator/BrlR - + + PA0424 mexR Multidrug resistance operon repressor MexR 0 - + PA0149  Probable sigma-70 factor, ECF subfamily 0 - - PA0472 fiuI RNA polymerase sigma factor/FiuI 0 - - PA3899 fecI RNA polymerase sigma factor/FecI 0 - - PA2468 foxI ECF subfamily RNA polymerase sigma-70 factor/ECF sigma factor FoxI 0 - - PA2426 pvdS Extracytoplasmic-function sigma-70 factor/sigma 0 - -  37 factor PvdS PA0179 cheY Chemotaxis protein, CheY 0 - + PA0448 gcdR LysR family transcriptional regulator, GcdR 0 + - PA4165   Probable transcriptional regulator 0 + - PA4132   Conserved hypothetical protein 0 + + PA4112   Probable sensor/response regulator hybrid 0 - - PA3995   Probable transcriptional regulator 0 - + PA3973   Probable transcriptional regulator 0 - + PA3921   Probable transcriptional regulator 0 - - PA3782   Probable transcriptional regulator 0 - - PA3721 nalC Transcriptional regulator/NalC 0 - + PA3385 algZ Alginate and motility regulator Z 0 - + PA3341 slyA MarR family transcriptional regulator/probable transcriptional regulator 0 - + PA3260   Probable transcriptional regulator 0 - + PA3160 wzz O-antigen chain length regulator 0 + + PA3034   Probable transcriptional regulator 0 - + PA3006 psrA Transcriptional regulator PsrA 0 - + PA2931 cifR Transcriptional regulator/CifR 0 - + PA2846   LysR family transcriptional regulator 0 - + PA2718   MerR family transcriptional regulator 0 - + PA2696   Probable transcriptional regulator 0 - + PA2663 ppyR Pyoverdine operon regulator, PpyR 0 - + PA2586 gacA Response regulator GacA 0 - + PA2572   Probable two-component response regulator 0 - + PA2523 czcR Two-component response regulator/CzcR 0 + - PA2519 xylS Transcriptional regulator XylS 0 - + PA2511 antR Transcriptional regulator/AntR 0 - + PA2376   Probable transcriptional regulator 0 - + PA2127 cgrA CupA gene regulator A, CgrA 0 + + PA2005 hbcR Transcriptional regulator/HbcR 0 + - PA1998 dchR LTranscriptional regulator, DchR 0 - + PA1911 femR Sigma factor regulator, FemR 0 - - PA1760   Probable transcriptional regulator 0 - - PA1430 lasR Transcriptional regulator LasR 0 - + PA1399   Probable transcriptional regulator 0 - + PA1290   Probable transcriptional regulator 0 - + PA1269   Probable transcriptional regulator 0 - + PA1196 ddaR Transcriptional regulator DdaR 0 - + PA0942   Probable transcriptional regulator 0 - + PA0929 pirR Two-component response regulator 0 - - PA0877   Probable transcriptional regulator 0 - + PA0876   Probable transcriptional regulator 0 - + PA0873 phhR/ Transcriptional regulator PhhR 0 - + PA0839   Probable transcriptional regulator 0 - +  38 PA4296 pprB Two-component response regulator, PprB 0 - + NA pvrR Two-component response regulator 0 - + PA4596 nfxB Transcriptional regulator NfxB 0 + + PA4726 cbrB Two-component response regulator CbrB 0 - + PA4776 pmrA Two-component regulator system response regulator PmrA 0 - + PA4777 pmrB Two-component regulator system signal sensor kinase PmrB 0 - + PA4856 retS RetS (Regulator of Exopolysaccharide and Type III Secretion) 0 + - PA5274 rnk Nucleoside diphosphate kinase regulator 0 + + PA5324 sphR Sphingosine-responsive Regulator, SphR 0 - - PA5342   Probable transcriptional regulator 0 + - PA5356 glcC Transcriptional regulator GlcC 0 + - PA5382 yeiE Probable transcriptional regulator 0 + - PA5438   Probable transcriptional regulator 0 - + PA5499 np20 Transcriptional regulator np20 0 - + PA1351   RNA polymerase ECF-subfamily sigma-70 factor + - 0 PA0367   Probable transcriptional regulator + - 0 PA3346 hsbR HptB-dependent secretion and biofilm regulator HsbR + - 0 PA2917   Probable transcriptional regulator - - 0 PA2588   Probable transcriptional regulator + - 0 PA2032 yjiR Transcriptional regulator + - 0 PA1836   Probable transcriptional regulator + - 0 PA1431 rsaL Regulatory protein RsaL + - 0 PA5059 phaD TetR family transcriptional regulator + + 0 PA5437   Probable transcriptional regulator - + 0  3.4 Mutant screens revealed more than 100 surfing-essential genes  From the screen of the PA14 transposon mutant library (Liberati et al., 2006), 5,307 mutants were screened under surfing conditions, i.e. on SCFM medium with 0.3-5% agar and 0.4% mucin. An initial large plate screen yielded 320 mutants that exhibited irregular surfing as shown in Figure 3-2. When retested using regular Petri dishes, none of the mutants initially identified as hypersurfers showed any significant difference in their motility zone when compared to the wild-type. Non-surfing behaviour was observed for 192 mutants, which exhibited either a complete lack of motility, alternative forms of motility such as swimming or swarming, or one-directional motility (Figure 3-2). Among these 192 mutants exhibiting surfing deficiency, 44 mutants belonged to regulatory genes as listed in Table 3-2. There were 13 regulators belonging to two-component regulatory systems, 10 regulators belonging to one of the four known quorum sensing  39 systems, and 4 regulators involved in chemotaxis.   Figure 3-2. Sequential screening of the strain PA14 transposon mutant library revealed 44 regulators required for surfing motility. The library consisted of 5,664 mutants, 94% of which proved viable. Initial large plate screening yielded 320 mutants exhibiting irregular surfing (i.e. hypersurfing or surfing deficiency). Retesting on regular Petri dishes, led to the verification of 192 mutants as surfing deficient, exhibiting either not motility, alternative forms of motility (e.g. swarming), or one-directional (1D) motility phenomena. Among the 192, there were 44 mutants in regulatory genes.  Table 3-2. Regulators for which transposon mutant variants exhibited surfing deficiency. Surfing deficiency included no motility, a different form of surfing such as swimming or swarming, or One-directional (1D) motility. The mutant library screen was performed as described in Fig. 3.2. RNA-Seq data was collected from edge and centre cells of surfing colonies grown in SCFM with 0.5% agar/0.4% mucin using wild-type PA14. RNA-Seq fold-change had a cut-off of ± 1.5. Descriptions and gene annotations are from www.pseudomonas.com (Winsor et al., 2016).  Gene Fold Change RNA-Seq Gene Product Description Surfing phenotype in Library Screen Centre Edge cbrA NC NC Two-component sensor No motility cbrB NC -1.95 Two-component response regulator No motility cheA 3.04 NC Chemotaxis protein No motility cheW 3.14 NC Chemotaxis protein No motility  40 cheZ NC NC Chemotaxis protein No motility cueR NC NC Copper-responsive transcriptional regulator Swimming cysB NC NC Transcriptional regulator No motility czcS NC NC Putative heavy metal sensor histidine kinase No motility dipA -1.98 -2.14 Putative sensory box protein Swimming fleQ NC NC Transcriptional regulator 1D motility fleR NC NC Probably two-component response regulator No motility fleS NC NC Two-component sensor Swimming flgM -2.94 -3.21 Flagellin biosynthesis negative regulator FlgM No motility gacA NC NC Response regulator No motility gacS NC NC Sensor/response regulator hybrid No motility lasI 2.24 NC Autoinducer synthesis protein No motility nirQ -2.38 -2.27 Regulatory protein 1D motility PA0034 NC NC Probably two-component response regulator No motility PA0475 NC NC Probable transcriptional regulator No motility PA1157 NC NC Probable two-component response regulator No motility PA1463 NC NC Hypothetical protein No motility PA2276 NC NC Probable transcriptional regulator 1D motility PA2882 NC NC Probably two-component sensor No motility PA3197 NC NC Hypothetical protein No motility PA3348 NC NC Probable chemotaxis protein methyltransferase 1D motility PA3599 NC -1.81 Probably transcriptional regulator No motility PA3921 NC NC Probable transcriptional regulator 1D motility PA4398 NC NC Two-component sensor No motility PA4831 NC NC Probably transcriptional regulator 1D motility PA5392 NC NC Conserved hypothetical protein No motility pfeS NC NC Two-component sensor Swimming pqsA -3.03 3.29 Probable coenzyme A ligase No motility pqsB -3.46 4.57 2-heptyl-4(1H)-quinolone synthase subunit B No motility pqsC -2.72 5.56 2-heptyl-4(1H)-quinolone synthase subunit C No motility pqsD -3.12 5.14 3-oxoacyl-ACP synthase No motility pqsE -2.33 5.12 Quinolone signal response protein No motility pqsH NC NC FAD-dependent monooxygenase No motility pqsR NC NC Transcriptional regulator (Also called mvfR) No motility rcsB NC NC Probable response regulator No motility rhlI 3.44 NC Autoinducer synthesis protein No motility rhlR NC NC Transcriptional regulator No motility rocA1 NC NC Two-component response regulator Swimming rpoN NC NC RNA polymerase sigma-54 No motility rsmA NC 2.22 Regulator of secondary metabolite No motility  Non-regulatory or effectors genes are listed in Appendix Table A-2. The majority of genes required for surfing were hypothetical or metabolic genes, including several genes involved in biotin synthesis, bioA, bioD, bioF, PA0503. Among the effectors found to be important for  41 surfing, algX, an alginate biosynthesis gene involved in chronic infections during the transition from non-mucoidal to mucoidal variant, was identified. Virulence factors included a hydrogen cyanide synthase gene, hcnC, serine protease, mucD, and phospholipase C, plcB. Mutants in twitching motility genes, namely pilT, pilU, pilW, and fimX previously untested by Yeung et al. (2012) were also found to be surfing deficient raising the issue as to the potential for an ancilliary role for twitching in surfing. Previously identified flagellar genes were also confirmed. Additional flagellar genes found to be important for surfing included fliK and fliL. There were also mutants in two cell division proteins that exhibited surfing deficiency, rrmJ and minD. There were four surfing deficient mutants in genes involved in resistance to ROS including the oxidoreductases rmd, PA0545, PA3489, and PA1127. Other critical resistance genes included the following porins and efflux pumps: oprO, PA2454, and PA4455. There were also three tonB-dependent receptors found to be important for surfing: PA4168, PA2089, and PA1271.  3.5 Surfing was mediated by interactions between 44 regulators To identify interdependent regulation (so-called regulatory hierarchy) among the essential regulators identified from the mutant library screen, RT-qPCR was performed for 39 regulator genes, in mutants affecting 29 regulators [NB. only the first gene from any particular operon or pathway was screened in order to avoid redundancy, e.g. both chemotaxis genes, cheZ and cheA, were screened as a representatives of their respective operons). Mutants from each pathway were selected based on the consistency of the surfing deficient phenotype. Figure 3-3 summarizes the results of this analysis as heatmaps clustered based on similar dysregulation patterns among mutants and among regulators, and a graph summarizing the total log fold-change among the other regulators that resulted in response to each disruption mutation. Among the tested regulators, major (also termed master) regulators found higher up in the regulatory hierarchy/network could be identified as those whose mutant resulted in the most dysregulation of the other regulators and regulators which themselves were relatively unaffected by disruption mutations in the other regulators. More specific regulators lower in the regulatory network were those that were most affected by mutants in other regulators while their own mutants demonstrated little to no dysregulation of the other tested regulators occurred. Patterns of dysregulation among the mutants when comparing results at the edge and centre varied greatly (Figure 3-3C,D). At both the centre and edge, mutants in gacA, cbrA, PA1463, pfeS, and rpoC were the five that demonstrated the greatest influence on expression of other regulators. In  42 contrast there was substantial variation in the influence of genes found lower in the regulatory network.   43  Figure 3-3. Gene expression profiles (RT-qPCR) of 39 regulators in mutants in 29 individual regulators required for surfing. Gene expression relative to that assessed in the centre of wild-type surfing colonies is shown in (A) and (C). Gene expression relative that  44 assessed at the edge of wild-type surfing colonies is shown in (B) and (D). (A) and (B) Heatmaps are hierarchically clustered based on similar gene expression profiles within each mutant and between each regulator. Green represents up-regulation and red represents down-regulation. (C) and (D) show the sum of the log fold change number of other regulators that change expression in each respective mutant, as identified in this checkerboard RT-qPCR assay. Regultors affecting the greatest number of other regulators influence these reglators and thus the highest bars are highest in the hierarchy.  3.6 Discussion RNA-Seq revealed that surfing motility is a complex adaptation that involved large transcriptomic changes, with more than a thousand genes dysregulated. Surfing cells in the centre and at the edge exhibited distinct transcriptomic profiles. Analysis of these profiles were consistent with the possibility that cells in the centre appeared to be less metabolically active compared to those at the edge where cells appeared to be actively dividing and growing; this seems reasonable since nutrients would be more likely to have been consumed at the centre. Although motility genes specifically for flagella biosynthesis were generally expressed throughout a surfing colony, chemotaxis genes were more actively expressed in the centre. Quorum sensing genes were also more actively expressed in the centre which correlated with the higher density of cells in this region and the up-regulation of virulence factors such as elastases and lipases. However, edge cells were expressing more pyoverdine and pyochelin biosynthesis genes, indicating an active requirement for iron acquisition.  Among the approximate 106 regulators found to be dysregulated at the surfing edge, only 31 were found to also be dysregulated under swarming (as compared to data collected by Coleman) and 84 in biofilms (as compared to data collected by Pletzer) with 21 shared among the three conditions. Among the 21 shared regulators between the three conditions, 4 were dysregulated in the same direction (i.e. all down- or up-regulated). Two of the regulators were predicted sigma factors specifically from the sigma-70 family (FemI, PA4896). Sigma-70 or RpoD in P. aeruginosa is a major sigma factor involved in facilitating the recognition of diverse promoters by RNA polymerase. Sigma-70 sigma factors are divided into four different subgroups, homologous to RpoD (Potvin et al., 2008). The first group involves RpoD and sigma factors closely related to it, which all play similar functions and are essential for cell survival. The other groups, however, are non-essential and less homologous to RpoD. They play roles in stress responses, motility, and other adaptations (Potvin et al., 2008). FemI is an extracytoplasmic function (ECF) sigma factor that regulates the expression of a TonB-dependent transducer,  45 FemA (Llamas et al., 2008). FemA is involved in iron-uptake through the mycobactin siderophore produced by Mycobacterium species (Llamas et al., 2008). A lack of mycobactin in the system may have resulted in a shut-down of the FemI/FemA system. Both of these were down-regulated under surfing conditionsa ccording to the RNA-Seq data. RNA-Seq data also revealed that several other TonB-dependent receptors were down-regulated in surfing. The mutant library screen, however, revealed that surfing was dependent on 3 TonB-dependent receptors: PA4168, PA2089, and PA1271. Therefore, the three conditions, surfing, swarming, and biofilms, may have exhibited a similar shut down of the FemI/FemA system involved in iron acquisition and may rely on other mechanisms of high affinity iron acquisition.  The 2 other regulators similarly dysregulated among the three conditions were a sensor/response hybrid, PA1243, and a two-component response regulator, GcbA. PA1243 was up-regulated in all three conditions while GcbA was down-regulated. PA1243 was found to be a part of the sigma-22 or AlgT regulon (Wood and Ohman, 2012); however, no other studies on PA1243 have been performed. AlgT is an ECF sigma factor that is involved in the production of alginate and the transition of P. aeruginosa during chronic infection from non-mucoid to a mucoid variant (Wood and Ohman, 2012). GcbA, which was found to be down-regulated in all three conditions, is a diguanylate cyclase involved in the production of the secondary messenger, c-di-GMP (Petrova et al., 2014). Although it is found to be involved in the initial attachment stage of biofilms, it is also found to be non-essential for biofilm formation (Petrova et al., 2014). It is found to be involved in flagella-dependent motility and the switch between motile to sessile lifestyle (Petrova et al., 2014). Interestingly, these genes involved in alginate production and c-di-GMP synthesis were similarly dysregulated in all three distinct adaptations.  Among the 63 additional regulators dysregulated in both surfing and biofilm but not swarming, 14 shared the same direction of dysregulation and only two shared up-regulation, the O-antigen chain length regulator, wzz, and transcriptional regulator, nfxB. Wzz regulates the length of the O-antigen before it is attached to the LPS core (Daniels et al., 2002). NfxB is a repressor of the multidrug efflux system, mexCD-oprJ (Purssell and Poole, 2013). An up-regulation of NfxB results in reduced expression of mexCD-oprJ. The other down-regulated genes found in surfing and biofilms included several sigma factors such as FiuI, FecI, FoxI, and PvdS, as well as several predicted or putative transcriptional regulators, and the known regulators SphR and FemR. Surfing also shared an inverse dysregulation of 49 regulators  46 compared to biofilms. These regulators included NalC, GacA, LasR, and CbrA which are regulators found through the mutant library screen to be essential for mediating surfing motility.  Surfing shared 10 additional regulators with swarming but not biofilm cells. Among the ten, only two shared the same direction of dysregulation. PhaD was up-regulated in both swarming and surfing. PhaD (PA5059) in Pseudomonas oleovorans was found to be involved in synthesis of poly(3-hydroxyalkanoates) (PHAs), as a means of storing carbon under nutrient limiting conditions (Klinke et al., 2000). Nine of the regulators found to be dysregulated in both surfing and swarming exhibited an inverse relationship. A majority were putative transcriptional regulators and the known regulator RsaL. RsaL is a regulatory protein involved in regulating the expression of virulence genes through the regulation of quorum sensing (De Kievit et al., 2001). This gene was found to be up-regulated in swarming but down-regulated in surfing edge cells.   The mutant library screen revealed 192 mutants with an altered surfing phenotype. Among the 192 were approximately 40 mutants in regulator genes with several in overlapping operons. Surfing was found to be dependent on three quorum sensing systems, Rhl, Las, and Pqs as well as global regulatory systems such as the GacAS and CbrAB two-component systems. The screen also identified 13 uncharacterized or hypothetical regulators. Regulators found to be essential for surfing motility through the mutant library screen were subsequently tested for dysregulation in mutants of the other regulators using RT-qPCR. Results revealed a large distinction between the overall dysregulation when compared to wild-type edge and centre cells. Although relative to centre cells there was a mixture of dysregulated levels, relative to the edge, many of the regulators exhibited mostly up-regulation compared to the wild-type. In order to distinguish a potential hierarchy in regulation, a set of criteria was established. Regulators belonging higher up in the regulatory network had the following characteristics:  1. Mutants of these regulators resulted in a large fold-change relative to the wild-type 2. Regulator expression was relatively unaffected in a large number of mutants 3. Mutants of these regulators affected the expression levels of a large number of surfing-essential regulators  The following regulatory network was proposed based on these criteria as shown in Figure 3-4. Not all of the 40 regulators could be placed in the network due to contradicting characteristics at the edge and centre or contradicting results in relation to the proposed criteria shown above (i.e. mutants caused dysregulation in many genes but were also dysregulated in  47 many mutants). Therefore, Figure 3-4 highlights regulators that shared similar patterns of dysregulation relative to both centre and edge cells and that could be matched to the proposed criteria. Among the 13 regulators which shared similar effects in both the centre and edge, three regulators were identified as having the largest influence in the expression pattern of other surfing-essential regulators, therefore, higher up in the regulatory network. These regulators were PfeS, CbrA, and PA1463.   Figure 3-4. Putative surfing regulatory network based on regulators that had the same expression patterns in centre and edge cells. Each tier represents regulators whose mutant variant exhibited relatively the same number of dysregulated regulators and whose regulator’s expression levels were affected in the same number of mutants. Tier 1 represents regulators who had the most effect on the expression of other regulators, exhibited the highest sum logFC, and which were the least affected in mutants of other regulators. Tier 4 represents the regulators whose mutants had the least effect on the expression of other regulators (0-3), were dysregulated in the greatest number of mutants, and which had a relatively low logFC relative to other mutants. Only 13 regulators exhibited a similar expression pattern between the centre and edge cells as shown here.    Tier 1 • pfeS, cbrA, PA1463Tier 2 • PA2276, rocA1, PA4398, PA3197Tier 3 • rcsB, gacS, PA2882Tier 4 • PA0034, pqsA, PA2524 48 Chapter 4. PA1463 and PfeR as major regulators of surfing motility  4.1 Introduction In Chapter 3, major regulators involved in surfing were identified through a mutant library screen. Despite expression profiles being different at the edge and centre, there was consensus on the importance of 3 major regulators: PfeS, CbrA, and PA1463. CbrA is a two-component sensor cognate to CbrB, the response regulator. The CbrAB two-component system regulates genes for carbon and nitrogen catabolism (Li and Lu, 2007). It was also found to be essential for mediating swarming motility, biofilm formation, and cytotoxicity (Yeung et al., 2014). However, CbrA was found to be involved in virulence during acute infections and antibiotic resistance independent of CbrB (Yeung et al., 2014, 2011). Consistent with its reduced virulence CbrA grown in amoeba was found to be involved in regulating the expression of virulence, iron acquisition, and redox response genes (Yeung et al., 2014). Here I found that transposon mutants in both cbrA and cbrB were surfing deficient and, therefore, essential for mediating surfing (Chapter 3 Table 3-2). The role of CbrAB has been extensively studied as a global regulator involved in swarming motility (Yeung et al., 2011). The role of PfeS, a sensor kinase, and PA1463, a putative chemotaxis regulator, however, had not been well established and, here I investigated PfeS and PA1463 as major regulators involved in surfing motility.  PfeRS is a two-component regulatory system known to regulate the expression of the ferric enterobactin receptor, pfeA (Dean & Poole, 1993). PfeA is normally involved in iron acquisition through the siderophore, enterobactin which is produced by E. coli. P. aeruginosa is known to produce two types of siderophores, pyoverdine (pyo) and pyochelin (pch) (Dean et al., 1996). However, Pseudomonas can also recognize and use siderophores produced by other bacterial species including enterobactin, ferrioxamine B, and aerobactins (Cornelis et al., 1987; Poole et al., 1990). Here it was shown that knocking out pfeR or pfeA resulted in surfing deficiency. However, given the lack of enterobactin in the experiments, it is proposed that PfeA may be responsive to other molecules, such as other types of iron chelators.  Here I found that PfeRS appeared to be involved in regulating a large subset of genes specifically under surfing motility. RT-qPCR data (Figure 3.3) revealed that the pfeS disruption mutant exhibited dysregulation of several other surfing-essential regulators. Here I performed RNA-Seq on a pfeR surfing-deficient knock-out mutant that revealed 1,856 genes dysregulated under surfing conditions indicating that this gene encoded a global regulator. The presence of a  49 homologous Fur binding site upstream of the pfeRS operon indicates that pfeRS expression is iron regulated (Dean et al., 1996). Consistent with this I showed here that surfing had a dependency on iron, whereby low iron conditions resulted in reduced surfing which could be restored to some extent by overexpressing the pfeRS operon.  PA1463 is an uncharacterized hypothetical chemotaxis protein based on its possession of a conserved CheW domain. It is in a predicted operon of 3 genes, PA1463-PA1464-PA1465 and the latter two genes have also been predicted to be involved in chemotaxis (Mao et al., 2009). It was recently proposed that PA1463 is homologous to the Vibrio sp. ParP protein, which plays a role in the localization of chemotactic proteins within the cell (Reinhardt and Bardy, 2018; Ringgaard et al., 2014). Specifically, Reinhardt and Bardy (2018) found that PA1463 regulates the localization of CheA and DipA, directly interacting with DipA. Mutants of both dipA and parP were found to be deficient in swimming and biofilm dispersal (Reinhardt and Bardy, 2018). DipA is a phosphodiesterase involved in the regulation of c-di-GMP levels (Roy et al., 2012). It reduces c-di-GMP levels and promotes a shift from sessile to motile lifestyle, thus, playing a key role in biofilm dispersion. Therefore, dipA mutants exhibit high levels of c-di-GMP, reduced swarming motility, and increased biofilm formation (Roy et al., 2012). Here I found that the PA1463 operon also played a key role in regulating several other regulators, and knocking-out the PA1463 operon resulted in the dysregulation of 827 genes under surfing conditions indicating its potential role as a master regulator.  4.2 Surfing is highly dependent on the pfeRS and PA1463 operons   Chromosomal deletions, in the PAO1 strain of P. aeruginosa, of pfeR, the cognitive response regulator of pfeS, and pfeA, an enterobactin receptor regulated by PfeRS, were obtained from Dr. Keith Poole, Queen’s University (Dean et al., 1996); NB the pfe mutants were generated in a spontaneous pyoverdine deficient mutant (PAO6609). PA1463 is the first gene in a 3 gene operon PA1463-PA1464-PA1465 (Mao et al., 2009). A chromosomal deletion mutant of the whole PA1463 operon (PA1463o) was constructed in P. aeruginosa PA14. Both mutants in pfeR and PA1463o exhibited complete inhibition of surfing motility in SCFM/0.5% agar with mucin (Figure 4-1). Complementation using a high copy plasmid containing each respective operon restored the wild-type surfing phenotype. Conversely both a spontaneous pyoverdine deficient mutant (PAO6609) and a chromosomal deletion mutant of all pyo/pch (pyoverdine and pyochelin) genes continued to exhibit wild-type-like surfing, albeit lacking the normal blue/green  50 pigmentation in the centre of the motility zone. A mutant in pfeA, one of the genes known to be regulated by PfeRS, was also found to be surfing deficient when knocked-out. No substantial growth deficiencies were observed for any of the mutants tested, and although the pfeA knock-out exhibited slower growth during the exponential phase it reached the same OD600 as the other strains after 13 hours (Figure 4-1).  Figure 4-1. Surfing was inhibited in knock out mutations in pfeR or the PA1463 operon. All strains were grown on SCFM/0.5% agar with 0.4% mucin and incubated at 37°C for 18 hours. Knock-out mutants of pfeR and pfeA in the PAO6609 strain and PA1463o in the PA14 strain exhibited surfing deficiency. Mutants complemented with the cloned operons exhibited either wild-type surfing and in the case of DpfeR/pfeRS+ led to a modest increase in surfing motility (greater surface coverage in the same amount of time). Pyoverdine and pyochelin mutants exhibited similar surfing to the wild-type but lacked pigmentation. The complemented strain DPyo/pch exhibited an irregular motility zone shape. Growth curves were generated in regular LB media over a period of 16 hours.  4.3 PfeR and PA1463 as master regulators in surfing As previously shown in Chapter 3, both pfeS and PA1463 were found to be important for mediating surfing motility and their transposon mutants were responsible for the dysregulation of  51 a large subset of other surfing-essential regulators (Figure 3-3). Table 4-1 shows the RT-qPCR data collected on surfing-essential regulators in the context of the pfeR and PA1463o knock-out mutants and their complemented strains. In the pfeR mutant, 13 regulators were found to be down-regulated and two up-regulated among the 32 tested. Among these 15, 9 exhibited wild-type expression levels in the complemented strain. In the PA1463o knock-out, 14 regulators were down-regulated with only one significantly up-regulated gene. Thirteen of the dysregulated genes exhibited wild-type levels of expression in the complemented strain. These two mutants had different dysregulation patterns for the tested regulators. For example, dipA was significantly dysregulated in the PA1463o mutant but not in the pfeR mutant. It is also worthy of note that PA1463 was significantly down-regulated in the pfeR mutant but the complemented strain still exhibited significant down-regulation. PfeS, on the other hand, was not significantly dysregulated in the PA1463o mutant.  Table 4-1. Expression of surfing-essential regulators in the pfeR and PA1463 operon mutants and complements. RT-qPCR was performed on RNA collected from the pfeR and PA1463o knock-outs under surfing conditions (SCFM/0.5% agar with 0.4% mucin) relative to their respective wild-type/parent strains (PA14 for PA1463 and PAO6609 for pfeR) also grown under surfing conditions. A fold-change cut-off of ±2 was considered used as a cut-off as indicated with *. RpoD was used as the house-keeping gene.  DpfeR DPA1463o Gene FC Mutant FC Complement FC Mutant FC Complement cbrA -2.91* 1.42 -1.96 2.31* cheZ 1.02 -1.80 -1.12 1.09 cueR -2.70* -5.02* -1.98 1.14 cysB -2.35* -2.75* -1.55 1.05 czcS -4.39* -1.38 -5.98* -1.45 dipA -1.05 1.06 -2.57* 1.57 fleQ -1.50 1.03 -2.17* -1.30 fleR 1.69 -1.26 1.32 -2.18* flgM -1.73 1.02 -1.43 1.10 gacA -2.22* 1.29 -4.19* 1.50 lasI -2.42* -13.12* 1.24 1.08 motB -1.03 -1.99 1.17 1.33 nirQ -2.65* -1.5 -5.86* 1.37 PA0034 -1.25 -1.15 -1.41 1.50 PA0475 -2.95* -1.07 -5.84* -1.31 PA1157 1.13 -1.07 -2.85* -1.51 PA1463 -4.08* -3.25* - -  52 PA2276 -3.67* 1.47 -3.40* -1.05 PA2882 -2.82* -1.97 -1.04 1.82 PA3197 1.09 -1.19 -2.63* -1.52 PA3348 -1.30 -2.92* -1.51 -1.02 PA3599 1.10 1.02 -4.59* -1.55 PA3921 1.67 -1.07 -1.71 1.15 PA4398 2.75* 1.2 1.30 4.25* PA4831 -1.32 -1.15 -3.18* -2.08* PA5392 -2.45* -1.22 -2.75* 1.03 pfeS - - -1.41 1.50 pqsA 1.67 -3.18* 4.31* -2.25* pqsR 1.51 -2.42* -2.27* -1.91 rcsB -11.67* -3.67* -1.46 -1.02 rhlR 2.15* -2.92* 1.25 -1.61 rocA1 1.9 1.27 1.40 1.79 rpoN 1.51 -1.33 -2.07* -1.40  4.4 PfeR and PA1463 regulated a large subset of Pseudomonas genes RNA-Seq performed on the PAO6609 (derived from the PAO1 WT) surfing motility edge relative to swimming cells revealed 499 significantly dysregulated genes as summarized in Appendix Table A-3. In comparison, as was shown in Chapter 3, the PA14 strain had 1,094 dysregulated genes in the surfing motility edge relative to swimming cells (NCBI GEO accession number GSE110044). The surfing-deficient DpfeR mutant relative to the PAO6609 WT under surfing conditions demonstrated 1,856 dysregulated genes (1,177 upregulated, 679 downregulated) where 6 genes exhibited inverse dysregulation compared to wild-type surfing as summarized in Table 4-2. Conversely, the complemented mutant demonstrated only 390 dysregulated genes relative to the WT.  Among the 1,856 genes found to be both dysregulated in the mutant relative to the wild-type under surfing conditions (Appendix Table A-4), there were several virulence genes including phosphatases, phospholipases, phenazines, and elastases. There were also several redox genes known to be involved in the oxidative stress response. There was a large subset of iron transport genes up-regulated including several pyochelin synthesis genes and fptA, the pyochelin-iron receptor. Several other iron transporters, including fpvB, the alternative pyoverdine transporter, were found to be down-regulated. TonB, involved in energization of siderophore-mediated iron acquisition in Pseudomonas (Poole et al., 1996), was found to be up-regulated. Also among the  53 genes dysregulated in the mutant were the rhlI and rhlR genes, that mediated the Rhl quorum sensing regulation, which had previously been shown to be essential for mediating surfing (Table 3-3). Table 4-2 shows 48 genes previously found to be essential for surfing initation through the mutant library screen (Chapter 3) that were dysregulated in the DpfeR mutant compared to the parent strain including genes involved in alginate biosynthesis (algX) and fimbriae assembly (cupA, cupE) which were ~4 fold down-regulated. Table 4-2. Surfing-essential effectors dysregulated in the △pfeR and △PA1463o mutants. RNA-Seq was performed on the surfing deficient DpfeR and DPA1463 mutants compared to the parent strain PAO6609 and wild-type PA14 surfing respectively in SCFM/0.5% agar with 0.4% mucin. A log fold-change cut-off of ± 1.5 and p-value < 0.05 was used. Surfing-essential genes were found through the PA14 Tn mutant library screen as described in Chapter 3. Those genes were matched up to the dysregulated genes in both mutants to reveal 48 surfing-essential effector genes dysregulated in the DpfeR mutant and 17 in the DPA1463 mutant. Descriptions and gene annotations are from www.pseudomonas.com (Winsor et al., 2016). Gene ID Gene Name Description LogFC Mutant/ WT surf pfeR/PAO6609 PA0104  Hypothetical protein -2.52 PA0504 bioD Dethiobiotin synthase -1.57 PA0545  Hypothetical protein -2.59 PA0551 epd D-erythrose 4-phosphate dehydrogenase 1.56 PA0663  Hypothetical protein 1.55 PA0718  Hypothetical protein of bacteriophage Pf1 -2.44 PA0766 mucD Serine protease 1.60 PA0817  Probable ring-cleaving dioxygenase -2.30 PA1119 yfiB YfiB 1.96 PA1187  Probable acyl-Coa dehydrogenase -2.17 PA1271  Probable TonB-dependent receptor 1.75 PA1547  Hypothetical protein -2.66 PA1875  Probable outer membrane protein precursor 4.30 PA1935  Hypothetical protein -2.55 PA1982 exaA Quinoprotein ethanol dehydrogenase -1.91 PA2009 hmgA Homogentisate 1,2-dioxygenase 2.68 PA2089  Hypothetical protein -4.26 PA2120  Hypothetical protein -2.43 PA2130 cupA3 Usher cupa3 -4.35 PA2195 hcnC Hydrogen cyanide synthase 1.56 PA2576  Hypothetical protein -2.15 PA2685 vgrG4 Vgrg4 1.59 PA2693  Conserved hypothetical protein -2.18  54 PA2969 plsX Fatty acid biosynthesis protein 2.14 PA3324  Probable short-chain dehydrogenase -2.64 PA3325  Conserved hypothetical protein -2.12 PA3387 rhlG Beta-ketoacyl reductase -2.02 PA3546 algX Alginate biosynthesis protein -4.73 PA3573  Probable major facilitator superfamily (MFS) transporter -1.63 PA3735 thrC Threonine synthase 1.52 PA3749  Probable major facilitator superfamily (MFS) transporter -3.83 PA3818  Extragenic suppressor protein 2.54 PA3884  Hypothetical protein -3.15 PA4001 sltB1 Soluble lytic transglycosylase B 1.53 PA4006 nadD1 Nicotinate mononucleotide adenylyltransferase 1.76 PA4144  Probable outer membrane protein precursor -2.24 PA4168 fpvB Second ferric pyoverdine receptor 1.76 PA4210 phzA1 Probable phenazine biosynthesis protein 5.38 PA4471  Hypothetical protein 5.22 PA4612  Conserved hypothetical protein -2.27 PA4650 cupE3 Pilin subunit 2.04 PA4743 rbfA Ribosome-binding factor A 2.91 PA4753  Conserved hypothetical protein 2.00 PA4981 lysP Lysine-specific permease -2.39 PA5015 aceE Pyruvate dehydrogenase 1.71 PA5192 pckA Phosphoenolpyruvate carboxykinase 1.78 PA5323 argB Acetylglutamate kinase 2.48 PA5399 dgcB Dimethylglycine catabolism -2.93 PA5555 atpG ATP synthase gamma chain 1.92 PA1463o/PA14 WT PA0062  Lipoprotein 1.95 PA0298 spuB Glutamine synthetase -2.07 PA0545  Hypothetical protein -1.75 PA0766 mucD Serine protease 1.73 PA1838 cysI Sulfite reductase 1.53 PA2120  Hypothetical protein -2.29 PA2693  Hypothetical protein -1.77 PA2927  Hypothetical protein 1.82 PA3324  Short chain dehydrogenase -1.51 PA3730  Hypothetical protein -2.04 PA4144  Outer membrane protein 2.38 PA4552 pilW Type 4 fimbrial biogenesis protein 2.11 PA4616  C4-dicarboxylate-binding protein -1.66 PA4838  Hypothetical protein 4.61 PA5109  Hypothetical protein 1.99 PA5323 argB Acetylglutamate kinase 1.64 PA5399  Ferredoxin -3.49  55  RNA-Seq on the DPA1463o mutant relative to the PA14 WT under surfing conditions revealed 827 significantly dysregulated genes and only 81 that were dysregulated in the PA1463o complement strain relative to the wild-type. Of interest, four chemotaxis genes, namely cheW, cheA, PA0236, and PA0176, were up-regulated in the PA1463o mutant relative to WT surfing (Table 4-2, A-5). CheW and PA01776 exhibited a ~1.6-1.7 fold upregulation in the mutant whereas they were found to be down-regulated in wild-type surfing (Table 4-2). The mutant also exhibited significant downregulation in the PQS quorum sensing system (Appendix Table A-5), which had also been shown through the mutant library screen to be essential for mediating surfing motility (Table 3-2). More specifically, the mutant exhibited an up-regulation of the pqsABCDE operon and pqsH, which are involved in synthesizing the final autoinducer, PQS, under surfing conditions relative to the WT. Among the 827 dysregulated genes in the mutant, 17 were previously found to be essential for surfing initiation through the mutant library screen (Chapter 3) as summarized in Table 4-2 including genes involved in alginate biosynthesis (mucD) and pili assembly (pilW). 4.5 Surfing dependence on iron and PfeRS regulation  Due to the known involvement of the PfeRS system in iron acquisition (Dean et al., 1996; Dean & Poole, 1993), the dependency of surfing on iron was investigated. Dipyridyl is a chelator that binds iron to derecase its availability to bacteria. Titrating dipyridyl into SCFM/0.3% agar with mucin resulted in an initial shift from surfing occurring on the surface to swimming which occurred within the agar (Figure 4-2). Increasing the concentration of dipyridyl from 25µM to 50µM resulted in a shift from surfing to swimming. Increasing concentrations of dipyridyl or decreasing concentrations of iron resulted in reduced swimming, with a complete abolishment of motility and growth at 500µM dipyridyl. Adding iron (FeSO4) into a system where surfing had been prevented (50µM dipyridyl) resulted in a gradual increase in surfing motility. Surfing was completely restored by adding 10µM FeSO4. In 500µM of dipyridyl that completely inhibited motility and growth, addition of 200µM FeSO4 restored wild-type surfing. Increasing iron concentrations led to an increase in the density of surface growth. Overexpression of pfeRS in the wild-type abolished the switch to swimming when dipyridyl was titrated into the system indicating a role for this transport system in efficiently assimilating iron in the context of surfing motility. In this case, surfing motility only became deficient at 250µM dipyridyl and growth was  56 absent at 500µM dipyridyl.   Figure 4-2. Surfing motility was dependent on iron and surfing persisted under more extreme iron-limiting conditions when pfeRS was overexpressed. Iron was removed from the medium using 2,2-dipyridyl. Cells were inoculated and grown at 37°C for 15 hours in SCFM with 0.3% agar and 0.4% mucin.  4.6 Discussion   The mutant library screen (Table 3-2) identified 44 regulators found to be essential for mediating surfing motility. Among these regulators, a checkerboard RT-qPCR assay measuring the expression levels of 39 of these regulators in transposon mutants in 29 of these regulators revealed 3 that appeared to act as master regulators affecting the expression levels of a large subset of other essential surfing regulators. Among the three, pfeS and PA1463 had not been previously investigated as master regulators. RNA-Seq revealed that knock out either the pfeR  57 regulator in PAO6609 or the PA1463 operon in PA14 resulted in the dysregulation of a large number of genes, 827 and 1,856 respectively. Both RT-qPCR (Table 4-1) and RNASeq (Table A-4, A-5) also revealed that these mutants in pfeR and PA1463o affected the expression levels, mainly down-regulating, of several essential surfing regulators (Table 3-2). Similarly, compared to wild type surfing cells, the DpfeR and DPA1463o mutants both exhibited down-regulation in the master regulator, gacA, the denitrification regulator, nirQ (Hayashi et al., 1998), the two-component sensor found to be involved in carbapenem resistance, czcS (Perron et al., 2004), and several hypothetical regulators, PA0475, PA2276, and PA5392. Interestingly, although the expression levels of pfeS and pfeR were unaffected in the PA1463o mutant, PA1463 was significantly down-regulated in the pfeR mutant. A DpfeR mutant affected the expression of more than twice the number of genes compared to DPA1463, including affecting the expression of PA1463 itself. Therefore, PfeRS appeared to be higher in the hierarchy of regulation than PA1463. Among the genes dysregulated in the mutants as found through RNA-Seq, 344 genes were similarly dysregulated between the mutants. This constitutes approximately half of the dysregulated genes in the PA1463 mutant.  PA1463, as previously mentioned, is potentially a homolog of the ParP partitioning protein and contains a homologous CheW domain, which functions in CheW in the localization of CheA and DipA (Ringgaard et al., 2014). Interestingly according to the RNA-Seq data, chemotaxis genes including cheA and cheW were found to dysregulated in the DPA1463o mutant, which indicates that PA1463o might also be involved in regulating the expression of cheA and cheW. Notably, cheW exhibited an inverse dysregulation in the mutant compared to the wild-type. DipA expression was also significantly down-regulated in the PA1463o mutant as also found through RT-qPCR (Table 4-1), which indicates that PA1463 also regulates dipA expression. DipA, in turn, regulates several adaptions by regulating the levels of c-di-GMP. Based on the RT-qPCR data, knock out of PA1463o also found affected the expression of several known master regulators including down-regulating the expression of gacA and nirQ. RNA-Seq revealed that several Pqs genes were also dysregulated in the DPA1463o mutant including the entire pqsABCDE operon and pqsH, all involved in the synthesis of the autoinducer PQS. As shown below, I found that the Pqs quorum sensing system played a key role in mediating surfing motility.   The knock-out of pfeR resulted in a massive dysregulation of >1,800 genes including the Rhl  58 quorum sensing regulators, rhlI and rhlR. In addition to the known regulation of pfeA, an iron acquisition receptor, it was found here to affect the expression of several other siderophore receptors as well as the genes mediating the synthesis of pyoverdine and pyochelin. Overexpressing the pfeRS operon in the PAO1 WT resulted in more resilient surfing in iron-limiting conditions, speaking to its role in mediating high-affinity iron acquisition by as yet unknown mechanisms. Although the role of PfeRS in iron-acquisition through PfeA has been extensively studied (Dean et al., 1996; Dean & Poole, 1993; Poole et al., 1990), it has not yet been shown to be involved in regulating other siderophores. Its role as a master regulator has also not been explored. Here I showed that knocking out pfeR resulted in massive transcriptional dysregulation including affecting the expression levels of several known global regulators like GacA, CbrA, RcsB, and PA1463 in the context of surfing. In addition to the many surfing-essential regulators found to be dysregulated in the two mutants, DpfeR and DPA1463o, several surfing-essential effectors were also found to be dysregulated, 48 in the pfeR mutant and 17 in the PA1463o knock-out. Both shared a dysregulation in genes involved in alginate and pili/fimbriae biosynthesis, which may suggest an important role for regulators pfeRS and PA1463 in the initiation of surfing motility.     59 Chapter 5. Broad-spectrum adaptive antibiotic resistance associated with Pseudomonas aeruginosa mucin-dependent surfing motility  5.1 Introduction The rise of antibiotic resistance is a global concern. As the number of new antibiotics being discovered declines and the extensive and sometimes inappropriate use of antibiotics continues, more patients suffer and die from infections caused by antibiotic resistant bacteria (Bassetti et al., 2013; Ventola, 2015). As mentioned in Chapter 1, P. aeruginosa can deploy intrinsic, acquired and adaptive resistance mechanisms (Breidenstein et al., 2011; Taylor et al., 2014). Adaptive resistance refers to resistance that occurs due to environmental circumstances (e.g. exposure to stresses including antibiotics, complex adaptive growth states such as swarming or biofilm formation, etc.) and is thought to be largely due to transcriptional changes in genes that determine resistance/susceptibility and is reversible when environmental circumstances are reversed (Taylor et al., 2014).  Here I expanded on the original observation (Yeung et al., 2012) that surfing cells were polymyxin resistant to demonstrate that surfing cells exhibited multi-drug adaptive resistance, dependent on the complex adaptive changes that accompanied this motility phenotype. Compared to swimming, surfing adaptive cells were significantly more resistant to several classes of antibiotics including aminoglycosides, polymyxins, flouroquinolones, and carbapenems. Screening mutants in resistome genes that were found by me to be dysregulated under surfing conditions revealed changes in susceptibility under surfing conditions that may account for their contribution to the observed resistance.  5.2 Surfing cells exhibited broad-spectrum antibiotic resistance  Disk diffusion assay results (Figure 5-1), assessing how close surfing and swimming cells approached an antibiotic disk, revealed a significant decrease in the zone of inhibition (i.e. increased resistance) under surfing conditions (SCFM/0.3% agar, 0.4% mucin) when compared to swimming (SCFM/0.3% agar). This was observed for 12 of the 17 antibiotics tested with the exceptions of 3 of the β-lactams and 2 macrolides. Compared to swimming bacteria, and disk diffusion assays on solid 1.5% agar plates, surfing cells exhibited significant adaptive resistance to the tested aminoglycosides, carbapenems, polymyxins, fluoroquinolones, trimethoprim, tetracycline, and chloramphenicol, with complete resistance to 3 different aminoglycosides, imipenem, clarithromycin, and the polymyxins (Figure 5-1).  60   Figure 5-1. Multi-drug adaptive resistance of surfing colonies. The zones of inhibition (mm) under swimming (0.3% agar) and surfing (0.3% agar 0.4% mucin) conditions in SCFM were obtained using the motility disk diffusion method with 17 different antibiotics. Statistical significance between swimming and surfing was determined using two-way ANOVA based on 3 independent experiments: * p<0.05, ** p<0.01, *** p< 10-3, **** p<10-4. 5.3 Antibiotic incorporation assays to confirm adaptive resistance  To further investigate the adaptive resistance of surfing colonies, 5 selected antibiotics were incorporated into growth plates to determine how they affected the initiation and propagation of motility colonies. Four of the selected antibiotics, polymyxin B, imipenem, tobramycin and norfloxacin were chosen as representatives of their antibiotic classes that showed the greatest (or complete) resistance under surfing conditions compared to swimming, and no effect of mucin on MICs in liquid media (Table A-6). Tetracycline was chosen since disk diffusion results were more consistent when compared to trimethoprim and chloramphenicol. Antibiotic incorporation assays, revealed a concentration-dependent inhibition of surfing motility and showed that surfing motility proceeded at antibiotic concentrations that completely inhibited swimming (Figure 5-2).   61  Figure 5-2. Antibiotic concentration dependent inhibition of surfing motility. Surfing motility colonies of wild-type PA14 were assessed with the antibiotic at varying concentrations incorporated into 25 mL of SCFM/0.3% agar containing 0.4% mucin (surfing) or no mucin (swimming). Incorporation assay results in part A are described as the % plate coverage, relative  62 to the control with no antibiotics, measured using Image J. Surfing colonies are represented by the black bars and swimming by the grey bars. Statistical significance between surfing and swimming was assessed using two-way ANOVA. * p<0.05, ** p<0.01, *** p< 10-3, **** p<10-4  For example, surfing on the agar surface occurred on 0.1 µM imipenem whereas swimming in-agar was completely abolished at this concentration. As the imipenem concentration increased, there was a clear reduction in the size of the surfing colony and at a concentration of 1 µM imipenem both surfing and swimming were completely inhibited. Indeed, for all five antibiotics tested, inhibition of surfing occurred with increasing concentrations but still occurred to some extent at concentrations much higher than those inhibiting swimming. 5.4 Adaptive antibiotic resistance was not due to the presence of mucin alone To show that the observed resistances were attributable to the surfing adaptation rather than the presence of mucin, I examined the effect of mucin on antibiotic activity. Mucin itself could conceivably influence antibiotic diffusion or susceptibility. Therefore, as one control I assessed the effect of mucin on antibiotic susceptibility by testing its effects in a disk diffusion format using 1.5% agar, under which conditions surfing did not occur. I observed that for 9 out of the 12 antibiotics for which surfing cells demonstrated resistance (and 3 of 5 for which they did not demonstrate resistance), there were no significant differences in the diffusion zone between agar plates with and without mucin, indicating that per se mucin had a minimal influence on antibiotic susceptibility (Table 5-1). For those that show a mucin dependent alteration of susceptibility on 1.5% agar plates, tobramycin revealed increased susceptibility in the presence of mucin, the opposite of the effect of surfing conditions, while amikacin showed a partial but much lesser effect cf. surfing, and ciprofloxacin showed a significant reduction in susceptibility. From this, I concluded that, with the possible exception of ciprofloxacin, the surfing adaptation rather than altered antibiotic diffusion was responsible for the observed multidrug adaptive resistance phenotype.  I also assessed the broth dilution MIC of P. aeruginosa PA14 (Appendix Table A-6) in the presence and absence of mucin and observed increased MIC values accompanying mucin addition for gentamicin, amikacin and colistin, but no differences for other antibiotics in the same classes (tobramycin, polymyxin B). Conversely, for tetracycline mucin actually increased susceptibility by 4-fold. Overall these data suggested that the observed resistances for most antibiotics (Figure 5-1 and 5-2) was likely due to the adaptation accompanying surfing motility  63 rather than the presence per se of mucin. For this reason, I investigated these adaptive changes in greater detail.  Table 5-1. Mucin addition had little impact on antibiotic susceptibility at hiogher agar concentrations that prevent surfing motility. Disk diffusion assays were performed on SCFM with 1.5% agar in the absence or presence of 0.4% mucin. Bacterial cultures were spread as lawn and antibiotic disks applied on top. The zones of inhibition (mm) were measured after overnight incubation at 37°C. P-values were calculated using 2-way ANOVA. Antibiotic Zone of clearing on SCFM plates 1.5% agar 1.5% agar + 0.4% mucin P-value Gentamicin 8.7 8.0 1.0 Tobramycin 5.3 10.7 <10-4 Amikacin 7.3 3.7 <10-4 Imipenem 4.3 4.7 >1.0 Meropenem 10.7 9.0 0.6 Ceftazidime 10.7 9.3 0.7 Erythromycin 4.0 9.3 <10-4 Clarithromycin 2.7 6.3 <10-4 Aztreonam 6.3 6.7 >1.0 Piperacillin 8.0 6.7 0.7 Polymyxin B 3.0 3.0 >1.0 Colistin 4.0 2.3 0.6 Norfloxacin 5.0 3.7 0.7 Ciprofloxacin 9.7 6.3 0.0003 Trimethoprim 5.7 5.7 >1.0 Tetracycline 9.3 8.3 1.0 Chloramphenicol 9.7 10.3 1.0 5.5 Surfing-mediated antibiotic resistance is associated with multiple resistome genes RNA-Seq data (NCBI GEO Accession: GSE110044), comparing the surfing colony edge and centre (SCFM 0.3% agar, 0.4% mucin) to swimming in agar (SCFM 0.3% agar), as previously discussed in Chapter 3, revealed that the surfing adaptation strongly affected gene expression. In total, when compared to swimming in liquid media, there were 1,467 genes dysregulated at the edge and 2,078 genes in the centre, with 816 genes commonly dysregulated between the two, while differences were consistent with the strong phenotypic differences in the blue-green centre and at the thick white edge of surfing colonies (Yeung et al., 2012). In particular these global gene expression data confirmed that the surfing adaptation was considerably different from swarming in that, out of the 1,467 genes dysregulated at the edge of  64 the surfing colony, only 215 (14.6%) matched those previously (Overhage et al., 2008) found to be dysregulated during swarming, while out of the 2,078 dysregulated in the centre, 217 (10.5%) overlapped with those from swarming cells.   To examine the possibility that adaptive resistance during surfing motility was due to the dysregulation of genes that influenced resistance, literature searches were conducted. This revealed 119 genes that when mutated led to increased susceptibility (intrinsic resistance genes) and 252 genes that when mutated mediated antibiotic resistance; collectively these form the resistomes for various antibiotics (Alvarez-Ortega et al., 2010; Breidenstein et al., 2008; Dötsch et al., 2009; Fernández et al., 2010; Gallagher et al., 2011; Schurek et al., 2008; Wiegand et al., 2008). Among the resistome genes, 65 were identified, through RNA-Seq gene expression data from surfing cells, that matched the direction of dysregulation of expression levels expected if they were to have a potential role in surfing mediated resistance. Thus to be potentially important for surfing-mediated resistance, one would expect genes for which mutants led to resistance to be transcriptionally downregulated, while intrinsic resistance genes for which mutants led to supersusceptibility would be expected to be upregulated.  Available transposon mutants of these 65 resistome genes were tested for changes in susceptibility to certain antibiotics.  Table 5-2 shows the resistome genes dysregulated at the edge and/or centre for which transposon mutants showed a change in susceptibility, under surfing motility conditions, to at least one of the 5 tested antibiotics based on a disk diffusion assay. The mean zone of inhibition measurements are presented in Appendix Table A-7. Several of these genes showed a change in susceptibility to more than one antibiotic, possibly illustrating a contribution to broad-spectrum resistance under surfing conditions.  Table 5-2. Resistome genes and their corresponding changes, when mutated, in antibiotic susceptibility relative to the wild-type under surfing conditions. This group included 8 resistome genes similarly regulated in both the centre and at the edge. A further 10 resistome genes were dysregulated only at the edge of surfing colonies and affected in such a way as to influence resistance or susceptibility. Twenty resistome genes, dysregulated only in the centre of surfing colonies, were affected in such a way as to influence antibiotic resistance or susceptibility. Gene Gene function (Winsor et al., 2016) RNA-Seq Fold Change Antibiotic susceptibility, under surfing conditions, of mutant relative to WTa Centre Edge armR Anti-repressor for MexR -3.2 -5.1 TOBR, IMIR, PXBR, NFXR, TETR atpB ATP synthase A chain -2.1 NCb TOBR, NFXR  65 braB Branched chain amino acid transporter -4.2 NC NFXR ccmF Cytochrome C-type biogenesis protein -2.2 NC TOBR, PXBR, NFXR ccoO1 Cytochrome c oxidase, cbb3-type, CcoO subunit -3.5 NC TOBR, NFXR clpS ATP-dependent Clp protease adaptor protein NC -2.3 TOBR, IMIR, PXBR, NFXR cycH Cytochrome c-type biogenesis protein NC 2.2 TOBS, PXBS ddaH Dimethylarginine dimethylaminohydrolase 4.9 3.4 IMIS, TETS etfA Electron transfer flavoprotein α-subunit -6.2 NC TOBR, PXBR, NFXR gidA Glucose-inhibited division protein A -2.2 NC NFXR htpX Heat shock protein -2.0 NC NFXR mutS DNA mismatch repair protein  -2.5 NC TOBR, PXBR nalC Transcriptional regulator -5.3 -2.7 TOBR, PXBR, NFXR nuoB NADH dehydrogenase I chain B -2.8 NC TOBR, IMIR, PXBR, NFXR nuoF NADH dehydrogenase I chain F -2.4 NC TOBR nuoG NADH dehydrogenase I chain G -2.1 NC TOBR, NFXR PA1348 Hypothetical protein NC -3.4 IMIR, NFXR PA1428 Conserved hypothetical protein -3.4 NC TOBR, NFXR PA1513 Hypothetical protein NC -3.0 TETR PA2047 Probable transcriptional regulator NC -2.0 TOBR, NFXR PA2566 Conserved hypothetical protein NC -5.0 NFXR PA2571 Probable two-component sensor NC -2.7 TOBR PA3233 Hypothetical protein 2.5 NC NFXS PA3576 Hypothetical protein NC -2.9 TOBR, NFXR, TETR PA3667 Probable pyridoxal-phosphate dependent enzyme -1.7 -2.5 TETR PA4292 Probable phosphate transporter -6.7 NC TOBR, IMIR, PXBR, NFXR PA4429 Probable cytochrome c1 precursor -2.3 NC TOBR, PXBR, NFXR PA4766 Conserved hypothetical protein NC -2.3 TOBR PA4781 Cyclic di-GMP phosphodiesterase NC -2.9 TOBR, NFXR PA5130 Conserved hypothetical protein NC 2.4 TOBS, IMIS, PXBS, NFXS, TETS pchF Pyochelin synthetase -2.2 NC TOBR pckA Phosphoenolpyruvate carboxykinase -2.6 NC TOBR recG ATP-dependent DNA helicase 1.9 2.1 TOBS, PXBS, NFXS, TETS rph Ribonuclease PH -2.3 NC TOBR serA D-3-phosphoglycerate dehydrogenase -12.4 NC TOBR, PXBR, NFXR thiG Thiamine biosynthesis protein, thiazole moiety -2.9 NC IMIR, NFXR a Antibiotic abbreviations are IMI - imipenem, TET - tetracycline, PXB - polymyxin B, TOB - tobramycin, NFX - norfloxacin. Superscript R indicates resistant; superscript S indicates supersusceptible. bNC = no change in gene expression under the given condition Five of the tested mutants, DrecG, DddaH, DarmR, DnalC, and DPA3667, were similarly  66 dysregulated in the centre and edge of a surfing colony, with recG and ddaH both up-regulated and armR, nalC, and PA3667 down-regulated. Complementation of selected resistome mutants with the respective cloned genes showed that this broad-spectrum effect could be significantly reversed either partially, completely or excessively (Table 5-3), while overexpression of some of these genes also revealed a change in susceptibility to other antibiotics as shown in Table 5-3. RT-qPCR data (Table A-9) verified the direction of dysregulation shown in the RNA-Seq data for selected resistome genes. Table 5-3. Complementation of selected resistome mutants that showed broad spectrum changes in surfing-dependent susceptibility led to restoration of antibiotic susceptibility. Results show the average zones of inhibition of each mutant and its complemented equivalent against five antibiotics (n=3) cf. wild-type (n=6). Mutants of up-regulated resistome genes were tested against 10 µg/disk of antibiotic and down-regulated against 100 µg/disk. Standard deviations ranged between 0 and 2.5 mm. Statistical significance relative to wild-type was determined using two-way ANOVA. * p<0.05, ** p<0.01, *** p< 10-3, **** p<10-4  Strain Zone of Inhibition (mm) Imipenem Tetracycline Polymyxin B Tobramycin Norfloxacin 10 µg/disk antibiotic concentration Wild-type 5.7 5.0 5.6 3.3 1.0 ΔrecG 7.3 8.7* 9.7** 12.5**** 7.3**** ΔrecG/recG+ 6.0 5.7 3.7 6.7* 3.0 ∆ddaH 9.0** 0*** 5.3 3.0 2.3 ∆ddaH/ddaH+ 6.3 3.3 5.7 6.3 2.0 100 µg/disk antibiotic concentration Wild-type 12.3 6.7 10.3 12.0 14.7 ΔPA1428 12.7 7.7 8.0 7.0*** 0.0**** ΔPA1428/PA1428+ 9.0* 7.3 9.0 11.6 13.3 ΔPA2047 12.3 7.0 5.7 7.3** 9.7*** ΔPA2047/PA2047+ 9.7 7.0 7.3 11.3 12.0 ΔthiG 6.3**** 6.7 7.0 8.7 10.3** ΔthiG/thiG+ 9.0* 8.7 8.3 11.0 15.0 ΔatpB 9.7 4.0 8.0 8.3* 9.7*** ΔatpB/atpB+ 10.3 7.3 8.3 12.0 14.0 ΔPA3667 15.7 0.0**** 7.7 10.0 12.0 ΔPA3667/PA3667+ 12.0 9.3 11.3* 11.0 9.7*** ΔPA3576 12.0 3.0* 6.0 8.3* 10.7* ΔPA3576/PA3576+ 10.7 6.3 7.3 9.0* 12.3 ΔPA3721 10 2** 14.5**** 0**** 10** ΔPA3721/PA3721+ 11.7 8.3 9.3 11.3 13.0 ΔclpS 8.3* 6.3 15**** 6.7*** 8.3**** ΔclpS/clpS+ 11.3 13.0**** 11.5 11.7 12.3 ΔarmR 0**** 0**** 1.0**** 6.3**** 0****  67 ΔarmR/armR+ 12.3 12.3**** 10.3 12.7 15.0 5.6 Discussion P. aeruginosa is a highly adaptable organism that exhibits diverse lifestyles from coordinated forms of motility like swarming to community-based sessile structures like biofilms. Another lifestyle includes P. aeruginosa surfing motility that occurs on the surface of agar plates under artificial cystic fibrosis-like conditions where the mucin content is high (Yeung et al., 2012). In Chapter 3, I presented data that confirmed the role of this motility form as a complex adaptation influencing expression of hundreds of genes. Here I demonstrated that this novel form of motility is associated with multidrug adaptive resistance. Both disk diffusion and antibiotic incorporation assays revealed that cells undergoing surfing were significantly more resistant to multiple antibiotics compared to swimming, and the same concentrations of antibiotics that completely abolished swimming were found to be much less effective against surfing cells.  Antibiotic susceptibility was generally unaffected by mucin in the presence of high agar concentrations at which swimming and surfing do not occur, indicating that mucin had a minimal effect on susceptibility to most antibiotics (Table 5-1). MIC assays also confirmed that the observed adaptive resistance was dependent on surface growth associated with the surfing adaption, and not merely due to the presence of mucin. Indeed experiments measuring the resistance of surfing and swimming colonies to antibiotics incorporated into agar plates not only confirmed that surfing cells were considerably more resistant to the 5 tested antibiotics (Figure 5-2), but also that surfing cells could grow at concentrations above the liquid MICs in the presence of mucin (Table 5-S1), again consistent with the concept of adaptive resistance. RNA-Seq data on cells collected from the centre of a surf colony revealed 10 genes, ccoO1, atpB, nuoB, PA4429, eftA, serA, ccmF, thiG, nuoF, and pckA, involved in metabolism and energy production, that were down-regulated and for which mutants exhibited increased resistance to certain antibiotics. Three of these genes, ccoO1, atpB, and PA4429, have also been shown to be dysregulated under swarming conditions as described previously  . Mutants for these 10 metabolic genes that were down-regulated at the centre of surfing colonies showed an increased resistance to norfloxacin and/or tobramycin. Aminoglycosides are taken up by energy dependent mechanisms (Bryan & Kwan, 1983), and reduced metabolic activities have previously been shown in P. aeruginosa biofilms to contribute to resistance to tobramycin (Walters et al., 2010). Although norfloxacin has been shown to affect animal metabolism through interactions  68 with cytochrome P450 (McLellan et al., 1996), it has not been shown to affect metabolism in P. aeruginosa and the effects on susceptibility could reflect reduced DNA replication (involving the target topoisomerases) in these metabolically-challenged cells. Here I have demonstrated that reduced expression levels of certain metabolic resistome genes in the surf centre may contribute to adaptive resistance against tobramycin and/or norfloxacin.  To explain the mechanisms behind surfing-mediated resistance, I explored the contribution of resistome genes found to be dysregulated in surfing through RNA-Seq and transposon mutant screens. In total, 36 resistome genes were identified as being dysregulated under surfing conditions and exhibiting a change in susceptibility to certain antibiotics when mutated. Among the 36 resistome genes, there were 5 that showed the same direction of dysregulation (i.e. both down or up-regulated) in both the centre and edge of a surfing colony. For example, recG and ddaH were both up-regulated in the surfing centre and edge, and their mutants exhibited similar reduced resistance to tetracycline. The mutant in recG (encoding an ATP-dependent DNA helicase) also exhibited increased susceptibility to polymyxin B, tobramycin, and norfloxacin. Tetracycline and tobramycin target protein synthesis through the 30S ribosomal submit while polymyxin B targets the cell membrane, and norfloxacin targets DNA replication. The broad-spectrum activity observed by recG as a resistome gene against such diverse antibiotics may arise from its regulatory nature, since it is known that RecG transcriptionally regulates OxyR-controlled genes in P. putida (Yeom et al., 2012). Genes identified in the RecG regulon of P. putida included porins (oprE, oprD, PP0883) and thioredoxin reductase (trxB) involved in stress coping mechanisms (Yeom et al., 2012).  There were 3 genes, armR, nalC, and PA3667, that were down-regulated in both regions of the surf colony. NalC is known to negatively regulate the expression of armR, and ArmR inhibits the DNA binding activity of MexR (Starr et al., 2012; Wilke et al., 2008). MexR negatively regulates expression of the mexAB-oprM operon, which encodes for a major efflux pump in P. aeruginosa, involved in intrinsic and mutational broad-spectrum antibiotic resistance (Wilke et al., 2008). ArmR allosterically binds to MexR to alleviate its repression on the mexAB-oprM operon (Wilke et al., 2008). Interestingly, Starr et al. (2012) revealed that a knock-out mutant of armR still exhibited increased expression levels of the mexAB-oprM operon under certain conditions (Starr et al., 2012). Here I showed that mutants in armR and nalC, which are both down-regulated under surfing conditions, exhibited similar increases in resistance to tobramycin,  69 norfloxacin, and polymyxin B. The observed increases in resistance to these antibiotics might be attributed in part to increased expression levels of the mexAB-oprM operon.  There were 11 genes dysregulated at the edge and 20 genes at the centre of a surfing colony that exhibited a change in susceptibility to at least one of the tested antibiotics when mutated compared to the wild-type. PA5130 was a conserved hypothetical protein found to be up-regulated at the surfing edge and exhibited an increased susceptibility to all 5 of the tested antibiotics when mutated. The ATP-dependent protease adapter clpS which was downregulated at the edge, exhibited a significant increase in resistance to imipenem, polymyxin B, tobramycin, and norfloxacin. ClpS has been previously shown by our lab to contribute to antibiotic resistance, biofilm formation, and swarming motility (Fernández et al., 2012). More specifically, a transposon mutant variant of clpS was observed to have increased resistance to β-lactams through the increased expression of β-lactamase (Fernández et al., 2012). Here it was shown that clpS also had an effect on resistance to imipenem, polymyxin B, tobramycin, and norfloxacin under surfing conditions. Swarming is another complex form of motility exhibited by P. aeruginosa found to be involved with major transcriptional changes (Overhage et al., 2008; Wang et al., 2013), substantially distinct from the transcriptional profile of surfing cells. Swarming cells have also previously been shown to be resistant to multiple antibiotics including polymyxin B, ciprofloxacin, and gentamicin, and pvdQ mutants influenced swarming-specific resistance (Overhage et al., 2008; Wang et al., 2013). Here surfing motility was also found to be associated with resistance to these same antibiotics and several others. Among the resistome genes identified in this study to be dysregulated under surfing conditions that showed contributions to adaptive antibiotic resistance (Table 5-2), pchF (Overhage et al., 2008; Tremblay & Déziel, 2010), atpB, ccoO1, and PA4429 (Tremblay & Déziel, 2010) were also shown to be dysregulated under swarming conditions (Overhage et al., 2008; Tremblay & Déziel, 2010). However our preliminary studies of the swarming resistome (Coleman and Hancock, 2018, manuscript in preparation) have indicated major differences compared to the surfing-associated resistome described here, and less than 15% of dysregulated genes were in common for the two motility adaptations. Thus the mechanistic overlap in swarming and surfing mediated adaptive resistance would appear to be minimal.  The biofilm growth state also leads to adaptive resistance (Domitrovic et al., 2016; Høiby et  70 al., 2010). Biofilms represent a very different adaptation being sessile rather than motile communities. Our preliminary analyses of gene expression in biofilm bacteria have suggested that there are considerable differences compared to surfing bacteria (D. Pletzer, E. Sun and R.E.W. Hancock, unpublished data, Table 3-1) with only 22-34% commonly dysregulated genes, and thus I would again anticipate different adaptations were involved in resistance. One overlapping gene is nalC, identified here as being dysregulated in surfing and mediating surfing-associated polymyxin B, tobramycin and norfloxacin resistance. Mutants in nalC were found in biofilms formed by clinical strains of P. aeruginosa isolated from prosthetic valves, and the such isolates were resistant to fluoroquinolones and carbapenems (Domitrovic et al., 2016). Other surfing resistome genes identified in our study included nuoB, nuoF, and nuoG. The nuo operon, nuoA-N, involved in nitrate sensing, has previously been shown to be activated during biofilm formation and important for regulating biofilm formation as well as motility (Southey-Pillig et al., 2005; Van Alst et al., 2007), but its role in biofilm mediated adaptive resistance was not studied. The other genes identified in this study as important for surfing-mediated resistance have not yet been shown to be involved in biofilm formation or related resistance.  In conclusion, surfing motility is a novel form of motility that results in a mucin-triggered lifestyle adaptation. Here I demonstrated how surfing cells exhibit increased resistance that can be attributed to a variety of transcriptomic changes resulting from that adaptation.   71 Chapter 6. Surfing motility: A conserved yet diverse adaptation among motile bacteria  6.1 Introduction Bacteria are found in a broad array of dynamic abiotic and biotic environments. They can lead to both positive (biodegradation, normal flora, probiotics) and negative (infections, diseases) implications in humans. In order to thrive in so many different changing environments, bacteria must adapt. Motility is critical to their ability to colonize certain sites, to move towards more favorable environments and away from unfavorable conditions, and to form complex multicellular surface-associated structures such as biofilms (Harshey, 2003). Bacterial motility is also important to pathogenicity since it is involved in movement between body compartments, host cell adherence, colonization, formation of biofilms, and survival. It is often coupled with metabolism and the expression of virulence factors (Belas & Suvanasuthi, 2005; Haiko & Westerlund-Wikström, 2013; Rajagopala et al., 2007).   Here I examined whether surfing motility was conserved amongst other motile bacteria. Results revealed that the physical characteristics of surfing including rapid surface spreading and adaptation were observed in the investigated bacteria both under artificial cystic fibrosis host-like conditions and rich medium supplemented with mucin. However, other characteristics of surfing were found to be more variable.  6.2 Physical characteristics of surfing motility exhibited by multiple motile bacterial species  To determine if the physical characteristics of surfing were conserved in other Gram-negative motile bacteria, Enterobacter cloacae, Proteus mirabilis, Salmonella enterica, E. coli, and Vibrio harveyi were grown under the same conditions under which P. aeruginosa was originally reported to surf (i.e. artificial cystic fibrosis medium supplemented with mucin on semi-solid plates with 0.3% agar). The same basic physical characteristics of surfing were observed in all tested bacterial species (Figure 6-1). The addition of mucin to SCFM in 0.3% agar resulted in surface growth and a significantly larger area of spread in comparison to swimming without mucin that occurred within the agar. In contrast, on 1.5% agar plates without mucin, most bacteria grew as punctuate colonies with almost no spread. Unlike the other tested species, P. mirabilis as observed previously (Rauprich et al., 1996) exhibited swarming motility characterized by concentric rings on 1.5% agar without mucin (Rauprich et al., 1996). On mucin-supplemented media, P. mirabilis did not exhibit the same concentric phenotype, instead  72 demonstrating a larger, thicker spread similar to that observed for P. aeruginosa surfing.   Figure 6-1. Mucin triggered rapid surface motility in a range of bacterial species. Bacterial strains were grown under swimming conditions (0.3% agar), surfing conditions (0.3% agar in the presence of 0.4% mucin), and solid medium conditions (1.5% agar) in SCFM medium. The rate of motility zone growth, depicted on the right graphs, was assessed as the diameter of the motility zone (mm) over 10 hours of incubation at 37°C and surfing is represented by the continuous lines and swimming by the dashed lines (N=3). Overall, the physical characteristics of surfing first observed for P. aeruginosa were also observed for other motile Gram-negative bacterial species including E. cloacae, P. mirabilis, S. enterica, E. coli, and V. harveyi. The rate of motility zone growth was consistently faster in the presence of mucin and the motility zone eventually filled the plate (within ~10-15 hours). Although S. enterica, E. coli and V. harveyi exhibited more rapid swimming motility than P.  73 aeruginosa, their swimming zones (within agar) were marginally less than their surfing zones (surface-localized) at the same incubation time. Even though other species did not show the differential pigmentation observed during P. aeruginosa surfing (Yeung et al., 2012), surface growth on mucin supplemented media was quite thick throughout, as also observed for P. aeruginosa surfing.  6.3 Surfing motility demonstrated adaptability to various medium viscosities P. aeruginosa surfing motility is not as stringent compared to other forms of motility such as swarming and swimming (Yeung et al., 2012). Swarming often occurs at a limited range of medium viscosities (e.g. 0.4-0.7% agar for Pseudomonas), and is dependent on specific medium conditions (not occurring in rich medium or with ammonium as an N source), while swimming is limited to very low viscosity media (≤ 0.3% agar) (Yeung et al., 2012). Agar titration assays in both minimal SCFM (Figure 6-2) and nutrient-rich LB (Appendix Figure A-2) media revealed that surfing was generally less dependent on growth conditions compared to swarming and swimming in all tested species, since for most it occurred at a broad variety of agar concentrations and in both nutrient-rich LB and defined SCFM media. In general, there was a decrease in the size of surfing colonies as agar concentration increased, however, surfing still occurred to a significant extent at high agar concentrations in all except E. cloacae in SCFM with mucin. E. cloacae did, however, exhibit significant surfing at up to 0.5% agar in LB (Appendix Figure A-3), in contrast to SCFM where surfing was only observed at 0.3% agar. Interestingly, although E. cloacae surfing was reduced at higher concentrations, at 1.0% agar it began to exhibit dendritic surface spread (a swarming like behavior) under conditions containing mucin. P. mirabilis had no significant change in the area of surfing from 0.3%-1.0% agar in SCFM and LB with mucin. Swimming, in general, was completely inhibited at concentrations higher than 0.3% in all except P. mirabilis that exhibited swimming in 0.3% and 0.5% agar, although swimming was completely inhibited at ³ 0.8% agar. P. mirabilis also exhibited a difference in the conditions under which swarming (concentric rings) was observed. P. mirabilis began exhibiting a swarming phenotype at 0.8% agar in LB which was not observed in SCFM. However, at 1.5% agar in SCFM without mucin, swarming was indeed observed (Figure 6-1). In general, I observed that surfing manifested somewhat differently in each of the different bacterial species but tended to occur at higher agar concentrations than those that supported swimming and swarming.   74  Figure 6-2. Effect of medium viscosity on surfing motility. Bacterial strains were point inoculated onto SCFM medium at varying agar concentrations, with and without mucin, and grown for 18 hours at 37°C to test the effects on surfing (Surf) and Swimming (Swim) motility. Percent plate coverage as a function of agar concentration was measured using ImageJ (N=3) and graphs appear on the left with representative images of motility zones on the right. Corresponding images are presented in Appendix Figure A-3.  6.4 Consistent surfing-like motility was not observed in alternative wetting agents  Yeung et al (2012) previously tested the role of mucin as a wetting agent by demonstrating that surfing-like phenotypes were observed in PA14 under artificial cystic fibrosis conditions containing Tween 20 detergent or carboxymethylcellulose. However, the observed surfing phenotypes were somewhat different from those observed under mucin conditions (Yeung et al., 2012). Here I demonstrated (Figure 6-3) that rich media containing either carboxymethylcellulose (CMC) or Tween-20 promoted distinct rapid surface motility in P. aeruginosa at the highest concentrations tested by Yeung et al (2012). CMC, despite being at a higher concentration (1.0% wt/vol) than Tween-20 (0.01%) or mucin (0.4%), was unable to promote any form of motility in E. cloacae, S. enterica, E. coli, or V. harveyi. For P. mirabilis,  75 CMC promoted a distinct spotty phenotype, quite different from surfing observed under mucin conditions (which displayed an even, thick circular motility zone). Tween-20, however, appeared to promote surfing-like motility to various extents in all of the tested bacteria except E. coli. Minimal surfing was observed in E. coli but there was an increased motility zone of growth with increased incubation time and increased Tween-20 concentration (data not shown). Tween-20 was able to promote surfing in some tested bacteria at the very low concentration of 0.01% wt/vol.   Figure 6-3. Effect of alternative wetting agents on surfing motility. Mucin was substituted with carboxymethyl cellulose (CMC) at 1% wt/vol or Tween-20 at 0.01% added into 0.3% agar LB.  6.5 Surfing cells exhibited distinct multiple antibiotic resistance  Surfing is a complex adaptive lifestyle in Pseudomonas causing large changes in gene expression and virulence properties (Yeung et al., 2012, Chapter 3). As with other complex lifestyle adaptations including swarming motility and biofilm formation (Drenkard, 2003; Overhage et al., 2008), P. aeruginosa exhibits increased resistance to a series of antibiotics when undergoing surfing motility (Yeung et al., 2012, Chapter 5). Here I studied surfing mediated  76 resistance based on the distance of closest approach of motility colonies to antibiotic-containing disks in the context of surfing conditions (Table 6-1).  Table 6-1. Surfing motility mediated diverse adaptive multi-drug resistance in different bacterial species. Antibiotic screens were done using the disk diffusion assays on plates containing LB ± 0.4% mucin with 0.3% agar. Statistical analysis to determine relative susceptibility was performed using two-way ANOVA to compare surfing and swimming circumstances, whereby increased resistance represented a lower mean zone of inhibition. R indicates an increased resistance and S indicates an increased susceptibility under surfing motility conditions relative to swimming. Class Antibiotic Relative Susceptibility P. aeruginosa E. cloacae P. mirabilis S. enterica E. coli V. harveyi Aminoglycosides Gentamicin R   R R  Tobramycin R  R R  R Amikacin R   R   b-lactams Imipenem R R     Meropenem R R   R R Carbenicillin R    R  Piperacillin   R  R  Aztreonam  R R    Ceftazidime     R  Macrolides Erythromycin R S     Azithromycin R    R R Quinolones Ciprofloxacin R R  S R R Norfloxacin      R Polymyxins Polymyxin B R R  R R R Colistin R    R R Others Trimethoprim R R R R  R Tetracycline R R R   R Chloramphenicol R R    R  P. aeruginosa strain PA14 exhibited increased resistance to aminoglycosides, carbapenems, polymyxins, macrolides, carbenicillin, ciprofloxacin, trimethoprim, tetracycline, and chloramphenicol, when compared to susceptibility under swimming conditions (Chapter 5; summarized in Table 6-1). The other tested bacterial species also showed increased resistance to multiple antibiotics under surfing conditions when compared to swimming motility. However, the antibiotics to which surfing colonies exhibited resistance varied substantially in different bacterial species, but was broad spectrum, affecting 5 to 14 of the 18 antibiotics tested from diverse classes. Furthermore, resistance rarely affected all members of a given class of antibiotics, indicating that there were likely multiple resistance mechanisms triggered, as found  77 in Pseudomonas (Chapter 5). Thus, the patterns of susceptibility to particular classes of antibiotics, as observed for P. aeruginosa (e.g. resistance to all tested aminoglycosides, macrolides, carbapenem b-lactams, and polymyxins) were not generally observed in other species. For example, S. enterica showed increased resistance to all tested aminoglycosides as was seen in P. aeruginosa, but was only resistant to polymyxin but not colistin. Conversely, surfing E. coli and V. harveyi were resistant to both polymyxins but only one aminoglycoside each. E. cloacae was the only species among those tested to exhibit a similar surfing-mediated adaptive resistance to both carbapenems, imipenem and meropenem, as observed for P. aeruginosa, but did not demonstrate adaptive aminoglycoside resistance.   Some species also exhibited resistance to antibiotics for which P. aeruginosa demonstrated no surfing-mediated adaptive changes in susceptibility such as the β-lactams, piperacillin, aztreonam, and ceftazidime. E. cloacae, E. coli, and P. mirabilis exhibited increased resistance to at least one of these antibiotics under surfing conditions. Conversely, in the case of E. cloacae and S. enterica increased susceptibility was observed during surfing relative to swimming bacteria towards ciprofloxacin and erythromycin respectively (Table 6-1). 6.6 Surfing dependence on flagella was conserved  Yeung et al. (2012) previously demonstrated that mutants deficient in flagella biosynthesis genes in P. aeruginosa PA14 were surfing deficient. Here I also demonstrated (Figure 6-4) that this dependence on flagella was conserved in the following species: S. enterica, E. coli, and P. mirabilis. Mutants of flagella biosynthesis genes in each of these species exhibited complete inhibition of motility. On the other hand, pilus-deficient mutants of E. coli and P. mirabilis exhibited normal surfing, as was also observed in P. aeruginosa in this study (Figure 6-4) and previously (Yeung et al., 2012). An E. coli fim mutant did, however, exhibit slower growth of the surfing motility zone compared to the wild-type (data not shown) but still exhibited the physical characteristics of surfing motility. Therefore, surfing appeared to have a conserved dependence on flagella but not pili or fimbriae.   78  Figure 6-4. Surfing motility was dependent on flagella but not pili/fimbriae. Flagella deficient mutants in P. aeruginosa (∆fliC), S. enterica (∆fliC), P. mirabilis (∆flaD), and E. coli (∆flhDC) demonstrated complete inhibition of surfing motility in 0.3% agar SCFM supplemented with 0.4% agar after 13-15 hours of incubation. Pilus or fimbriae deficient mutants of P. aeruginosa (∆pilC), P. mirabilis (∆mrpA), and E. coli (∆fim) still exhibited surfing motility under the same conditions.  6.7 Dependence on quorum sensing of P. aeruginosa surfing motility was not conserved   Surfing motility in P. aeruginosa PA14 is dependent on the Rhl and Las quorum sensing systems based the inhibitory effects of transposon mutants in the rhlI and lasI genes, which could be complemented by the addition of their respective homoserine lactones (Yeung et al., 2012). Additional screens of quorum sensing mutants (Figure 6-5) revealed that P. aeruginosa mutants in genes involved in the PQS quorum sensing system (pqsABCDE, pqsR) also exhibited surfing-deficiency. Indeed, certain mutants such as pqsR and pqsB exhibited swarming motility rather than surfing motility in the presence of mucin. Genetic complements were generated for lasI and rhlI, the autoinducer synthesis proteins. Addition of their respective autoinducers or genetic complementation of lasI, rhlI, and pqsA restored wild-type surfing (Figure 6-5; (Yeung et al., 2012)).   79  Figure 6-5. P. aeruginosa surfing was dependent on quorum sensing. (A) Quorum sensing PA14 mutants (∆pqsA, ∆pqsB, ∆pqsC, ∆pqsD, ∆pqsE, ∆pqsR, ∆lasI, ∆rhlI, ∆rhlR) exhibited surfing deficiency as shown by the negative control (∆fliC) or conversion to swarming. Surface coverage was determined by analyzing the % surface coverage using ImageJ relative to wild-type PA14. (B) Complements of quorum sensing mutants (rhlI+, lasI+) exhibited complete or partial surfing restoration. Addition of exogenous autoinducer molecules restored surfing with a slight increase in motility zone compared to wild-type. Differences in plate coverage area of the mutants cf. the wild-type were singificant by 2-way ANOVA at p<0.0001 (****).  To test if this dependence on quorum sensing was conserved in the other motile bacteria, quorum sensing mutants were obtained for S. enterica (∆luxS), and V. harveyi (∆luxR). Each of these quorum sensing mutants still exhibited normal surfing in SCFM with mucin (Figure 6-6).   80   Figure 6-6. Surfing-dependence on quorum sensing did not extend to bacterial species other than P. aeruginosa. Motility assays were performed on SCFM containing 0.3% agar and 0.4% mucin (surfing), or 0.3% agar (swimming). Swimming for the three test species, P. aeruginosa, S. enterica, and V. harveyi, showed no dependence on quorum sensing since their respective quorum sensing mutants continued to exhibit wild-type swimming. Although the P. aeruginosa lasI mutant was surfing deficient, quorum sensing mutants from S. enterica (∆luxS) and V. harveyi (∆luxR) continued to show surfing.  6.8 Discussion Surfing is a mucin-dependent adaptation that was first observed in P. aeruginosa (Yeung et al., 2012). Here I showed that E. coli, S. enterica, P. mirabilis, and E. cloacae, which are known to associate with the mucosa during infections, as well as the marine bacterium V. harveyi, exhibited similar physical characteristics to those reported for P. aeruginosa on artificial cystic fibrosis semi-solid medium containing mucin. The bacterial species selected for this study with the exception of E. cloacae had been previously reported to exhibit more than one form of motile adaptation, including swimming and swarming (Armbruster et al., 2013; Böttcher et al., 2016; Hejazi & Falkiner, 1997; Kearns & Losick, 2003; Kim & Surette, 2003). The surface adaptation observed in the presence of mucin was distinct from the characteristics of swimming which occurs within agar and swarming (as summarized in Chapter 8, Table 8-2), and unlike both motility processes surfing was dependent on the presence of mucin. For all tested organisms, surfing was faster than swimming motility. Interestingly, the conditions under which surfing  81 occurred were also observed to be less stringent than the conditions needed to display other motility forms such as swarming or swimming. In particular, swimming motility was only observed at low viscosities (0.3% agar), whereas surfing was observed at a range of viscosities (0.3-1.0%) in both minimal and rich media. Overall several characteristics of surfing that have been catalogued in P. aeruginosa (Yeung et al., 2012), including rapid surface spread, adaptability to various media viscosities, minimal growth substrate requirements, dependence on flagella, and multidrug adaptive resistance were observed for all the tested Gram-negative motile bacteria.   Previously (Yeung et al., 2012), it was shown that P. aeruginosa exhibited surfing-like motility in SCFM agar plates with carboxymethyl cellulose or Tween-20 instead of mucin; however, the appearance of these motility colonies were different from those observed under mucin conditions. Here I tested these two alternative wetting agents in rich medium (LB) at the concentrations previously tested (Yeung et al., 2012) and observed distinct surface motility phenotypes in P. aeruginosa. Carboxymethyl cellulose (CMC) was found to be ineffective at promoting surfing in any of the other tested species, however, Tween-20 was able to promote surfing in all except E. coli and P. aeruginosa where a swarming-like phenotype was observed instead. Neither wetting agent was able to induce surfing in E. coli under the conditions in which mucin induced surfing. Mucin was, therefore, the only agent able to consistently promote distinctive surfing motility in all the tested species.   Surfing was initially reported to be dependent on intact flagella but not pili in P. aeruginosa (Yeung et al., 2012). In this study, these findings were corroborated for other species as shown in Figure 6.4. This dependence of surfing motility on flagella was also found to be conserved in P. mirabilis, E. coli, and S. enterica. Pili or fimbriae mutants of P. aeruginosa, E. coli, and P. mirabilis were also screened, but did not exhibit surfing deficiency. Surfing was observed to be slower in an E. coli fimbriae mutant, but it still occurred to a diminished extent unlike the flagella mutants which exhibited complete inhibition of surfing. The type IV pili in P. aeruginosa (Köhler et al., 2000) and type 1 fimbriae in E. coli (Inoue et al., 2007) were previously found to be important in swarming motility, but as shown in this study did not play an obligate role in surfing motility.   Many of the tested bacterial species are known to cause a wide range of infections that are often difficult to treat. With regards to mucosal infections by these bacteria, adaptive resistance  82 accompanying a motile lifestyle in the presence of mucin could exacerbate this. Here I demonstrated that the surfing motility adaptation led to increased resistance (and in two cases enhanced susceptibility) to specific antibiotics when compared to bacteria undergoing swimming motility. All tested bacterial species exhibited a certain level of broad-spectrum resistance under surfing conditions, although the antibiotics for which adaptive resistance was observed differed greatly.   In this study, I also tested the importance of quorum sensing which had been previously reported to be involved in P. aeruginosa surfing (Yeung et al., 2012). Using transposon mutants, A dependence of surfing on the N-acyl homoserine lactone (AHL) Rhl and Las quorum sensing systems was demonstrated in P. aeruginosa (Yeung et al., 2012), as confirmed here. Mutants deficient in rhamnolipid production genes regulated by the Rhl system, namely rhlA and rhlB mutants, necessary for swarming motility in P. aeruginosa, were found to exhibit wild-type-like surfing and thus surfing was confirmed to be independent of rhamnolipids (Appendix Figure A-4, Yeung et al., 2012). Here it was demonstrated that surfing was also dependent on the PQS system in P. aeruginosa. Mutants displaying surfing deficiency included those in the PQS operon, pqsABCDE, involved in synthesizing the autoinducer, PQS, and pqsR, the transcriptional regulator that binds to and mediates responses to PQS. Interestingly, such mutants, e.g. the pqsR mutant, often exhibited a swarming phenotype rather than surfing in medium supplemented with mucin, possibly explaining the surface coverage observed previously (Yeung et al., 2012). Complementing the quorum sensing transposon mutants with the respective wild-type genes, as well as the addition of their respective autoinducers exogenously restored surfing to the wild-type-like level. Indeed high concentrations of the autoinducers actually further enhanced surfing to a level greater than that of the wild-type (i.e. demonstrating increased surface coverage in less time), as shown here and previously for the Rhl and Las autoinducers (Yeung et al., 2012). Therefore, it appears that each of the Rhl, Las, and PQS systems are required for surfing motility in P. aeruginosa. Although these data confirmed and extended information on the importance of quorum sensing in P. aeruginosa surfing, I did not observe this dependence on the AHL-based quorum sensing systems of S. enterica and V. harveyi. However, each of these AHL-based quorum sensing systems involved distinct autoinducers and have distinct regulons.   To further examine how conserved surfing motility is in other bacteria, I also tested the Gram-positive bacterium, Bacillus subtilis (Appendix Figure A-5). B. subtilis exhibited similar  83 surface spread as was observed in the other tested bacteria under conditions involving SCFM agar supplemented with mucin. In contrast, B. subtilis swimming occurred within the agar and at 1.5% agar exhibited no spread (Appendix Figure A-5a). B. subtilis mucin-dependent motility also exhibited similar characteristics as observed for the other bacteria including faster spreading than swimming, broad-spectrum antibiotic resistance, and adaptability to various agar concentrations. Indeed B. subtilis did exhibit surfing-like phenotypes at a range of viscosities (0.3-1.0% agar) in both LB and SCFM media supplemented with mucin, but it also exhibited significant surface spread at higher agar concentrations without mucin, especially on LB agar (Appendix Figure A-5d). This might reflect the type of swarming motility described by Kearns and Losick (2003), who previously described B. subtilis swarming at 0.5-0.7% agar. However, because B. subtilis swarming did not exhibit any visible features distinct from surfing, it was difficult to distinguish between the two forms of motility. There was, indeed a clear shift from embedded agar motility (swimming) at 0.3% agar to surface spread (potentially swarming) at higher agar concentrations in medium without mucin. In contrast in the presence of mucin, only surface motility was observed. The mucin-promoted motility was found to be partially dependent on flagella in that a flagellar mutant exhibited dendritic rather than circular surface spread, but no dependence on the Com quorum sensing system (mutants exhibited wild-type-like surfing) (Appendix Figure A-5b).   In conclusion, I observed that surfing motility demonstrated conserved features in other motile, mucosa-associated pathogens and was associated with broad-spectrum antibiotic resistance. However, the surfing adaptation could be differentially regulated in different bacterial species.     84 Chapter 7. Role of the stringent stress response in Pseudomonas surfing motility  7.1 Introduction  The stringent stress response plays a key adaptational role in Pseudomonas survival and virulence. The stringent response is regulated by the secondary messenger nucleotide, ppGpp, produced and hydrolyzed by the RelA and SpoT enzymes. RelA-induced ppGpp production is triggered by amino acid starvation, and a SpoT-induced response is triggered by environmental stress conditions such as limiting iron conditions, or carbon and fatty acid starvation (Vogt et al., 2011). Activation of the stringent stress response results in global transcriptomic changes that divert energetically costly processes such as growth and cell division to stress response and coping mechanisms (Boutte and Crosson, 2013). Vogt et al. (2011) previously showed that the stringent stress response plays an important role in Pseudomonas virulence in the rat lung model, while Pletzer et al (2017) showed its importance in a mouse abscess model, as well as expression of spoT and relA in abscess infections. A stringent response mutant (DrelADspoT) exhibited attenuated virulence and ability to survive under conditions of heat shock and oxidative stress (Pletzer et al., 2017; Vogt et al., 2011). The CF lung environment is a complex niche of stress factors that favour highly adaptable organisms like P. aeruginosa. It is often associated with high levels of inflammation, reactive oxygen species, and a diverse microbial community simultaneously producing synergistic and antagonistic compounds (Cifani et al., 2013; Harrison, 2007). Therefore, the stringent response is likely crucial for Pseudomonas survival and colonization of the CF lungs (Xu et al., 2016). The stringent stress response regulates oxidative stress tolerance (Khakimova et al., 2013), production of virulence factors during acute infections through the regulation of quorum sensing (van Delden et al., 2001; Schafhauser et al., 2014), as well as swarming and biofilm formation (Fuente-Núñez et al., 2014; Xu et al., 2016).  Since the stringent response has been proposed to play a key role in adaptability in the CF lung environment (Xu et al., 2016), I explored here its role in surfing motility. Mucin is a key component found in large abundances in the CF lung (Quinton, 2008) and a critical important inducer of surfing motility. Here I demonstrated the essential role of the stringent response in both strain PAO1 and the CF-adapted Liverpool epidemic strain LESB58.  LESB58 was first isolated in 1996 from chronically infected CF patients (Cheng et al., 1996). It promotes a stronger chronic infection than PAO1 or PA14 in mouse models (Fothergill et al.,  85 2012). Sequencing of its genome in 2009 revealed a number of genomic prophage islands which were found to be important in chronic infectivity, allowing LESB58 to out-compete other P. aeruginosa strains in chronic infection models in mice (Winstanley et al., 2009). Here, I explored the dependence of surfing motility on the stringent stress response in LESB58 and PAO1 and provided evidence it occurred through the regulation of the quorum sensing regulator, pqsH, and copper-resistance regulator, cueR. Both pqsH and cueR had been identified through the mutant library screen (Table 3-2) as essential regulators for surfing motility.  7.2 The stringent stress response regulated swarming and surfing, but not swimming Three forms of motility, swarming, surfing and swimming, were investigated in the LESB58 and PAO1 wild-type and stringent response mutants (DrelADspoT) (Figure 7-1; Appendix Figure A-6).   Figure 7-1. Stringent response mutants exhibited surfing inhibition but wild-type swimming. Stringent response mutants and complements in PAO1 (B) and LESB58 (A) under  86 swimming and surfing conditions. Surfing colonies were grown in KB media with 0.3% agar and 0.4% mucin at 37°C for 15 hours. Swimming colonies were grown in SCFM media with 0.3% agar at 37°C for 36 hours. Both the LESB58 and PAO1 wild-type isolates exhibited all three forms of motility. However, the LESB58 strain exhibited extremely poor swimming motility, significantly less than either strains PAO1 and PA14, showing minimal swimming or in-agar motility zone growth even after 36 hours of incubation. Compared to the wild-type, the stringent response mutant exhibited a normal swimming motility phenotype in contrast to what was observed for surfing and swarming motility. The double-mutant exhibited complete inhibition of surfing in LESB58 and almost complete attenuation in strain PAO1 (Figure 7-1), and attenuation of swarming in both strains (Appendix Figure A-6; swarming performed by Daniel Pletzer). Complementing the mutant with either relA or spoT partly restored the swarming and surfing phenotypes, but to a lesser extent than the wild-type, whereby the two complemented isolates covered less surface area, while the relA complemented mutant formed a less structured swarming colony. Under surfing conditions, the spoT+ complemented mutant also exhibited a stronger pigmentation level as it appeared bright blue-green. Therefore, the stringent response mutant exhibited inhibition of surfing and swarming but not swimming motility in both the PAO1 and LESB58 strains.  7.3 Differential gene expression in the stringent response mutant during motility The expression of motility genes and surfing-essential regulators were explored by RT-qPCR in the context of swarming, swimming, and surfing in the LESB58 wild-type and DrelADspoT stringent response mutant as shown in Table 7-1. All three forms of motilities were dependent on flagellar biosynthesis; therefore, I determined the expression levels of key flagellar and chemotaxis regulators in the mutant vs. the wild-type. Under swarming conditions, the chemotaxis gene, cheY, and two-component regulator found to be involved in regulating flagellar biosynthesis genes, fleR, were found to be significantly down-regulated in the stringent response mutant relative to the wild-type. In addition, the rhamnolipid biosynthesis gene, rhlB, was also found to be down-regulated by 5.8-fold in the mutant. Swarming motility is highly dependent on the production of rhamnolipids as surfactants (Caiazza et al., 2005). Major quorum sensing regulators, including RhlR which regulates the expression of rhamnolipid biosynthesis genes, were found to be down-regulated in the mutant under swarming conditions. Under surfing conditions, pqsH, which is involved in the synthesis of the PQS autoinducer, as well as the  87 copper resistance regulator, cueR, were found to be significantly down-regulated in the stringent response mutant that exhibited surfing deficiency. Under swimming conditions, however, it did not appear to be affected in the stringent response mutant. In contrast under swimming conditions I still observed dysregulation between the mutant and wild-type in rhlR, rhlB, and cueR, which were all found to be down-regulated.  Table 7-1. Differential gene expression levels of key surfing- and swarming-essential regulators and effectors in LESB58. RT-qPCR was performed on RNA collected from the swarming (by Pletzer), swimming, and surfing edge cells in the stringent response mutant (ΔrelA/ΔspoT) and wild-type. Dysregulation was observed in different regulators among the three forms of motility. A fold-change cutoff of ± 2.0 fold was considered to impute a meaningful change. Analysis was performed by comparison with the house-keeping gene, RpoD.  Gene  Fold change in ΔrelA/ΔspoT compared to WT LESB58 Swarming Swimming Surfing fleQ -1.0 1.1 1.7 fleR -3.5 -1.4 1.1 cheY -3.8 -1.5 1.7 rhlB -5.8 -2.1 3.2 lasR -4.3 1.8 2.1 rhlR -3.5 -2.2 2.2 pqsH -2.4 -1.1 -2.8 pqsR 1.3 -1.0 1.7 cueR -1.9 -3.1 -4.4 7.4 Cross-complementation with the copper-transport regulator cueR or the quinolone synthase pqsH restored surfing motility in a stringent response mutant  As previously shown through the mutant library screen (Table 3-2), surfing is dependent on cueR and pqsH. These two surfing-essential regulators were also found to be down-regulated in the DrelADspoT mutant which exhibited surfing deficiency. To determine whether hierarchical regulation existed among these regulators, the cueR and pqsH genes were complemented into the DrelADspoT mutant. Surfing motility was restored to a wild-type-like level in the mutant complemented with wild type cueR and pqsH genes in LESB58 as shown in Figure 7-2.   88  Figure 7-2. Overexpression of pqsH and cueR in the stringent response mutant restored surfing in the LESB58 strain. Surfing assays were done in SCFM with 0.3% agar and 0.4% mucin, grown at 37°C for 18 hours. PqsH and cueR were cloned into a high copy plasmid (pBBR5).  7.5 Discussion  The stringent response is a key adaptive mechanism used by bacteria like P. aeruginosa and plays an important role in adapting to diverse niches. Here I have shown that a mutant deficient in the production of ppGpp, the second messenger involved in regulating genes under the stringent stress response, is also deficient in certain forms of motility, particularly surfing (Figure 7-1), and swarming (Figure A-6). Complementing either relA or spoT, both of which can synthesize ppGpp, partially restored surfing and swarming motility. The relA complemented strain, for example, exhibited surfing but to a lesseer extent than the wild-type, while the spoT complement also exhibited reduced surfing and hyperpigmentation. These observations were made in both the LESB58 and PAO1 strains of P. aeruginosa. Swimming motility, although observed to be much slower in the LESB58 compared to PAO1, was not affected by the stringent response mutant, but the somewhat more substantative phenotype in the LESB58 strain might have been due to this general limitation in motility. Swarming, on the other hand, like surfing, was found to be attenuated in the stringent response mutant. Therefore, the stringent response appeared to play an important role in mediating surfing and swarming.   To determine the down-stream effects of the stringent response regulators on other key regulatory systems, the expression levels of surfing essential regulators were measured in the stringent response mutants compared to the wild-type. RT-qPCR analysis (Table 7-1) showed that the double mutant under swarming conditions exhibited down-regulation in several regulators for flagella synthesis, chemotaxis, and quorum sensing including the production of rhamnolipids which is required for swarming motility. Under surfing conditions, however, two  89 key regulators were found to be significantly down-regulated, the Pqs quorum sensing gene, pqsH, and the copper responsive regulator, cueR. Overexpressing these regulators in the double-mutant restored surfing motility, speaking to the potential hierarchical effect of the stringent response on these regulators. Therefore, the stringent response influenced the expression levels of pqsH and cueR which, as previously shown through the mutant library screen in Chapter 3, were essential regulators mediating surfing motility.   CueR is a dimeric transcriptional regulator with two domains: a sensor domain that responds to environmental stimuli such as the presence of copper and a DNA-binding domain (Bagchi, 2015). CueR was found to directly regulate the expression of several genes involved in drug resistance, copper resistance and virulence as well as several uncharacterized proteins (Bagchi, 2015; Thaden et al., 2010). It was previously found that cueR expression is regulated by the Las quorum sensing system (Thaden et al., 2010). Here I showed that it was likely also regulated by the stringent stress response. The cueR transposon mutant was shown in Chapter 3 to be surfing deficient. Complementation of cueR in the transposon mutant restored surfing to a wild-type-like state (Appendix Figure A-7). Therefore, cueR is an essential regulator for surfing motility found to be dysregulated in the stringent response mutant. Overexpressing the cueR regulator in the stringent response mutant restored surfing motility.  Similar data was obatined for pqsH whereby a pqsH disruption mutant in PA14 was found to be surfing deficient, but surfing was restored by complementing with the wild type pqsH gene (Figure A-7). PqsH plays a role in converting the precursor of the autoinducer HHQ to its final form, PQS. Previous studies showed that the stringent response negatively regulates the production of PQS (Schafhauser et al., 2014). However, here I found a down-regulation of pqsH under surfing conditions, whereas Schafhauser et al. (2014) reported an observed up-regulation in both pqsA and pqsH during stationary phase in liquid culture conditions, resulting in an accumulation of HHQ and PQS. Therefore, the stringent response appeared to be contextually regulating PQS production. Under surfing, which was reliant on the Pqs quorum sensing system (Figure 6-5), pqsH down-regulation was observed in the stringent response mutant which could limit the conversion of HHQ to PQS leading to low PQS levels. Surfing was highly dependent on PQS-regulated gene expression (Figure 6-5), but swimming was not dependent on the PQS system (Schafhauser et al. 2014). This again confirmed that stringent response regulation of the PQS system was context dependent. In conclusion, I observed that pqsH and cueR were both  90 down-regulated in the stringent response mutant and overexpressing either regulator in the mutant recovered surfing motility, indicating a role for the stringent stress response as a global regulatory system that potentially mediates surfing through cueR and pqsH.      91 Chapter 8. Conclusions 8.1 Surfing in comparison to other motile lifestyles  Surfing is a novel motile adaptation first discovered in the opportunistic pathogen, P. aeruginosa grown on 0.3-1% agar plates under artificial CF-like growth conditions in the presence of mucin (Yeung et al., 2012). P. aeruginosa is the leading cause of death in CF patients, accounting for more than half of all CF infections in adults. Motility is known to be important for P. aeruginosa virulence since strains deficient in flagella or pili biosynthesis exhibit significantly lower virulence in acute infection models as well as an attenuated ability to form biofilms (Drake and Montie, 1988; O’Toole and Kolter, 1998). It has been previously shown that under conditions of excess mucin P. aeruginosa exhibits a novel rapid form of surface motility, phenotypically distinct from other forms of motility (Yeung et al., 2012). Table 8-1 highlights key differences among the various motility adaptations. Table 8-1. Differences between motile adaptations in P. aeruginosa. Observations were taken from cited papers or studies conducted in this thesis.  Property Swimming Swarming Twitching Surfing Sliding In vitro appearance Circular, spread within agar Dendritic (PA14, LESB58) or solar flared (PAO1), on surface Semi-circular, spread in interstitial space between agar and plate Semi-circular, on surface, blue-green centre and white outer edge1 Concentric and dendritic, on surface2 Viscosity requirements (% agar wt/vol) ≤ 0.3% 0.5-0.8% 1.5% 0.3-1.0% 0.3%-0.7%2 Appendage requirements Flagella Flagella and Type IV pili3 Type IV pili4  Flagella1 None Involved in adaptive resistance No Yes5 Untested Yes Untested Required for virulence in vivo Untested Untested Yes6 Untested Untested Dependence on quorum sensing No Yes3 Yes7 Yes1 Untested 1.  (Yeung et al., 2012) 2.  (Murray and Kazmierczak, 2008) 3.  (Köhler et al., 2000)  92 4.  (Bradley, 1980) 5.  (Overhage et al., 2008) 6.  (Alarcon et al., 2009) 7.  (Beatson et al., 2002)  Previously, Yeung et al. (2012) found that surfing motility, like swarming and swimming, is dependent on the presence of intact flagella. However, unlike swarming and twitching, it is not dependent on the type IV pili (Yeung et al., 2012). However, here I found, through a transposon mutant library screen, that surfing was dependent on three previously untested twitching genes, pilT, pilU, and pilW. PilT and PilU, normally involved in pili retraction, are also involved in adherence and cytotoxicity during acute infections (Comolli et al., 1999). Both pilT and pilU mutants exhibit a hyperfimbrial phenotype due to their inability to retract (Whitchurch and Mattick, 1994). Despite that, they are still deficient in twitching motility (Whitchurch and Mattick, 1994). PilW, on the other hand, is a membrane-bound protein found to be involved in protein secretion during type IV pili biosynthesis (Alm et al., 1996). Therefore, pilW mutants exhibit a type IV pilus deficient phenotype (Siryaporn et al., 2014). Like the pilU and pilT mutants, the pilW mutant also exhibits significantly reduced cell adherence and virulence (Siryaporn et al., 2014). Interestingly, it has been found that not all pilus deficient mutants, namely DpilB and DpilC, exhibit reduced virulence (Siryaporn et al., 2014). Therefore, among the many pilus biosynthesis and twitching genes, pilU, pilT, and pilW play key roles in colonization through surface adherence (Comolli et al., 1999; Siryaporn et al., 2014). Therefore, although surfing is not dependent on the structural pilus genes as previously described (Yeung et al., 2012), it was found to be dependent on key twitching genes involved in adherence and secretion.   Besides differential dependence of pili and flagellar genes, surfing motility also exhibited less stringent nutritional and environmental requirements compared to other forms of motility, being mainly dependent on mucin as a wetting agent. Swarming normally occurs on semi-aqueous conditions (0.5-0.8% wt/vol agar) with a poor nitrogen source such as casamino acids and is inhibited by ammonium. Swimming occurs in aqueous conditions (less than 0.3% agar) in the presence of ammonium as a nitrogen source. Twitching occurs on solid surfaces or the interstitial space between the agar and the plate in rich media. Sliding motility occurs on solid surfaces in strains that are deficient in both flagella and pili. Surfing, however, was found to be relatively flexible in medium viscosity. Surfing was observed in 0.3-1.0% agar in P. aeruginosa  93 whereas swimming was only observed at 0.3% agar (Figure 6-2). Surfing was also found to occur in both rich and minimal conditions in the absence and presence of ammonia (Yeung et al., 2012, Figure 6-2, A-2). The only key requirement for surfing was the presence of mucin since it was also found that mucin, as a wetting agent, uniquely induced surfing motility in P. aeruginosa. Tween-20, was also able to act as a substitute in inducing surfing-like behaviour in E. cloacae, P. mirabilis, S. enterica, and V. harveyi. However, in P. aeruginosa, substituting mucin with alternative wetting agents such as CMC or Tween-20 induced more swarming-like phenotypes. CMC was also ineffective at relatively high concentrations in being able to induce surfing among bacteria other than Pseudomonas.   Various conditions induce different adaptations that are regulated by distinct sets of regulators. Compared to other forms of motile adaptations like swarming and sessile lifestyles like biofilms, surfing was found to be dependent on and exhibited dysregulation of a unique cohort of regulators. RNA-Seq data collected from surfing, swarming, and biofilm cells revealed that the three adaptations only share dysregulation in 21 regulatory genes, not all in the same direction of dysregulation. Surfing and swarming cells exhibited dysregulation of 10 regulators not dysregulated in biofilms, while surfing cells and biofilms exhibited dysregulation in 63 other regulators not dysregulated under swarming conditions. Therefore, surfing had more dysregulated regulators in common with biofilm cells than swarming. Among the regulators found to be dysregulated in all three adaptations, two sigma-70 family regulators and a two-component sensor, gcbA, were found to be commonly down-regulated, while the putative two-component regulator, PA1243, was found to be up-regulated. Among the 10 regulators dysregulated in both swarming and surfing cells but not biofilms, only one regulator exhibited the same direction of dysregulation in the two motility forms. PhaD was up-regulated in both motile adaptations. PhaD is involved in polyhydroxyalkanoates synthesis in P. oleovorans, but its role in P. aeruginosa has not been well established (Klinke et al., 2000). Therefore, surfing and swarming might share a dependence on PHA production, which can be used for carbon storage during nutrient limiting conditions. Compared to biofilm cells, surfing exhibited a similar direction of dysregulation to 14 regulators in addition to the ones found to be similarly dysregulated in all three conditions. Among these 14 genes, surfing and biofilm cells shared similar expression level of regulators for various multidrug resistance genes and several sigma factors. However, surfing also shared an inverse dysregulation compared to biofilm cells for  94 major regulators like GacA, LasR, and CbrA. Therefore, these different adaptations appeared to have relatively unique regulatory networks and cascades that mediate them.  8.2 Regulation of surfing motility   Surfing was found to result in the dysregulation of 1,094 genes in cells collected from the edge of a surfing colony and 1,172 genes from the centre relative to swimming, and 1,617 genes were similarly dysregulated between the two. Cells in the centre and edge shared the same direction of dysregulation of flagellar biosynthesis genes, but they exhibited differential regulation on pilus genes. Primarily, core pilus genes were up-regulated at the edge but down-regulated in the centre. Alternative pilus genes, on the other hand, were inversely dysregulated. The centre and edge also exhibited inverse dysregulation in pyoverdine and pyochelin biosynthesis, chemotaxis, quorum sensing, energy production and cell division genes. Although centre genes exhibited an up-regulation of chemotaxis and quorum sensing regulators and effectors, which were relatively down-regulated in edge cells, centre cells may have been dividing and growing less compared to those at the edge, as suggested by an up-regulation of energy production, cell division and protein synthesis genes.   RNA-Seq revealed that surfing resulted in massive dysregulation of genes, which differed between centre and edge cells. The mutant library screen, on the other hand, revealed key genes involved in mediating the initiation of surfing motility. Transposon mutants in 192 genes were identified and verified as being surfing deficient, exhibiting either complete inhibition of motility, alternative motile phenotypes, or irregular growth patterns under surfing conditions. Among those genes, approximately 40 regulators were identified including known global regulators such as GacAS, CbrA, FleQ, LasI, PqsR, RhlR, and RpoN. Looking at the gene expression levels of each of the surfing-essential regulators in each of the transposon mutants for those same regulators revealed that three regulators consistently exhibited a large influence on the expression levels of the other regulators in both centre and edge cells, namely PfeS, PA1463 and CbrA. As CbrA had already been extensively studied for its role in motility and virulence as a global regulator (Yeung et al., 2011), here I focused on PfeS and PA1463 as master regulators in surfing motility.   Knocking out either the PfeR regulator or the PA1463 operon resulted in complete abolishment of surfing. Complementing either with its respective operon led to rescue of wild-type-like surfing phenotype. Both knock-out mutants were found to affect a large cohort of  95 surfing-essential regulators. Thus pfeR mutant exhibited dysregulation of 9 essential regulators while the PA1463o mutant led to changes in 13 regulators among 32 tested genes for which wild-type expression could be restored through complementation. Additionally, the pfeR mutant also revealed significant down-regulation (4.1-fold) of PA1463, whereas pfeRS was not significantly dysregulated in the PA1463o knock-out. Therefore, the pfeRS system may regulate the expression of the PA1463 operon. Five regulators were found to be dysregulated in both mutants, specifically czcS, gacA, nirQ, PA2276, and PA5392. RNA-Seq performed in these surfing deficient mutants revealed 827 genes dysregulated in the PA1463o mutant and 1,856 genes in the pfeR mutant relative to wild-type surfing cells. Therefore, both regulators have relatively large regulons (defined as all genes found to be dysregulated in the mutant, whether regulated either directly or indirectly), including regulating the expression of several key surfing-essential regulators.  8.3 Surfing as a conserved complex adaptation Due to the known role of the PfeRS system in regulating iron acquisition, the dependence of surfing on iron was investigated. Titrating out iron from the system resulted in a reduction of surfing, and a switch from surfing to swimming. Exogenous addition of iron restored surfing motility. Overexpressing the pfeRS operon in the PAO1 wild-type resulted in increased persistence of surfing under iron limiting conditions, requiring a 5-fold higher concentration of the iron chelator dipyridyl to abolish surfing. The swich to swimming observed in the wild-type was also not observed in the pfeRS overexpression strain.  Surfing, as previously mentioned, is clearly a complex adaptation that involves several regulatory systems and a cascade of genes that mediate initiation and progression of the motility. Here I also explored surfing motility in the context of antibiotic resistance and how conserved it is among other motile bacterial species. Table 8-2 summarizes the major differences in motile adaptations among different bacterial strains. Table 8-2. Comparison of motility in diverse species. Observations were taken from the cited papers or from the studies presented in Chapter 6. Type of Motility Pseudomonas aeruginosa Enterobacter cloacae Proteus mirabilis Salmonella enterica Escherichia coli Vibrio harveyi Physical appearance on motility plates Swim Within agar circular pattern1,2 Within agar circular pattern Within agar circular pattern3 Within agar circular pattern Within agar circular pattern Within agar circular pattern  96 Swarm Surface motility, dendritic or flared pattern2 Not described Surface motility, concentric rings/terrace4 Surface motility, circular5 Surface motility, circular5 Surface motility, circular for related Vibrio sp.6 Surf Surface motility, thick, circular pattern, blue-green centre and white outer edge1 Surface motility, circular pattern, thick throughout Surface motility, circular pattern, thick throughout Surface motility, circular pattern, thick throughout Surface motility, circular pattern, thick throughout Surface motility, circular pattern, thick throughout Viscosity requirements (agar concentration %) Swim ≤ 0.31 0.3 0.3-0.43 0.3 0.3 0.3 Swarm 0.5-0.77,8 0.5-0.89 1.5-39 0.5-0.85,9 0.5-0.89 1.5-39 Surf 0.1-1.01 0.3-0.5 0.3-1.0 0.3-1.0 0.3-1.0 0.3-1.0 Media/growth requirements Swim Plates not dried Plates not dried Plates not dried Plates not dried Plates not dried Plates not dried Swarm Poor N source not NH4, minimal media, plates dried Not described High glutamine10 Glucose as C source5, Plates dried9 30°C5 30°C for related Vibrio sp.6 Surf Mucin1 Mucin Mucin Mucin Mucin Mucin Dependence on Quorum Sensing Swim No Not described   Yes11  Swarm Yes8 Not described No12   Yes for related Vibrio sp.6 Surf Yes1 Untested Untested No Untested No Rate of motility Swim 0.9mm/h 1.6mm/h 1mm/h 4mm/h 2.3mm/h 7.3mm/h Swarm Not described Not described 1.2mm/h4 1.5mm/h9 Untested Untested Surf 2.9mm/h 4.9mm/h 2.3mm/h 5.9mm/h 4.9mm/h 9mm/h Multidrug Resistance Swim No No No No No No Swarm Yes13 Not described Not described Yes9,14 Not described Yes for related Vibrio sp.6 Surf Yes Yes Yes Yes Yes Yes Dependence on flagella Swim Yes2 Not described No15 Yes16 Yes11 Yes for related Vibrio sp.17 Swarm Yes2 Not described Yes15 Yes5,18 Yes5 Yes6,17  97 Surf Yes1 Untested Untested Yes Untested Untested Dependence on Biosurfactant Swim       Swarm Rhamnolipid19 Untested Capsular polysacch-aride20 None21 Capsular polysacch-aride22 Untested Surf None1 Untested Untested Untested Untested Untested 1. (Yeung et al., 2012) 2. (Rashid and Kornberg, 2000) 3. (Liaw et al., 2000) 4. (Rauprich et al., 1996) 5. (Harshey and Matsuyama, 1994) 6. (Jaques and McCarter, 2006) 7. (Overhage et al., 2008) 8. (Köhler et al., 2000) 9. (Butler et al., 2010) 10. (Armbruster et al., 2013) 11. (Sperandio et al., 2002) 12. (Schneider et al., 2002) 13. (Overhage et al., 2007) 14. (Kim and Surette, 2003) 15. (Gygi et al., 1997) 16. (Lockman and Curtis, 1990) 17. (Belas and Colwell, 1982) 18. (Stafford and Hughes, 2007) 19. (Caiazza et al., 2005) 20. (Gygi et al., 1995) 21. (Chen et al., 2007) 22. (Takeda et al., 2001) Surfing P. aeruginosa cells were found to be more resistant to several classes of antibiotics including aminoglycosides, macrolides, polymyxins, and quinolones compared to swimming cells. Analgous but unique broad-spectrum resistance was also observed in several other bacterial species that exhibited surfing motility in the presence of mucin, including E. coli, P. mirabilis, S. enterica, E. cloacae, V. harveyi, and B. subtilis. Analysis of the P. aeruginosa resistome revealed 36 genes that when disrupted exhibited a change in susceptibility to certain test antibiotics. Among these resistome genes, 25 mutants exhibited a change in susceptibility to more than one antibiotic tested, speaking to their role in broad-spectrum resistance. Although various other bacterial species also exhibited surfing-mediated broad-spectrum resistance, the antibiotics to which resistance was observed varied greatly between species. Some species even showed increased susceptibility under surfing conditions to certain antibiotics that was not observed in P. aeruginosa. Besides antibiotic resistance, the different tested species also exhibited similar flexibility in medium conditions and dependence on flagella but not pili, as was found in P. aeruginosa surfing (Yeung et al., 2012). However, a dependence on quorum sensing  98 was not observed in the tested species, S. enterica and V. harveyi.  8.4 Concluding remarks In conclusion, surfing motility, a novel form of bacterial motility dependent on mucin, first established in P. aeruginosa, was shown to be conserved in other motile bacteria, both Gram-negative and Gram-positive. Surfing mediated broad-spectrum adaptive resistance, also conserved in other bacteria. It is less stringent than other forms of motility in terms of viscosity and nutrient availability but was dependent on iron. Surfing is a complex motile lifestyle adaptation that involved a large array of regulators that coordinately work to mediate surfing motility. Surfing was found to be dependent on quorum sensing and the stringent stress response. PfeRS, a two-component system, and PA1463, a putative chemotaxis regulator, were found to be master regulators and essential for mediating surfing motility through the regulation of other surfing-essential genes.  8.5 Clinical implications  Surfing motility, as presented in this thesis, is a novel social adaptation of the pathogen, P. aeruginosa, that was first discovered using an artificial model of the cystic fibrosis lung sputum. This model mimics several key characteristics of the CF lung including abundant amino acid content and high levels of mucin. Mucin is an important component of the CF sputum that contributes to the disease phenotype by inhibiting mucociliary function and regulating the mucosal viscosity that uniquely promotes surfing. Other wetting agents tested showed poor consistently in promoting surfing motility in P. aeruginosa. Mucin, therefore, has unique properties that promote surfing as an adaptation that may optimally contribute to the survival of P. aeruginosa under CF conditions. Many characteristics of surfing also reflect P. aeruginosa acute infections including the up-regulation of several virulence factors, mainly those secreted by the type 2 secretion system as well as a dependence on key motility appendages. In additon, surfing shared more genetic and physical characterisitcs with biofilms rather than swarming. Biofilms are adaptations that occur in relatively high frequency under chronic CF lung infections. Therefore, key adaptive features that promote Pseudomonas survival in the CF lung conserved in biofilms may also be reflected in surfing motility as a consequence of increased survivability in the CF lung environment. 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Characterization of a carbapenem-hydrolyzing enzyme, PoxB, in Pseudomonas aeruginosa PAO1.  119 Antimicrobial Agents and Chemotherapy, 60(2), 936–945. https://doi.org/10.1128/AAC.01807-15     120 Appendix Figure A-1. Disk diffusion assay plate set-up. Mid-log phase (OD600=0.4-0.5) cultures are inoculated at 1uL around the antibiotic disk at equal distances. Four-point inoculation was used for swim and surf antibiotic disk assays. Disk diffusion control assays were done using a bacterial lawn spread with 50uL of mid-log phase culture and dried antibiotic disks were applied to the centre of each plate.   Figure A-2. Effect of viscosity in rich medium on surfing motility. Bacterial strains were point inoculated onto LB medium at varying agar concentrations with and without mucin and grown for 18 hours at 37°C to test the effects on surfing (Surf) and Swimming (Swim) motility. Percent plate coverage was measured using ImageJ (N=3). 050100Pseudomonas aeruginosaPlate Coverage (%)050100Enterobacter cloacaePlate Coverage (%)Surf (LB+mucin) Swim (LB)050100Plate Coverage (%)Proteus mirabilis050100Salmonella enterica Plate Coverage (%)050100Escherichia coliPlate Coverage (%)Surf (LB+mucin) Swim (LB)050100 Vibrio harveyiPlate Coverage (%)0.3% agar0.5% agar0.8% agar1.0% agar 121  Figure A-3. Effect of medium viscosity on surfing motility. Bacterial strains were point inoculated onto SCFM medium at varying agar concentrations with and without mucin and grown for 18 hours at 37°C to test the effects on surfing (Surf) and Swimming (Swim) motility.  Figure A-4. Surfing motility of PA14 in rhamnolipid deficient mutants. Transposon mutants in P. aeruginosa PA14 of rhlA and rhlB were grown on SCFM with 0.3% agar and 0.4% mucin and incubated at 37°C for 13 hours. Both mutants still exhibited wild-type-like surfing motility.     122  Figure A-5. Bacillus subtilis exhibited rapid surface growth under high mucin conditions and surfing-mediated broad-spectrum antibiotic resistance. (A) B. subtilis exhibited rapid surface motility under surfing (SCFM+0.4% mucin) conditions that was slightly faster than swimming according to a 10 hour motility zone growth assay. (B) Quorum sensing mutants, DcomA and DcomQXP, continued to show wild-type like surfing. A flagellar mutant, Dhag, exhibited swarming-like patterns under surfing conditions and no swimming or swarming under their respective conditions. The DcomA mutant also exhibited attenuated swarming at 0.5% agar SCFM without ammonium. (C) B. subtilis exhibited broad-spectrum resistance to multiple tested antibiotics and increased susceptibility to Azithromycin relative to swimming. (D) Surface motility of B. subtilis in LB and SCFM at various agar concentration with and without mucin. Swarming (high agar concentration without mucin) and surfing (+ mucin) were indistinguishable and could occur at varying levels of viscosity.   123  Figure A-6. Effect of stringent response knock-outs on swarming in LESB58 and PAO1. Swarming assays were performed by Daniel Pletzer on KB media with 0.5% agar grown at 37°C for 18 hours.   Figure A-7. Surfing and swarming dependence on pqsH and cueR in PA14. PA14 transposon mutants of pqsH and cueR were complemented with their respective genes on a high-copy plasmid, pUCp18. Surfing assays were performed in SCFM with 0.3% agar and 0.4% mucin. Swarming assays were performed on SCFM with 0.5% agar. Plates were incubated at 37°C for 18 hours.    124 Table A-1. Concentrations of the antibiotics in the disk diffusion assay as well as their solvents. Ten µL of each antibiotic was added per disk and dried prior to application onto agar surfaces. MeOH – methanol. DMSO – dimethyl sulfoxide. Antibiotic Concentration (µg/disk) Solvent Gentamicin 10 Water Tobramycin 10 Water Amikacin 5 Water Imipenem 10 Water Meropenem 5 Water Ceftazidime 5 Water Aztreonam 30 Water Erythromycin 1000 MeOH Clarithromycin 500 Water Polymyxin B 10 Water Colistin 10 Water Norfloxacin 5 Water Ciprofloxacin 10 Water Trimethoprim 1000 DMSO Tetracycline 10 MeOH Chloramphenicol 5 Water Table A-2. Effectors genes whose transposon mutant variants exhibited surfing deficiency (no motility, an alternative form of motility, and one-directional motility). Transposon mutants come from the PA14 transposon mutant library (Liberati et al., 2006). Gene annotations and descriptions come from www.pseudomonas.com (Winsor et al., 2016). PAO1 Homolog PA14 Locus Tag/Gene Name Description PA5015 aceA Pyruvate dehydrogenase, E1 component PA3546 algX Alginate biosynthesis protein  PA4930 alr Biosynthetic alanine racemase PA5323 argB Acetylglutamate kinase PA3556 arnT 4-amino-4-deoxy-L-arabinose lipid A transferase PA5555 atpG ATP synthase gamma chain PA5561 atpI ATP synthase protein I PA0420 bioA Adenosylmethionine-8-amino-7-oxononanoate aminotransferase PA0504 bioD Dethiobiotin synthase PA0501 bioF 8-amino-7-oxononanoate synthase PA1073 braD Branched-chain amino acid transport protein brad PA4758 carA Carbamoyl-phosphate synthase small chain PA2904 cobI Precorrin-2 methyltransferase cobi PA2130 cupA3 Usher PA1483 cycH Cytochrome c-type biogenesis protein PA1838 cysI Sulfite reductase PA1124 dgt Deoxyguanosinetriphosphate triphosphohydrolase  125 PA0551 epd D-erythrose 4-phosphate dehydrogenase PA1982 exaA PQQ-linked alcohol dehydrogenase PA4959 fimX Conserved hypothetical PA1077 flgB Flagellar basal-body and rod protein PA1078 flgC Flagellar basal-body and rod protein PA1092 fliC Flagellin type B PA1094 fliD Flagellar capping protein PA1441 fliK Flagellar hook-length control protein PA1442 fliL Flagellar basal-body associated protein PA3583 glpK Glycerol kinase PA4724 gltX Putative glutamate-tRNA synthetase PA5203 gshA Glutamate-cysteine ligase PA2195 hcnC Hydrogen cyanide synthase PA1512 hcpA Secreted protein Hcp PA2009 hmgA Homogentisate 1,2-dioxygenase PA5193  hslO Putative chaperon PA4695 ilvH Acetolactate synthase isozyme III small subunit PA5277 lysA Diaminopimelate decarboxylase PA5025 metY Homocysteine synthase PA3244 minD Cell division inhibitor minD PA0766 mucD Serine protease mucD precursor PA4006 nadD NadD nicotinic acid mononucleotide adenylyltransferase PA4566 obg GTP-binding protein, GTP1/Obg family PA4208 opmD Outer membrane protein PA3280 oprO Pyrophosphate-specific outer membrane porin PA0062 PA14_00740 Putative lipoprotein PA0066 PA14_00780 Putative carbonic anhydrases PA0104 PA14_01270 Hypothetical NA PA14_03370 Hypothetical PA0307 PA14_04020 Conserved hypothetical PA0394 PA14_05160 Putative PLP dependent enzyme PA0406 PA14_05300 Putative tonB domain protein PA0428 PA14_05560 Putative ATP-dependent RNA helicase, DEAD box family PA0429 PA14_05580 Conserved hypothetical protein PA0462 PA14_06040 Hypothetical PA0503 PA14_06540 Putative biotin synthesis protein PA0545 PA14_07070 Putative reductase PA0568 PA14_07380 Hypothetical PA0583 PA14_07600 Putative 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase PA0584 PA14_07620 TrnA nucleotidyl transferase PA0624 PA14_08100 Conserved hypothetical PA0663 PA14_08490 Conserved hypothetical protein PA4233 PA14_09190 Putative MFS transporter PA4168 PA14_09970 Putative tonB-dependent receptor  126 PA4144 PA14_10330 Putative outer membrane protein precursor PA4137 PA14_10440 Putative porin PA4130 PA14_10550 Putative sulfite or nitrite reductase PA4072 PA14_11210 Putative amino acid permease PA4069 PA14_11250 Putative dtdp-4-rhamnose reductase-related protein PA4023 PA14_11790 Putative amino acid transporter PA3975 PA14_12410 Possible phosphomethylpyrimidine kinase PA3958 PA14_12670 Possible nuclease or phosphotase PA3892 PA14_13560 Putative fusaric acid resistance protein PA3884 PA14_13670 Hypothetical PA3858 PA14_14100 Putative amino-acid ABC transporter binding protein PA3836 PA14_14390 Putative ABC-type transport protein PA3818 PA14_14680 Inositol-1-monophosphatase PA3783 PA14_15140 Conserved hypothetical PA3749 PA14_15920 Putative major facilitator family transporter PA3730 PA14_16160 Hypothetical PA3697 PA14_16580 Hypothetical PA3649 PA14_17140 Putative membrane-associated zinc metalloprotease PA3641 PA14_17250 Putative Na+/alanine symporter PA3631 PA14_17370 Putative transport permease protein PA3628 PA14_17410 Putative esterase PA3573 PA14_18090 Putative major facilitator subfamily transporter protein PA3526 PA14_18720 Putative outer membrane protein precursor, OmpA family PA3489 PA14_18950 Putative NADH:ubiquinone oxidoreductase PA3488 PA14_18960 Hypothetical protein PA0243 PA14_19170 Putative lipoprotein PA3342 PA14_20840 Hypothetical PA3325 PA14_21040 Putative hydrolase PA3324 PA14_21050 Putative short-chain dehydrogenase NA PA14_24360 Putative serine protease PA3057 PA14_24570 Hypothetical PA2936 PA14_26070 Putative cytochrome b561 PA2927 PA14_26190 Hypothetical proteins PA2918 PA14_26310 Putative short-chain dehydrogenase PA2779 PA14_28140 Hypothetical PA2747 PA14_28600 Conserved hypothetical protein PA2454 PA14_29220 Putative porin PA2693 PA14_29290 Putative long-chain acyl-CoA thioester hydrolase PA2685 PA14_29390 Conserved hypothetical PA2618 PA14_30260 Putative arginyl-tRNA:protein arginylyltransferase PA2576 PA14_30790 Putative permease NA PA14_30910 Hypothetical PA2120 PA14_37150 Conserved hypothetical PA2089 PA14_37490 Putative tonB-dependent receptor PA2002 PA14_38610 Putative short-chain fatty acid transporter  127 PA1875 PA14_40250 Putative outer membrane protein precursor PA1547 PA14_44460 Putative membrane protein PA1509 PA14_44920 Conserved hypothetical PA1442 PA14_45810 Putative flagellar protein FliL PA1441 PA14_45830 Putative flagellar hook-length control protein FliK NA PA14_46610 Putative methyltransferase PA1271 PA14_47800 Putative tonB-dependent receptor PA1239 PA14_48210 Putative hydrolase PA1210 PA14_48650 Putative porin protein PA0718 PA14_48990 Hypothetical protein of bacteriophage Pf1 PA1187 PA14_49080 Probable acyl-coA dehydrogenase PA1127 PA14_49800 Probable oxidoreductase PA1120 PA14_49890 None PA1119 PA14_49900 Probable outer membrane protein precursor PA1045 PA14_50840 Putative DNA helicase PA1037 PA14_50920 Conserved hypothetical protein PA1033 PA14_50970 Probable glutathione S-transferase PA0974 PA14_51690 Conserved hypothetical PA0848 PA14_53300 Probable alkyl hydroperoxide reductase PA0817 PA14_53700 Probable ring-cleaving dioxygenase PA0794 PA14_53970 Probably aconitate hydratase PA4333 PA14_56300 Putative fumarase PA4431 PA14_57570 Putative cytochrome c reductase, iron-sulfur subunit PA4455 PA14_57870 Putative toluene tolerance ABC efflux transporter PA4471 PA14_58040 Hypothetical PA4511 PA14_58540 Conserved hypothetical protein PA4518 PA14_58620 Conserved hypothetical NA PA14_59000 Conserved hypothetical NA PA14_59410 Hypothetical NA PA14_59950 Conserved hypothetical PA0982 PA14_59960 Putative protein-disulfide isomerase PA1935 PA14_60080 Conserved hypothetical PA4612 PA14_61020 Ankyrin-like protein PA4616 PA14_61080 Putative C4-dicarboxylate-binding protein PA4650 PA14_61520 Conserved hypothetical PA4734 PA14_62640 Conserved hypothetical protein PA4753 PA14_62880 Putative RNA-binding protein PA4838 PA14_63970 Putative membrane protein PA4975 PA14_65760 NAD(P)H quinone oxidoreductase PA4981 PA14_65850 Putative amino acid ABC transporter, permease protein PA5076 PA14_67050 Putative amino acid ABC transporter, periplasmic amino acid-binding protein PA5109 PA14_67470 Conserved hypothetical PA5174 PA14_68360 Putative beta-ketoacyl synthase PA5376 PA14_71000 Put. lysine betaine/L-proline ABC transporter, ATP-binding subunit  128 PA5399 PA14_71280 Putative ferredoxin PA4729 panB 3-methyl-2-oxobutanoate hydroxymethyltransferase PA5192 pckA Phosphoenolpyruvate carboxykinase PA0773 pdxJ Pyridoxal phosphate biosynthetic protein  PA4050 pgpA Phosphatidylglycerophosphatase A PA4210 phzA1 Probable phenazine biosynthesis protein PA0395 pilT Twitching motility protein  PA0396 pilU Twitching motility protein  PA4552 pilW Type 4 fimbrial biogenesis protein  PA0026 plcB Phospholipase C PA2969 plsX Fatty acid/phospholipid synthesis protein PA5368 pstC Phosphate ABC transporter, permease protein PA1013 purC Phosphoribosylaminoimidazole-succinocarboxamide synthase PA4855 purD Phosphoribosylamine-glycine ligase PA4854 purH Phosphoribosylaminoimidazolecarboxamide transferase PA3763 purL Phosphoribosylformylglycinamidine synthase PA3050 pyrD Dihydroorotate dehydrogenase PA5331 pyrE Orotate phosphoribosyltransferase PA4743 rbfA Ribosome-binding factor A PA3387 rhlG Beta-ketoacyl reductase PA1396 RL112 Conserved hypothetical PA5454 rmd Oxidorectase PA4752 rrmJ Cell division protein  PA0966 ruvA Holliday junction DNA helicase PA4332 sadC Conserved hypothetical proteins PA4001 sltB1 Soluble lytic transglycosylase B PA0298 spuB Glutamine synthetase PA0594/PA0595 surA/ostA Peptidyl-prolyl cis-trans isomerase/organic solvent tolerance protein ostA precursor PA5070 tatC Sec-independent protein translocase PA3976 thiE Possible thiamin-phosphate pyrophosphorylase PA0381 thiG Thiamine biosynthesis protein, thiazole moiety PA3735 thrC Threonine synthase PA2832 tpm Thiopurine methyltransferase PA0849 trxB2 Thioredoxin reductase 2     129 Table A-3. Genes dysregulated in PAO6609 wild-type surfing relative to swimming. RNA-Seq was performed on PAO1 WT cells collected from a surfing edge and swim colony. 499 genes were found to be dysregulated under surfing relative to swimming. A log fold-change cut-off of ± 1.5 and p-value < 0.05 was used. Gene annotations and descriptions come from www.pseudomonas.com (Winsor et al., 2016). Gene ID Gene Name Description Log FC PA0017  Conserved Hypothetical Protein 1.68 PA0026 plcB Phospholipase C, PlcB 2.25 PA0027  Hypothetical Protein 1.99 PA0028  Hypothetical Protein 2.28 PA0045  Hypothetical Protein 1.81 PA0046  Hypothetical Protein 1.75 PA0048  Probable Transcriptional Regulator 2.78 PA0049  Hypothetical Protein 4.33 PA0051 phzH Potential Phenazine-Modifying Enzyme 2.68 PA0062  Hypothetical Protein -1.53 PA0122 rahU RahU -1.66 PA0125  Hypothetical Protein -1.65 PA0129 bauD Amino Acid Permease 2.31 PA0130 bauC 3-Oxopropanoate Dehydrogenase 1.51 PA0132 bauA Beta-Alanine:Pyruvate Transaminase 1.96 PA0144  Hypothetical Protein 2.71 PA0170  Hypothetical Protein -1.99 PA0171  Hypothetical Protein -1.71 PA0187  Hypothetical Protein -2.85 PA0188  Hypothetical Protein -3.05 PA0229 pcaT Dicarboxylic Acid Transporter PcaT -1.56 PA0234  Hypothetical Protein -1.77 PA0241  Probable Major Facilitator Superfamily (Mfs) Transporter -2.20 PA0247 pobA P-Hydroxybenzoate Hydroxylase -2.04 PA0258  Hypothetical Protein -1.76 PA0279  Probable Transcriptional Regulator -2.51 PA0281 cysW Sulfate Transport Protein CysW 1.87 PA0282 cysT Sulfate Transport Protein CysT 1.97 PA0283 sbp Sulfate-Binding Protein Precursor 3.10 PA0284  Hypothetical Protein 2.52 PA0349  Hypothetical Protein -1.72 PA0417 chpE Probable Chemotaxis Protein 1.79  130 PA0433  Hypothetical Protein -1.90 PA0434  Hypothetical Protein -2.56 PA0435  Hypothetical Protein -2.17 PA0439  Probable Oxidoreductase -2.25 PA0441 dht Dihydropyrimidinase -1.66 PA0451  Conserved Hypothetical Protein -1.61 PA0452  Probable Stomatin-Like Protein -2.11 PA0457  Hypothetical Protein 2.44 PA0476  Probable Permease -2.90 PA0497  Hypothetical Protein -2.93 PA0518 nirM Cytochrome C-551 Precursor 1.83 PA0523 norC Nitric-Oxide Reductase Subunit C 4.29 PA0524 norB Nitric-Oxide Reductase Subunit B 3.93 PA0525  Probable Dinitrification Protein Nord 2.93 PA0531  Probable Glutamine Amidotransferase -1.72 PA0578  Conserved Hypothetical Protein 1.55 PA0617  Probable Bacteriophage Protein 2.00 PA0618  Probable Bacteriophage Protein 1.69 PA0622  Probable Bacteriophage Protein 1.74 PA0628  Conserved Hypothetical Protein 1.54 PA0632  Hypothetical Protein 2.05 PA0634  Hypothetical Protein 1.53 PA0636  Hypothetical Protein 1.69 PA0639  Conserved Hypothetical Protein 1.67 PA0654 speD S-Adenosylmethionine Decarboxylase Proenzyme 1.57 PA0686 hxcR HxcR 1.79 PA0688 lapA Low-Molecular-Weight Alkaline Phosphatase A, LapA -2.49 PA0689 lapB Low-Molecular-Weight Alkaline Phosphatase B, LapB -1.52 PA0713  Hypothetical Protein -1.79 PA0717  Hypothetical Protein Of Bacteriophage Pf1 -2.64 PA0718  Hypothetical Protein Of Bacteriophage Pf1 -3.07 PA0719  Hypothetical Protein Of Bacteriophage Pf1 -2.34 PA0720  Helix Destabilizing Protein Of Bacteriophage Pf1 -1.90 PA0723 coaB Coat Protein B Of Bacteriophage Pf1 -1.80 PA0726  Hypothetical Protein Of Bacteriophage Pf1 -1.60 PA0728  Probable Bacteriophage Integrase -1.96 PA0730  Probable Transferase 2.18 PA0737  Hypothetical Protein -2.29 PA0781  Hypothetical Protein -4.67  131 PA0790  Hypothetical Protein -3.01 PA0844 plcH Hemolytic Phospholipase C Precursor 2.16 PA0845 cerN CerN 5.01 PA0850  Hypothetical Protein -1.91 PA0851  Hypothetical Protein 1.54 PA0852 cbpD Chitin-Binding Protein CbpD Precursor 2.22 PA0882  Hypothetical Protein -2.34 PA0979  Conserved Hypothetical Protein 2.44 PA0983  Conserved Hypothetical Protein -1.91 PA0985 pyoS5 Pyocin S5 1.73 PA0986  Conserved Hypothetical Protein 4.16 PA0987  Conserved Hypothetical Protein 2.63 PA1041  Probable Outer Membrane Protein Precursor -1.82 PA1130 rhlC Rhamnosyltransferase 2 -1.57 PA1151 imm2 Pyocin S2 Immunity Protein -1.60 PA1168  Hypothetical Protein 4.74 PA1217  Probable 2-Isopropylmalate Synthase -1.65 PA1221  Hypothetical Protein -1.75 PA1224  Probable Nad(P)H Dehydrogenase -2.06 PA1244  Hypothetical Protein -2.05 PA1251  Probable Chemotaxis Transducer -2.03 PA1266 lhpE D-Hydroxyproline Dehydrogenase Alpha-Subunit, LphE 2.14 PA1267 lhpB D-Hydroxyproline Dehydrogenase Beta-Subunit, LphB 2.17 PA1268 lhpA Hydroxyproline 2-Epimerase, LhpA 3.03 PA1275 cobD Cobalamin Biosynthetic Protein CobD 1.50 PA1291  Hypothetical Protein -2.07 PA1321 cyoE Cytochrome O Ubiquinol Oxidase Protein CyoE -1.77 PA1332  Hypothetical Protein -1.95 PA1333  Hypothetical Protein -2.33 PA1334  Probable Oxidoreductase 1.91 PA1346  Hypothetical Protein -2.18 PA1369  Hypothetical Protein -1.99 PA1370  Hypothetical Protein -2.06 PA1382  Probable Type Ii Secretion System Protein -1.51 PA1383  Hypothetical Protein -1.71 PA1384 galE Udp-Glucose 4-Epimerase -2.04 PA1385  Probable Glycosyl Transferase -1.84 PA1386  Probable Atp-Binding Component Of Abc Transporter -2.31 PA1387  Hypothetical Protein -1.55  132 PA1388  Hypothetical Protein -1.68 PA1390  Probable Glycosyl Transferase -1.93 PA1391  Probable Glycosyl Transferase -2.06 PA1393 cysC Adenosine 5'-Phosphosulfate (Aps) Kinase -1.78 PA1414  Hypothetical Protein -1.97 PA1418  Probable Sodium:Solute Symport Protein -1.82 PA1423 bdlA BdlA -1.68 PA1431 rsaL Regulatory Protein RsaL 4.53 PA1471  Hypothetical Protein -2.91 PA1499  Conserved Hypothetical Protein -1.92 PA1503  Hypothetical Protein -1.86 PA1507  Probable Transporter -1.91 PA1519  Probable Transporter -2.74 PA1525 alkB2 Alkane-1-Monooxygenase 2 -1.57 PA1565 pauB2 Fad-Dependent Oxidoreductase 2.06 PA1566 pauA3 Glutamylpolyamine Synthetase 1.78 PA1591  Hypothetical Protein 1.69 PA1600  Probable Cytochrome C -1.63 PA1617  Probable Amp-Binding Enzyme -1.57 PA1654  Probable Aminotransferase 1.73 PA1655  Probable Glutathione S-Transferase 1.51 PA1707 pcrH Regulatory Protein PcrH -2.07 PA1708 popB Translocator Protein Popb -1.92 PA1711 exsE ExsE -1.89 PA1739  Probable Oxidoreductase -1.90 PA1771 estX EstX 1.88 PA1873  Hypothetical Protein 2.21 PA1875  Probable Outer Membrane Protein Precursor 1.73 PA1877  Probable Secretion Protein 1.56 PA1887  Hypothetical Protein -3.00 PA1888  Hypothetical Protein -3.21 PA1892  Hypothetical Protein 1.85 PA1893  Hypothetical Protein 1.92 PA1894  Hypothetical Protein 1.76 PA1895  Hypothetical Protein 1.58 PA1897  Hypothetical Protein 1.68 PA1901 phzC2 Phenazine Biosynthesis Protein PhzC -4.69 PA1905 phzG2 Probable Pyridoxamine 5'-Phosphate Oxidase -5.50 PA1914  Conserved Hypothetical Protein 4.37  133 PA1920 nrdD Class Iii (Anaerobic) Ribonucleoside-Triphosphate Reductase Subunit, Nrdd -2.71 PA1921  Hypothetical Protein -6.45 PA1922  Probable Tonb-Dependent Receptor -7.63 PA1923  Hypothetical Protein -6.78 PA1924  Hypothetical Protein -7.49 PA1925  Hypothetical Protein -8.59 PA1927 metE 5-Methyltetrahydropteroyltriglutamate-Homocysteine S-Methyltransferase -1.80 PA1935  Hypothetical Protein -1.63 PA1937  Conserved Hypothetical Protein 3.94 PA1938  Conserved Hypothetical Protein 1.54 PA1939  Hypothetical Protein -1.95 PA1964  Probable ATP-Binding Component Of ABC Transporter 1.68 PA1970  Hypothetical Protein -1.65 PA1977  Hypothetical Protein -2.91 PA1979 eraS Sensor Kinase, EraS -1.55 PA1984 exaC Nad+ Dependent Aldehyde Dehydrogenase ExaC 4.03 PA2013 liuC Putative 3-Methylglutaconyl-Coa Hydratase 1.68 PA2014 liuB Methylcrotonyl-Coa Carboxylase, Beta-Subunit 1.52 PA2021  Hypothetical Protein -1.52 PA2030  Hypothetical Protein 2.19 PA2038  Hypothetical Protein 1.84 PA2073  Probable Transporter (Membrane Subunit) -1.60 PA2074  Hypothetical Protein -1.66 PA2089  Hypothetical Protein -1.76 PA2096  Probable Transcriptional Regulator -2.05 PA2099  Probable Short-Chain Dehydrogenase -1.66 PA2103  Probable Molybdopterin Biosynthesis Protein MoeB -1.98 PA2104  Probable Cysteine Synthase -2.00 PA2105  Probable Acetyltransferase -1.76 PA2106  Hypothetical Protein -1.88 PA2109  Hypothetical Protein 2.22 PA2110  Hypothetical Protein 5.25 PA2111  Hypothetical Protein 3.83 PA2112  Conserved Hypothetical Protein 3.79 PA2113 opdO Pyroglutamate Porin OpdO 4.98 PA2114  Probable Major Facilitator Superfamily (Mfs) Transporter 2.15 PA2116  Conserved Hypothetical Protein 2.38  134 PA2119  Alcohol Dehydrogenase (Zn-Dependent) -1.94 PA2147 katE Catalase Hpii 2.87 PA2167  Hypothetical Protein -1.90 PA2182  Hypothetical Protein -1.97 PA2183  Hypothetical Protein -1.71 PA2184  Conserved Hypothetical Protein -3.46 PA2185 katN Non-Heme Catalase KatN -2.64 PA2186  Hypothetical Protein -2.12 PA2188  Probable Alcohol Dehydrogenase (Zn-Dependent) -2.29 PA2190  Conserved Hypothetical Protein -2.94 PA2192  Conserved Hypothetical Protein -3.05 PA2204  Probable Binding Protein Component Of ABC Transporter 2.01 PA2217  Probable Aldehyde Dehydrogenase -1.90 PA2218  Hypothetical Protein -1.88 PA2221  Conserved Hypothetical Protein -1.65 PA2222  Hypothetical Protein -1.58 PA2224  Hypothetical Protein -1.68 PA2225  Hypothetical Protein -1.90 PA2226 qsrO QsrO -2.02 PA2229  Conserved Hypothetical Protein -2.50 PA2231 pslA PslA -1.63 PA2232 pslB PslB -1.65 PA2233 pslC PslC -1.68 PA2234 pslD PslD -1.73 PA2235 pslE PslE -1.75 PA2260  Hypothetical Protein 1.89 PA2294  Probable Atp-Binding Component Of ABC Transporter -2.44 PA2299  Probable Transcriptional Regulator 1.54 PA2304 ambC AmbC -1.54 PA2317  Probable Oxidoreductase 3.22 PA2318  Hypothetical Protein 2.77 PA2334  Probable Transcriptional Regulator -2.94 PA2343 mtlY Xylulose Kinase -1.95 PA2352  Probable Glycerophosphoryl Diester Phosphodiesterase 1.52 PA2381  Hypothetical Protein -2.48 PA2423  Hypothetical Protein 1.97 PA2429  Hypothetical Protein -2.06 PA2434  Hypothetical Protein -1.78 PA2437  Hypothetical Protein -2.16  135 PA2438  Hypothetical Protein -1.63 PA2439  Hypothetical Protein -2.29 PA2441  Hypothetical Protein -2.74 PA2458  Hypothetical Protein 1.67 PA2470 gtdA Gentisate 1,2-Dioxygenase -1.61 PA2504  Hypothetical Protein -1.67 PA2507 catA Catechol 1,2-Dioxygenase -2.84 PA2508 catC Muconolactone Delta-Isomerase -1.98 PA2511 antR AntR -2.22 PA2512 antA Anthranilate Dioxygenase Large Subunit -3.43 PA2513 antB Anthranilate Dioxygenase Small Subunit -3.12 PA2514 antC Anthranilate Dioxygenase Reductase -2.60 PA2516 xylZ Toluate 1,2-Dioxygenase Electron Transfer Component -2.04 PA2561 ctpH CtpH -2.33 PA2562  Hypothetical Protein -2.04 PA2567  Hypothetical Protein -1.76 PA2580  Conserved Hypothetical Protein -1.66 PA2587 pqsH Probable Fad-Dependent Monooxygenase 1.56 PA2588  Probable Transcriptional Regulator 1.70 PA2602  3-Mercaptopropionate Dioxygenase 2.52 PA2630  Conserved Hypothetical Protein 1.88 PA2636  Hypothetical Protein -1.63 PA2672  Probable Type Ii Secretion System Protein -2.00 PA2673  Probable Type Ii Secretion System Protein -1.63 PA2682  Conserved Hypothetical Protein -1.74 PA2700 opdB Proline Porin OpdB -1.90 PA2714  Probable Molybdopterin Oxidoreductase -2.95 PA2715  Probable Ferredoxin -2.44 PA2736  Hypothetical Protein -1.51 PA2747  Hypothetical Protein -1.79 PA2754  Conserved Hypothetical Protein -1.72 PA2759  Hypothetical Protein -2.71 PA2771  Diguanylate Cyclase With A Self-Blocked I-Site, Dcsbis -2.09 PA2779  Hypothetical Protein -1.95 PA2838  Probable Transcriptional Regulator -3.58 PA2847  Conserved Hypothetical Protein -1.52 PA2862 lipA Lactonizing Lipase Precursor -1.74 PA2863 lipH Lipase Modulator Protein -1.63 PA2911  Probable Tonb-Dependent Receptor -1.84  136 PA2912  Probable ATP-Binding Component Of ABC Transporter -1.65 PA2913  Hypothetical Protein -2.28 PA2914  Probable Permease Of ABC Transporter -1.58 PA2932 morB Morphinone Reductase 2.70 PA2934 cif Cftr Inhibitory Factor, Cif 1.78 PA2937  Hypothetical Protein -1.67 PA2938  Probable Transporter -2.12 PA2939  Probable Aminopeptidase 3.40 PA3000 aroP1 Aromatic Amino Acid Transport Protein AroP1 1.64 PA3032 snr1 Cytochrome C Snr1 -3.25 PA3062 pelC PelC -2.23 PA3137  Probable Major Facilitator Superfamily (Mfs) Transporter -1.78 PA3142  Integrase -1.58 PA3144  Transposase With Helix-Turn-Helix Hin Domain -1.55 PA3160 wzz O-Antigen Chain Length Regulator -2.02 PA3181  2-Keto-3-Deoxy-6-Phosphogluconate Aldolase 2.47 PA3182 pgl 6-Phosphogluconolactonase 2.44 PA3183 zwf Glucose-6-Phosphate 1-Dehydrogenase 2.84 PA3191 gtrS Glucose Transport Sensor, GtrS 1.82 PA3192 gltR Two-Component Response Regulator GltR 1.70 PA3193 glk Glucokinase 1.63 PA3194 edd Phosphogluconate Dehydratase 2.30 PA3195 gapA Glyceraldehyde 3-Phosphate Dehydrogenase 2.71 PA3229  Hypothetical Protein -2.46 PA3266 capB Cold Acclimation Protein B 1.79 PA3281  Hypothetical Protein -3.11 PA3282  Hypothetical Protein -3.89 PA3283  Conserved Hypothetical Protein -4.13 PA3284  Hypothetical Protein -4.72 PA3315  Probable Permease Of Abc Transporter -1.56 PA3319 plcN Non-Hemolytic Phospholipase C Precursor -1.68 PA3323  Conserved Hypothetical Protein -1.69 PA3327  Probable Non-Ribosomal Peptide Synthetase -2.11 PA3328  Probable FAD-Dependent Monooxygenase -2.12 PA3329  Hypothetical Protein -2.12 PA3330  Probable Short Chain Dehydrogenase -2.20 PA3331  Cytochrome P450 -1.86 PA3332  Conserved Hypothetical Protein -1.79 PA3333 fabH2 3-Oxoacyl-[Acyl-Carrier-Protein] Synthase III -1.71  137 PA3334  Probable Acyl Carrier Protein -1.88 PA3335  Hypothetical Protein -1.92 PA3336  Probable Major Facilitator Superfamily (Mfs) Transporter -1.99 PA3359  Hypothetical Protein -1.57 PA3360  Probable Secretion Protein -1.78 PA3361 lecB Fucose-Binding Lectin Pa-III -2.63 PA3390  Hypothetical Protein -1.51 PA3391 nosR Regulatory Protein NosR 3.11 PA3392 nosZ Nitrous-Oxide Reductase Precursor 2.60 PA3393 nosD NosD Protein 2.25 PA3415  Probable Dihydrolipoamide Acetyltransferase -1.75 PA3416  Probable Pyruvate Dehydrogenase E1 Component, Beta Chain -1.58 PA3417  Probable Pyruvate Dehydrogenase E1 Component, Alpha Subunit -1.67 PA3442  Probable Atp-Binding Component Of Abc Transporter 1.68 PA3445  Conserved Hypothetical Protein 1.80 PA3446  Conserved Hypothetical Protein 1.73 PA3450 lsfA 1-Cys Peroxiredoxin LsfA 3.39 PA3451  Hypothetical Protein -1.78 PA3467  Probable Major Facilitator Superfamily (Mfs) Transporter 1.85 PA3497  Hypothetical Protein -2.48 PA3498  Probable Oxidoreductase -2.05 PA3500  Conserved Hypothetical Protein -2.26 PA3506  Probable Decarboxylase -1.50 PA3510  Hypothetical Protein -1.63 PA3514  Probable ATP-Binding Component Of ABC Transporter -1.63 PA3518  Hypothetical Protein 2.15 PA3519  Hypothetical Protein 3.17 PA3520  Hypothetical Protein 2.23 PA3532  Hypothetical Protein 2.08 PA3535  Probable Serine Protease 4.39 PA3546 algX Alginate Biosynthesis Protein AlgX -2.12 PA3572  Hypothetical Protein -1.54 PA3577  Hypothetical Protein 1.59 PA3588  Probable Porin -3.81 PA3593  Probable Acyl-CoA Dehydrogenase -2.26 PA3597  Probable Amino Acid Permease -2.44 PA3598  Conserved Hypothetical Protein -1.73 PA3600  Conserved Hypothetical Protein -5.90 PA3601  Conserved Hypothetical Protein -5.13  138 PA3610 potD Polyamine Transport Protein PotD 1.52 PA3655 tsf Elongation Factor Tsf 1.60 PA3662  Hypothetical Protein -1.68 PA3724 lasB Elastase LasB 1.68 PA3741  Hypothetical Protein 1.63 PA3769 guaA Gmp Synthase 1.51 PA3784  Hypothetical Protein -1.63 PA3785  Conserved Hypothetical Protein -2.36 PA3789  Hypothetical Protein -1.76 PA3790 oprC Putative Copper Transport Outer Membrane Porin Oprc Precursor -2.56 PA3811 hscB Heat Shock Protein Hscb 1.53 PA3819  Conserved Hypothetical Protein -1.66 PA3841 exoS Exoenzyme S -2.00 PA3843  Hypothetical Protein -1.76 PA3877 narK1 Nitrite Extrusion Protein 1 1.83 PA3901 fecA Fe(Iii) Dicitrate Transport Protein FecA 2.75 PA3904  Hypothetical Protein 5.12 PA3905  Hypothetical Protein 6.43 PA3906  Hypothetical Protein 5.59 PA3907  Hypothetical Protein 5.70 PA3908  Hypothetical Protein 5.33 PA3935 tauD Taurine Dioxygenase 2.00 PA3938  Probable Periplasmic Taurine-Binding Protein Precursor 1.66 PA3940  Probable Dna Binding Protein 1.90 PA3967  Hypothetical Protein 2.01 PA4028  Hypothetical Protein -2.75 PA4062  Hypothetical Protein -1.61 PA4063  Hypothetical Protein -4.59 PA4065  Hypothetical Protein -3.59 PA4066  Hypothetical Protein -3.41 PA4071  Hypothetical Protein 3.67 PA4072  Probable Amino Acid Permease 2.77 PA4073  Probable Aldehyde Dehydrogenase 3.20 PA4100  Probable Dehydrogenase 4.78 PA4117 bphP Bacterial Phytochrome, BphP 1.77 PA4133  Cytochrome C Oxidase Subunit (Cbb3-Type) 2.01 PA4134  Hypothetical Protein 2.25 PA4139  Hypothetical Protein 2.37 PA4140  Hypothetical Protein 2.41  139 PA4152  Probable Hydrolase 1.50 PA4170  Hypothetical Protein -3.82 PA4175 piv Protease Iv 3.72 PA4178 eftM Sam-Dependent Methyltransferase , EftM 1.54 PA4181  Hypothetical Protein 2.22 PA4182  Hypothetical Protein 1.61 PA4187  Probable Major Facilitator Superfamily (Mfs) Transporter -3.00 PA4211 phzB1 Probable Phenazine Biosynthesis Protein -1.54 PA4218 ampP AmpP 2.36 PA4219 ampO AmpO 2.03 PA4220  Hypothetical Protein 1.96 PA4221 fptA Fe(Iii)-Pyochelin Outer Membrane Receptor Precursor 2.04 PA4222  Probable Atp-Binding Component Of Abc Transporter 2.05 PA4223  Probable Atp-Binding Component Of Abc Transporter 2.20 PA4224 pchG Pyochelin Biosynthetic Protein PchG 2.16 PA4225 pchF Pyochelin Synthetase 1.90 PA4226 pchE Dihydroaeruginoic Acid Synthetase 1.95 PA4228 pchD Pyochelin Biosynthesis Protein PchD 2.06 PA4229 pchC Pyochelin Biosynthetic Protein PchC 2.38 PA4230 pchB Salicylate Biosynthesis Protein PchB 2.66 PA4231 pchA Salicylate Biosynthesis Isochorismate Synthase 2.22 PA4271 rplL 50S Ribosomal Protein L7 / L12 1.69 PA4272 rplJ 50S Ribosomal Protein L10 1.73 PA4273 rplA 50S Ribosomal Protein L1 1.50 PA4274 rplK 50S Ribosomal Protein L11 1.52 PA4277 tufB Elongation Factor Tu 1.65 PA4298  Hypothetical Protein -1.59 PA4300 tadC TadC -1.60 PA4303 tadZ TadZ -1.54 PA4306 flp Type Ivb Pilin, Flp -2.17 PA4341  Probable Transcriptional Regulator -1.91 PA4355 pyeM PyeM -2.77 PA4364  Hypothetical Protein 2.69 PA4365 lysE Lysine Efflux Permease 2.83 PA4442 cysN ATP Sulfurylase GTP-Binding Subunit/Aps Kinase 1.79 PA4443 cysD ATP Sulfurylase Small Subunit 2.12 PA4500 dppA3 Probable Binding Protein Component Of ABC Transporter 1.72 PA4501 opdD Glycine-Glutamate Dipeptide Porin OpdP 2.08 PA4502 dppA4 Probable Binding Protein Component Of ABC Transporter 1.59  140 PA4505 dppD Dipeptide ABC Transporter Atp-Binding Protein DppD 1.55 PA4549 fimT Type 4 Fimbrial Biogenesis Protein Fimt -2.42 PA4568 rplU 50S Ribosomal Protein L21 1.73 PA4582  Conserved Hypothetical Protein -2.57 PA4584  Conserved Hypothetical Protein -1.63 PA4586  Hypothetical Protein -1.80 PA4590 pra Protein Activator 2.04 PA4607  Hypothetical Protein -2.01 PA4616  Probable C4-Dicarboxylate-Binding Protein 1.68 PA4620  Hypothetical Protein -1.57 PA4673  Conserved Hypothetical Protein 1.66 PA4677  Hypothetical Protein 1.66 PA4714  Conserved Hypothetical Protein 1.62 PA4724  Probable Aminoacyl-Transfer RNA Synthetase (Class I) 1.84 PA4740 pnp Polyribonucleotide Nucleotidyltransferase 1.66 PA4778 cueR CueR 2.15 PA4800  Hypothetical Protein 1.65 PA4834  Putative Nicotianamine Synthase -6.74 PA4835  Hypothetical Protein -6.11 PA4836  Hypothetical Protein -5.97 PA4837  Probable Outer Membrane Protein Precursor -6.26 PA4838  Hypothetical Protein -3.80 PA4869  Hypothetical Protein 2.14 PA4881  Hypothetical Protein -1.53 PA4888 desB Acyl-Coa Delta-9-Desaturase, DesB 3.56 PA4889  Probable Oxidoreductase 2.63 PA4903  Probable Major Facilitator Superfamily (Mfs) Transporter -2.57 PA4935 rpsF 30S Ribosomal Protein S6 1.53 PA4962  Conserved Hypothetical Protein 1.67 PA4973 thiC Thiamin Biosynthesis Protein ThiC 1.74 PA4979  Probable Acyl-Coa Dehydrogenase 1.80 PA4980  Probable Enoyl-Coa Hydratase/Isomerase 1.82 PA5001 ssg Cell Surface-Sugar Biosynthetic Glycosyltransferase, Ssg 1.55 PA5002 dnpA De-N-Acetylase Involved In Persistence, DnpA 1.68 PA5024  Conserved Hypothetical Protein 1.68 PA5101  Hypothetical Protein -1.58 PA5117 typA Regulatory Protein TypA 1.76 PA5139  Hypothetical Protein 2.04 PA5180  Conserved Hypothetical Protein -2.84  141 PA5181  Probable Oxidoreductase -2.75 PA5232  Conserved Hypothetical Protein 1.56 PA5274 rnk Nucleoside Diphosphate Kinase Regulator 1.66 PA5295  Hypothetical Protein 1.51 PA5325 sphA SphA 3.15 PA5326 sphD SphD 4.34 PA5327 sphC SphC 4.11 PA5328 sphB SphB 4.66 PA5352  Conserved Hypothetical Protein -2.79 PA5353 glcF Glycolate Oxidase Subunit GlcF -2.99 PA5354 glcE Glycolate Oxidase Subunit GlcE -2.48 PA5355 glcD Glycolate Oxidase Subunit GlcD -2.47 PA5383  Conserved Hypothetical Protein 4.47 PA5392  Conserved Hypothetical Protein 1.70 PA5396  Hypothetical Protein 2.24 PA5397  Hypothetical Protein 2.41 PA5406  Hypothetical Protein 1.65 PA5407  Hypothetical Protein 1.75 PA5410 gbcA GbcA 1.94 PA5415 glyA1 Serine Hydroxymethyltransferase 2.34 PA5416 soxB Sarcosine Oxidase Beta Subunit 2.13 PA5417 soxD Sarcosine Oxidase Delta Subunit 1.89 PA5418 soxA Sarcosine Oxidase Alpha Subunit 1.81 PA5419 soxG Sarcosine Oxidase Gamma Subunit 1.75 PA5420 purU2 Formyltetrahydrofolate Deformylase 2.46 PA5426 purE Phosphoribosylaminoimidazole Carboxylase, Catalytic Subunit 1.59 PA5434 mtr Tryptophan Permease 1.92 PA5437  Probable Transcriptional Regulator 1.86 PA5470  Probable Peptide Chain Release Factor -2.06 PA5498 znuA ZnuA -1.53 PA5499 zur Zinc Uptake Regulator, Zur -1.55 PA5532  Hypothetical Protein -2.32 PA5534  Hypothetical Protein -5.48 PA5535  Conserved Hypothetical Protein -5.69 PA5536 dksA2 Dksa2 -6.78 PA5537  Hypothetical Protein -3.76 PA5538 amiA N-Acetylmuramoyl-L-Alanine Amidase -6.84 PA5539  Hypothetical Protein -6.54 PA5540  Hypothetical Protein -5.79  142 PA5541 pyrQ Dihydroorotase -5.94 Table A-4. Genes uniquely dysregulated in the △pfeR mutant but not dysregulated under wild-type surfing conditions. RNA-Seq was performed on the △pfeR mutant relative to the PAO6609 WT under surfing conditions. 1572 genes were found to be uniquely dysregulated in the mutant. A log fold-change cut-off of ± 1.5 and p-value < 0.05 was used. Gene annotations and descriptions come from www.pseudomonas.com (Winsor et al., 2016).  Gene ID Gene Name Description Log FC PA0007   Hypothetical Protein 2.07 PA0009 glyQ Glycyl-Trna Synthetase Alpha Chain 1.88 PA0020 tsaP T4P Secretin-Associated Protein Tsap 1.99 PA0021   Conserved Hypothetical Protein -4.44 PA0030 cosX Cosx -2.99 PA0031 betC Choline Sulfatase -2.57 PA0037 trpI Transcriptional Regulator Trpi -1.97 PA0039   Hypothetical Protein 2.36 PA0041   Probable Hemagglutinin -2.05 PA0043   Hypothetical Protein -2.29 PA0044 exoT Exoenzyme T -2.03 PA0047   Hypothetical Protein 2.05 PA0050   Hypothetical Protein 3.36 PA0053   Hypothetical Protein -2.09 PA0056   Probable Transcriptional Regulator -2.94 PA0058 dsbM Dsbm -2.02 PA0059 osmC Osmotically Inducible Protein Osmc 2.59 PA0070 tagQ1 Tagq1 3.72 PA0072 tagS1 Tags1 -2.45 PA0073 tagT1 Tagt1 -1.61 PA0080 tssJ1 Tssj1 2.26 PA0083 tssB1 Tssb1 3.02 PA0084 tssC1 Tssc1 3.11 PA0085 hcp1 Hcp1 3.18 PA0091 vgrG1 Vgrg1 2.01 PA0099   Hypothetical Protein 1.55 PA0103   Probable Sulfate Transporter -2.49 PA0104   Hypothetical Protein -2.52 PA0112   Hypothetical Protein -1.59 PA0113   Probable Cytochrome C Oxidase Assembly Factor -1.57 PA0117   Probable Short Chain Dehydrogenase -1.87 PA0126   Hypothetical Protein 1.57 PA0134   Probable Guanine Deaminase -2.12 PA0135   Hypothetical Protein -3.51  143 PA0136   Probable Atp-Binding Component Of Abc Transporter -3.15 PA0145   Hypothetical Protein -1.89 PA0146   Conserved Hypothetical Protein -2.59 PA0150   Anti-Sigma Factor -1.77 PA0152 pcaQ Transcriptional Regulator Pcaq -2.32 PA0155 pcaR Transcriptional Regulator Pcar -2.09 PA0164   Probable Gamma-Glutamyltranspeptidase -2.67 PA0166   Probable Transporter -3.24 PA0172 siaA Siaa -2.03 PA0175   Probable Chemotaxis Protein Methyltransferase 2 PA0176 aer2 Aerotaxis Transducer Aer2 2.12 PA0177   Probable Purine-Binding Chemotaxis Protein 2.92 PA0178   Probable Two-Component Sensor 2.06 PA0181   Probable Transcriptional Regulator -1.9 PA0182   Probable Short-Chain Dehydrogenase -2.57 PA0183 atsA Arylsulfatase -2.83 PA0185   Probable Permease Of Abc Transporter -4.39 PA0186   Probable Binding Protein Component Of Abc Transporter -3.57 PA0189   Probable Porin -3.87 PA0194   Hypothetical Protein -2.57 PA0198 exbB1 Transport Protein Exbb -2.59 PA0199 exbD1 Transport Protein Exbd -1.58 PA0202   Probable Amidase -5.47 PA0203   Probable Binding Protein Component Of Abc Transporter -1.79 PA0206   Probable Atp-Binding Component Of Abc Transporter -3.21 PA0207   Probable Transcriptional Regulator -2.55 PA0213   Hypothetical Protein -1.73 PA0218   Probable Transcriptional Regulator -1.6 PA0222   Hypothetical Protein -1.69 PA0226   Probable Coa Transferase, Subunit A -2.74 PA0227   Probable Coa Transferase, Subunit B -3.47 PA0228 pcaF Beta-Ketoadipyl CoA Thiolase Pcaf -3.21 PA0230 pcaB 3-Carboxy-Cis,Cis-Muconate Cycloisomerase -2.18 PA0231 pcaD Beta-Ketoadipate Enol-Lactone Hydrolase -2.03 PA0235 pcaK 4-Hydroxybenzoate Transporter Pcak -2.62 PA0237   Probable Oxidoreductase -4.26 PA0238   Hypothetical Protein -3.71 PA0239   Hypothetical Protein -2.04 PA0240   Probable Porin -2.25 PA0242   Hypothetical Protein -4.44 PA0244   Hypothetical Protein -4.42 PA0245 aroQ2 3-Dehydroquinate Dehydratase -2.42 PA0246   Probable Major Facilitator Superfamily (Mfs) Transporter -1.58  144 PA0254 hudA Huda -2.11 PA0257   Hypothetical Protein -2.28 PA0263 hcpC Secreted Protein Hcp 1.75 PA0264   Hypothetical Protein -2.68 PA0268   Probable Transcriptional Regulator -2.52 PA0273   Probable Major Facilitator Superfamily (Mfs) Transporter -7.14 PA0278   Hypothetical Protein -2.66 PA0287 gpuP 3-Guanidinopropionate Transport Protein -4.25 PA0288 gpuA 3-Guanidinopropionase -3.36 PA0289 gpuR Transcriptional Activator Gpur -1.64 PA0311   Hypothetical Protein -4.26 PA0316 serA D-3-Phosphoglycerate Dehydrogenase 2.03 PA0320 carO Calcium-Regulated Ob-Fold Protein Caro -5.34 PA0322   Probable Transporter -1.66 PA0324   Probable Permease Of Abc Transporter -2.13 PA0325   Probable Permease Of Abc Transporter -2.43 PA0327 carP Calcium-Regulated Beta-Propeller Protein Carp -3.58 PA0334   Probable Major Facilitator Superfamily (Mfs) Transporter -2.4 PA0339   Hypothetical Protein -1.74 PA0345   Hypothetical Protein -1.7 PA0347 glpQ Glycerophosphoryl Diester Phosphodiesterase, Periplasmic -3.25 PA0348   Hypothetical Protein -2.79 PA0354   Conserved Hypothetical Protein 1.86 PA0355 pfpI Protease Pfpi 2.91 PA0368   Conserved Hypothetical Protein -1.73 PA0383   Conserved Hypothetical Protein -1.56 PA0389   Hypothetical Protein 1.87 PA0403 pyrR Transcriptional Regulator Pyrr -1.83 PA0404   Conserved Hypothetical Protein -1.69 PA0422   Conserved Hypothetical Protein 2.3 PA0423 pasP Pasp 4.13 PA0432 sahH S-Adenosyl-L-Homocysteine Hydrolase 1.53 PA0438 codB Cytosine Permease -2.12 PA0440   Probable Oxidoreductase -3.59 PA0443   Probable Transporter -1.8 PA0444   N-Carbamoyl-Beta-Alanine Amidohydrolase -2.18 PA0446   Conserved Hypothetical Protein 1.53 PA0447 gcdH Glutaryl-Coa Dehydrogenase 2.11 PA0450   Probable Phosphate Transporter -2.31 PA0453   Hypothetical Protein -2.63 PA0465 creD Inner Membrane Protein Cred -1.68 PA0472 fiuI Fiui 2.04 PA0489   Probable Phosphoribosyl Transferase -1.82  145 PA0496   Conserved Hypothetical Protein -1.92 PA0498   Hypothetical Protein -2.06 PA0499   Probable Pili Assembly Chaperone -3.14 PA0504 bioD Dethiobiotin Synthase -1.57 PA0505   Hypothetical Protein -2.02 PA0519 nirS Nitrite Reductase Precursor 2.98 PA0521   Probable Cytochrome C Oxidase Subunit -3.05 PA0526   Hypothetical Protein 1.61 PA0527 dnr Transcriptional Regulator Dnr 1.75 PA0530   Probable Class Iii Pyridoxal Phosphate-Dependent Aminotransferase -2.43 PA0534 pauB1 Fad-Dependent Oxidoreductase 2.08 PA0539   Hypothetical Protein -2.14 PA0541   Hypothetical Protein 1.88 PA0545   Hypothetical Protein -2.59 PA0546 metK Methionine Adenosyltransferase 4.2 PA0547   Probable Transcriptional Regulator 3.18 PA0548 tktA Transketolase 2.46 PA0551 epd D-Erythrose 4-Phosphate Dehydrogenase 1.56 PA0557   Hypothetical Protein -2.1 PA0563   Conserved Hypothetical Protein 2.37 PA0564   Probable Transcriptional Regulator -1.58 PA0567   Conserved Hypothetical Protein 2.83 PA0573   Hypothetical Protein -1.63 PA0576 rpoD Sigma Factor RpoD 2.52 PA0579 rpsU 30S Ribosomal Protein S21 3.78 PA0592 ksgA Rrna (Adenine-N6,N6)-Dimethyltransferase 1.90 PA0593 pdxA Pyridoxal Phosphate Biosynthetic Protein PdxA 1.67 PA0594 surA Peptidyl-Prolyl Cis-Trans Isomerase SurA 1.99 PA0595 lptD Lps-Assembly Protein LptD 2.50 PA0603 agtA AgtA 1.69 PA0607 rpe Ribulose-Phosphate 3-Epimerase 2.46 PA0619   Probable Bacteriophage Protein 1.74 PA0633   Hypothetical Protein 2.14 PA0635   Hypothetical Protein 1.95 PA0637   Conserved Hypothetical Protein -1.60 PA0642   Hypothetical Protein -3.38 PA0643   Hypothetical Protein -1.78 PA0644   Hypothetical Protein -2.62 PA0645   Hypothetical Protein -1.71 PA0649 trpG Anthranilate Synthase Component II 1.71 PA0651 trpC Indole-3-Glycerol-Phosphate Synthase 1.77 PA0652 vfr Transcriptional Regulator Vfr 1.82  146 PA0663   Hypothetical Protein 1.55 PA0666   Conserved Hypothetical Protein 1.73 PA0669   Probable DNA Polymerase Alpha Chain -1.87 PA0671   Hypothetical Protein -2.85 PA0672 hemO Heme Oxygenase 5.00 PA0673   Hypothetical Protein -2.68 PA0675 vreI Ecf Sigma Factor, VreI -2.19 PA0676 vreR Sigma Factor Regulator, VreR -2.26 PA0677 hxcW Hxcw -3.55 PA0679 hxcP Hxcp -2.46 PA0681 hxcT Hxct -2.83 PA0685 hxcQ Hxcq -5.15 PA0690 pdtA Phosphate Depletion Regulated Tps Partner A, PdtA -3.25 PA0692 pdtB Phosphate Depletion Regulated Tps Partner B, PdtB -2.67 PA0693 exbB2 Transport Protein ExbB2 -5.17 PA0696   Hypothetical Protein -3.74 PA0699   Probable Peptidyl-Prolyl Cis-Trans Isomerase -4.91 PA0701   Probable Transcriptional Regulator -3.97 PA0703   Probable Major Facilitator Superfamily (Mfs) Transporter -2.41 PA0706 cat Chloramphenicol Acetyltransferase 2.23 PA0709   Hypothetical Protein -1.57 PA0710 gloA2 Lactoylglutathione Lyase -1.59 PA0714   Hypothetical Protein -1.65 PA0715   Hypothetical Protein -1.87 PA0716   Hypothetical Protein -1.75 PA0724   Probable Coat Protein A Of Bacteriophage Pf1 -3.97 PA0727   Hypothetical Protein From Bacteriophage Pf1 -2.08 PA0729   Hypothetical Protein -2.11 PA0738   Conserved Hypothetical Protein -2.49 PA0739   Probable Transcriptional Regulator -2.8 PA0740 sdsA1 Sds Hydrolase Sdsa1 -3.56 PA0744   Probable Enoyl-Coa Hydratase/Isomerase 1.80 PA0745   Probable Enoyl-Coa Hydratase/Isomerase 2.09 PA0746   Probable Acyl-Coa Dehydrogenase 2.53 PA0747   Probable Aldehyde Dehydrogenase 2.10 PA0755 opdH Cis-Aconitate Porin OpdH 2.00 PA0758   Hypothetical Protein 1.84 PA0766 mucD Serine Protease MucD Precursor 1.60 PA0767 lepA Gtp-Binding Protein LepA 2.40 PA0768 lepB Signal Peptidase I 1.98 PA0770 rnc Ribonuclease Iii 1.69 PA0771 era Gtp-Binding Protein Era 1.67 PA0778 icp Inhibitor of Cysteine Peptidase 1.74  147 PA0779 asrA AsrA 2.56 PA0780 pruR Proline Utilization Regulator -1.54 PA0785 azoR1 Fmn-Dependent NADH-Azoreductase 1, Azor1 -2.46 PA0787   Hypothetical Protein -2.68 PA0791   Probable Transcriptional Regulator -1.54 PA0800   Hypothetical Protein -2.43 PA0806   Hypothetical Protein -2.47 PA0809   Probable Transporter -4.19 PA0811   Probable Major Facilitator Superfamily (Mfs) Transporter -3.29 PA0812   Hypothetical Protein -2.99 PA0813   Hypothetical Protein -3.07 PA0814   Conserved Hypothetical Protein -1.56 PA0816   Probable Transcriptional Regulator -2.00 PA0817   Probable Ring-Cleaving Dioxygenase -2.30 PA0824   Hypothetical Protein -3.42 PA0825   Hypothetical Protein -1.65 PA0828   Probable Transcriptional Regulator -2.99 PA0841   Hypothetical Protein -1.82 PA0842   Probable Glycosyl Transferase -3.43 PA0856   Hypothetical Protein 2.52 PA0857 bolA Morphogene Protein BolA 2.50 PA0864   Probable Transcriptional Regulator -2.37 PA0875   Conserved Hypothetical Protein -2.48 PA0876   Probable Transcriptional Regulator -2.05 PA0877   Probable Transcriptional Regulator -2.78 PA0878   Hypothetical Protein -3.64 PA0879   Probable Acyl-CoA Dehydrogenase -1.74 PA0883   Probable Acyl-CoA Lyase Beta Chain -3.99 PA0884   Probable C4-Dicarboxylate-Binding Periplasmic Protein -2.07 PA0886   Probable C4-Dicarboxylate Transporter -2.58 PA0888 aotJ Arginine/Ornithine Binding Protein AotJ 1.81 PA0889 aotQ Arginine/Ornithine Transport Protein AotQ 1.55 PA0892 aotP Arginine/Ornithine Transport Protein AotP 1.66 PA0894   Hypothetical Protein -2.00 PA0895 aruC N2-Succinylornithine 5-Aminotransferase (Soat) = N2-Acetylornithine 5-Aminotransferase (Acoat) 2.67 PA0896 aruF Subunit I Of Arginine N2-Succinyltransferase = Ornithine N2-Succinyltransferase 1.69 PA0897 aruG Subunit II of Arginine N2-Succinyltransferase = Ornithine N2-Succinyltransferase 1.80 PA0907 alpA Lysis Phenotype Activator, AlpA -2.10 PA0912   Hypothetical Protein -2.68 PA0913 mgtE MgtE 1.68  148 PA0918   Cytochrome B561 -1.79 PA0929   Two-Component Response Regulator 2.24 PA0931 pirA Ferric Enterobactin Receptor PirA 1.95 PA0938 wzz2 Wzz2 3.12 PA0939   Hypothetical Protein -1.69 PA0942   Probable Transcriptional Regulator -1.76 PA0945 purM Phosphoribosylaminoimidazole Synthetase 2.15 PA0955   Hypothetical Protein 1.75 PA0963 aspS Aspartyl-TrnA Synthetase 2.07 PA0964 pmpR PqsR-Mediated Pqs Regulator, PmpR 1.72 PA0973 oprL Peptidoglycan Associated Lipoprotein OprL Precursor 1.93 PA0977   Hypothetical Protein -1.75 PA0980   Hypothetical Protein -5.34 PA0981   Hypothetical Protein -2.76 PA0996 pqsA PqsA 2.43 PA0997 pqsB PqsB 2.32 PA0998 pqsC PqsC 2.47 PA0999 pqsD 3-Oxoacyl-[Acyl-Carrier-Protein] Synthase III 1.81 PA1000 pqsE Quinolone Signal Response Protein 1.67 PA1001 phnA Anthranilate Synthase Component I 1.72 PA1011   Hypothetical Protein 2.05 PA1016   Hypothetical Protein -2.03 PA1017 pauA Pimeloyl-CoA Synthetase -3.62 PA1019 mucK Cis,Cis-Muconate Transporter MucK -3.76 PA1020   Probable Acyl-CoA Dehydrogenase -3.43 PA1021   Probable Enoyl-CoA Hydratase/Isomerase -3.48 PA1022   Probable Acyl-CoA Dehydrogenase -2.05 PA1023   Probable Short-Chain Dehydrogenase -2.06 PA1024   NADH:Quinone Reductase -2.92 PA1025   Probable Porin -1.83 PA1028 amaA L-Pipecolate Oxidase -1.99 PA1029   Hypothetical Protein -2.23 PA1051   Probable Transporter -1.57 PA1052   Conserved Hypothetical Protein -3.42 PA1056 shaC ShaC -1.55 PA1065   Conserved Hypothetical Protein -2.09 PA1067   Probable Transcriptional Regulator -1.73 PA1077 flgB Flagellar Basal-Body Rod Protein FlgB 1.91 PA1078 flgC Flagellar Basal-Body Rod Protein FlgC 1.80 PA1079 flgD Flagellar Basal-Body Rod Modification Protein FlgD 1.94 PA1108   Probable Major Facilitator Superfamily (Mfs) Transporter -4.52 PA1109   Probable Transcriptional Regulator -1.80 PA1113   Probable ATP-Binding/Permease Fusion ABC Transporter -2.07  149 PA1119 yfiB YfiB 1.96 PA1122   Putative Peptide Deformylase -1.60 PA1123   Hypothetical Protein 1.72 PA1125   Probable Cobalamin Biosynthetic Protein -2.01 PA1129 fosA Fosfomycin Resistance Protein, FosA -1.89 PA1132   Hypothetical Protein 1.84 PA1133   Hypothetical Protein -2.58 PA1135   Conserved Hypothetical Protein -2.3 PA1136   Probable Transcriptional Regulator -2.44 PA1138   Probable Transcriptional Regulator -2.28 PA1139   Hypothetical Protein -2.17 PA1141   Probable Transcriptional Regulator -1.88 PA1143   Hypothetical Protein -3.77 PA1144   Probable Major Facilitator Superfamily (Mfs) Transporter -2.44 PA1147   Probable Amino Acid Permease -3.43 PA1148 toxA Exotoxin A Precursor -2.65 PA1150 pys2 Pyocin S2 -2.44 PA1152   Hypothetical Protein -3.55 PA1153   Hypothetical Protein -2.31 PA1154   Conserved Hypothetical Protein -2.11 PA1155 nrdB Nrdb, Tyrosyl Radical-Harboring Component of Class Ia Ribonucleotide Reductase 1.90 PA1156 nrdA Nrda, Catalytic Component of Class Ia Ribonucleotide Reductase 1.86 PA1165 pcpS PcpS -2.55 PA1178 oprH PhoP/Q and Low Mg2+ Inducible Outer Membrane Protein H1 Precursor -1.54 PA1179 phoP Two-Component Response Regulator PhoP -1.92 PA1182   Probable Transcriptional Regulator -2.81 PA1187   Probable Acyl-CoA Dehydrogenase -2.17 PA1188   Hypothetical Protein -2.10 PA1192   Conserved Hypothetical Protein 1.60 PA1193   Hypothetical Protein 1.56 PA1194   Probable Amino Acid Permease -2.36 PA1196 ddaR Transcriptional Regulator DdaR -2.02 PA1198   Conserved Hypothetical Protein 1.73 PA1199   Probable Lipoprotein 2.12 PA1200   Conserved Hypothetical Protein 1.75 PA1205   Conserved Hypothetical Protein -1.52 PA1209   Hypothetical Protein -2.14 PA1211   Hypothetical Protein -3.15 PA1212   Probable Major Facilitator Superfamily (Mfs) Transporter -2.29 PA1213   Hypothetical Protein -3.33  150 PA1214   Hypothetical Protein -3.31 PA1215   Hypothetical Protein -2.81 PA1219   Hypothetical Protein -3.64 PA1220   Hypothetical Protein -3.11 PA1223   Probable Transcriptional Regulator -2.12 PA1225   Probable NAD(P)H Dehydrogenase -2.99 PA1226   Probable Transcriptional Regulator -2.10 PA1227   Hypothetical Protein -2.14 PA1229   Probable Transcriptional Regulator -2.88 PA1234   Hypothetical Protein -1.62 PA1235   Probable Transcriptional Regulator -2.45 PA1240   Probable Enoyl-CoA Hydratase/Isomerase -2.18 PA1249 aprA Alkaline Metalloproteinase Precursor 4.64 PA1253 lhpG Alpha-Ketoglutaric Semialdehyde Dehydrogenase, LhpG -2.84 PA1257 lhpN Amino Acid ABC Transporter Membrane Protein, LhpN -2.19 PA1258 lhpM Permease of ABC Transporter, LhpM -1.59 PA1259 lhpH LhpH -1.84 PA1261 lhpR Transcriptional Regulator, LhpR -3.47 PA1262   Probable Major Facilitator Superfamily (Mfs) Transporter -4.01 PA1263   Hypothetical Protein -1.71 PA1264   Probable Transcriptional Regulator -2.61 PA1271   Probable TonB-Dependent Receptor 1.75 PA1274   Conserved Hypothetical Protein -2.11 PA1276 cobC Cobalamin Biosynthetic Protein CobC -1.83 PA1278 cobP Cobinamide Kinase -1.63 PA1281 cobV Cobalamin (5'-Phosphate) Synthase -2.33 PA1282   Probable Major Facilitator Superfamily (Mfs) Transporter -5.11 PA1284   Probable Acyl-CoA Dehydrogenase -2.08 PA1288   Probable Outer Membrane Protein Precursor 2.19 PA1296   Probable 2-Hydroxyacid Dehydrogenase -2.21 PA1300   Ecf Sigma Factor 2.13 PA1301   Probable Transmembrane Sensor 1.91 PA1310 phnW 2-Aminoethylphosphonate:Pyruvate Aminotransferase -3.59 PA1311 phnX 2-Phosphonoacetaldehyde Hydrolase -2.18 PA1312   Probable Transcriptional Regulator -2.02 PA1313   Probable Major Facilitator Superfamily (Mfs) Transporter -4.57 PA1316   Probable Major Facilitator Superfamily (Mfs) Transporter -3.83 PA1318 cyoB Cytochrome O Ubiquinol Oxidase Subunit I -1.84 PA1323   Hypothetical Protein 4.48 PA1324   Hypothetical Protein 5.08 PA1328   Probable Transcriptional Regulator -1.95 PA1330   Probable Short-Chain Dehydrogenase -2.42 PA1347   Probable Transcriptional Regulator -3.58  151 PA1351   Probable Sigma-70 Factor, Ecf Subfamily -1.79 PA1359   Probable Transcriptional Regulator -1.76 PA1360   Conserved Hypothetical Protein -3.92 PA1362   Hypothetical Protein -2.30 PA1363   Ecf Sigma Factor 1.93 PA1365   Probable Siderophore Receptor 1.61 PA1367   Hypothetical Protein -1.72 PA1368   Hypothetical Protein -1.65 PA1373 fabF2 3-Oxoacyl-Acyl Carrier Protein Synthase Ii -2.91 PA1374   Hypothetical Protein -1.95 PA1375 pdxB Erythronate-4-Phosphate Dehydrogenase -1.51 PA1379   Probable Short-Chain Dehydrogenase -2.75 PA1380   Probable Transcriptional Regulator -2.35 PA1389   Probable Glycosyl Transferase -2.29 PA1392   Hypothetical Protein -2.11 PA1395   Hypothetical Protein -1.72 PA1399   Probable Transcriptional Regulator -2.22 PA1400   Probable Pyruvate Carboxylase -3.47 PA1402   Hypothetical Protein -1.55 PA1403   Probable Transcriptional Regulator -2.51 PA1404   Hypothetical Protein 1.93 PA1405   Probable Helicase -1.8 PA1406   Hypothetical Protein -1.94 PA1409 aphA Acetylpolyamine Aminohydrolase -1.86 PA1411   Hypothetical Protein -2.5 PA1413   Probable Transcriptional Regulator -2.24 PA1416   Conserved Hypothetical Protein -3.83 PA1417   Probable Decarboxylase -2.96 PA1419   Probable Transporter -2.11 PA1424   Hypothetical Protein -3.45 PA1427   Hypothetical Protein -4.3 PA1433   Conserved Hypothetical Protein -1.58 PA1435   Probable Resistance-Nodulation-Cell Division (RND) Efflux Membrane Fusion Protein Precursor -3.06 PA1436   Probable Resistance-Nodulation-Cell Division (RND) Efflux Transporter -3.06 PA1437   Probable Two-Component Response Regulator -1.79 PA1438   Probable Two-Component Sensor -1.66 PA1451   Conserved Hypothetical Protein -1.60 PA1463   Hypothetical Protein -1.51 PA1464   Probable Purine-Binding Chemotaxis Protein -1.51 PA1467   Hypothetical Protein -1.77 PA1469   Hypothetical Protein -2.5  152 PA1479 ccmE Cytochrome C-Type Biogenesis Protein CcmE 2.08 PA1485   Probable Amino Acid Permease -1.69 PA1486 bapF Beta-Peptidyl Aminopeptidase -2.28 PA1487   Probable Carbohydrate Kinase -2.13 PA1488   Hypothetical Protein -2.95 PA1491   Probable Transporter -2.05 PA1498 pykF Pyruvate Kinase I -3.15 PA1500   Probable Oxidoreductase -3.46 PA1501   Conserved Hypothetical Protein -3.41 PA1502 gcl Glyoxylate Carboligase -3.63 PA1504   Probable Transcriptional Regulator 1.75 PA1506   Hypothetical Protein -2.07 PA1513   Hypothetical Protein -3.6 PA1514   Ureidoglycolate Hydrolaseybbt -3.42 PA1515 alc Allantoicase -3.56 PA1516   Hypothetical Protein -4.18 PA1517   Conserved Hypothetical Protein -3.78 PA1518   Conserved Hypothetical Protein -3.11 PA1537   Probable Short-Chain Dehydrogenase -1.78 PA1539   Hypothetical Protein -2.00 PA1541   Probable Drug Efflux Transporter -1.67 PA1545   Hypothetical Protein -1.88 PA1546 hemN Oxygen-Independent Coproporphyrinogen Iii Oxidase 2.71 PA1547   Hypothetical Protein -2.66 PA1555 ccoP2 Cytochrome C Oxidase, Cbb3-Type, CcoP Subunit 3.01 PA1555.1 ccoQ2 Cytochrome C Oxidase, Cbb3-Type, CcoQ Subunit 2.17 PA1556 ccoO2 Cytochrome C Oxidase, Cbb3-Type, CcoO Subunit 3.02 PA1557 ccoN2 Cytochrome C Oxidase, Cbb3-Type, CcoN Subunit 2.45 PA1558   Hypothetical Protein -2.06 PA1567   Conserved Hypothetical Protein -2.69 PA1569   Probable Major Facilitator Superfamily (Mfs) Transporter -3.79 PA1572   Conserved Hypothetical Protein -1.93 PA1573   Conserved Hypothetical Protein -1.71 PA1577   Hypothetical Protein -1.86 PA1578   Hypothetical Protein -2.05 PA1595   Hypothetical Protein -2.01 PA1596 htpG Heat Shock Protein HtpG 2.25 PA1598   Conserved Hypothetical Protein -3.25 PA1599   Probable Transcriptional Regulator -1.8 PA1601   Probable Aldehyde Dehydrogenase -1.91 PA1602   Probable Oxidoreductase -1.65 PA1603   Probable Transcriptional Regulator -1.59 PA1604   Hypothetical Protein -1.65  153 PA1615   Probable Lipase -1.53 PA1619   Probable Transcriptional Regulator -1.63 PA1621   Probable Hydrolase -1.97 PA1622   Probable Hydrolase -2.13 PA1626   Probable Major Facilitator Superfamily (Mfs) Transporter -3.36 PA1627   Probable Transcriptional Regulator -1.83 PA1628   Probable 3-Hydroxyacyl-Coa Dehydrogenase -2.84 PA1629   Probable Enoyl-CoA Hydratase/Isomerase -3.02 PA1630   Probable Transcriptional Regulator -1.50 PA1633 kdpA Potassium-Transporting ATPase, A Chain -3.41 PA1634 kdpB Potassium-Transporting ATPase, B Chain -4.49 PA1645   Hypothetical Protein -2.27 PA1656 hsiA2 HsiA2 3.3 PA1657 hsiB2 HsiB2 4.5 PA1658 hsiC2 HsiC2 4.33 PA1659 hsiF2 HsiF2 2.57 PA1660 hsiG2 HsiG2 1.74 PA1661 hsiH2 HsiH2 2.52 PA1664 orfX OrfX 1.66 PA1667 hsiJ2 HsiJ2 1.51 PA1668 dotU2 DotU2 1.79 PA1673   Hypothetical Protein 1.56 PA1676   Hypothetical Protein -1.70 PA1680   Hypothetical Protein -3.91 PA1686 alkA Dna-3-Methyladenine Glycosidase II -1.52 PA1688   Hypothetical Protein 1.74 PA1690 pscU Translocation Protein in Type III Secretion -3.18 PA1693 pscR Translocation Protein in Type III Secretion -2.61 PA1695 pscP Translocation Protein in Type III Secretion -4.35 PA1697   Atp Synthase in Type III Secretion System -2.75 PA1698 popN Type Iii Secretion Outer Membrane Protein PopN Precursor -3.16 PA1703 pcrD Type Iii Secretory Apparatus Protein PcrD -2.14 PA1705 pcrG Regulator in Type III Secretion -4.32 PA1706 pcrV Type Iii Secretion Protein PcrV -2.85 PA1709 popD Translocator Outer Membrane Protein PopD Precursor -3.06 PA1712 exsB Exoenzyme S Synthesis Protein B -3.10 PA1715 pscB Type Iii Export Apparatus Protein -2.9 PA1716 pscC Type Iii Secretion Outer Membrane Protein PscC Precursor -2.39 PA1717 pscD Type Iii Export Protein PscD -2.64 PA1722 pscI Type Iii Export Protein PscI -2.45 PA1723 pscJ Type Iii Export Protein PscJ -1.56 PA1735   Hypothetical Protein -1.67 PA1736   Probable Acyl-CoA Thiolase -1.66  154 PA1737   Probable 3-Hydroxyacyl-Coa Dehydrogenase -1.55 PA1738   Probable Transcriptional Regulator -1.60 PA1740   Hypothetical Protein -3.62 PA1746   Hypothetical Protein 1.69 PA1750   Phospho-2-Dehydro-3-Deoxyheptonate Aldolase 1.61 PA1755   Hypothetical Protein -2.29 PA1761   Hypothetical Protein -1.76 PA1764   Hypothetical Protein -1.77 PA1772   Probable Methyltransferase -2.09 PA1776 sigX Ecf Sigma Factor SigX 1.64 PA1778 cobA Uroporphyrin-Iii C-Methyltransferase -3.56 PA1779   Assimilatory Nitrate Reductase -2.8 PA1781 nirB Assimilatory Nitrite Reductase Large Subunit -2.81 PA1786 nasS Nass -1.58 PA1791   Hypothetical Protein 2.41 PA1800 tig Trigger Factor 2.20 PA1804 hupB DNA-Binding Protein Hu 1.60 PA1812 mltD Membrane-Bound Lytic Murein Transglycosylase D Precursor 1.76 PA1817   Hypothetical Protein -1.81 PA1825   Hypothetical Protein -1.81 PA1826   Probable Transcriptional Regulator -2.39 PA1827   Probable Short-Chain Dehydrogenase -3.00 PA1842   Hypothetical Protein 2.01 PA1843 metH Methionine Synthase 1.77 PA1845 tsi1 Tsi1 1.53 PA1847 nfuA Nfua -1.64 PA1848   Probable Major Facilitator Superfamily (Mfs) Transporter -4.98 PA1850   Probable Transcriptional Regulator -1.70 PA1852   Hypothetical Protein -1.60 PA1863 modA Molybdate-Binding Periplasmic Protein Precursor ModA 1.72 PA1866   Hypothetical Protein -1.90 PA1868 xqhA Secretion Protein XqhA -3.75 PA1869   Probable Acyl Carrier Protein 5.34 PA1870   Hypothetical Protein 2.40 PA1871 lasA Lasa Protease Precursor 4.48 PA1874   Hypothetical Protein 4.10 PA1876   Probable ATP-Binding/Permease Fusion ABC Transporter 2.05 PA1879   Hypothetical Protein -2.25 PA1884   Probable Transcriptional Regulator -1.99 PA1885   Conserved Hypothetical Protein -1.79 PA1899 phzA2 Probable Phenazine Biosynthesis Protein 4.48 PA1900 phzB2 Probable Phenazine Biosynthesis Protein 4.69 PA1907   Hypothetical Protein -2.25  155 PA1908   Probable Major Facilitator Superfamily (Mfs) Transporter -3.22 PA1912 femI Ecf Sigma Factor, Femi 3.66 PA1913   Hypothetical Protein 2.25 PA1916   Probable Amino Acid Permease -4.23 PA1919 nrdG Class Iii (Anaerobic) Ribonucleoside-Triphosphate Reductase Activating Protein, 'Activase', Nrdg -1.63 PA1928 rimJ Ribosomal Protein Alanine Acetyltransferase -1.95 PA1929   Hypothetical Protein -2.05 PA1936   Hypothetical Protein -2.91 PA1947 rbsA Ribose Transport Protein RbsA -1.56 PA1950 rbsK Ribokinase -2.19 PA1954 fapC FapC -2.12 PA1957   Hypothetical Protein -1.68 PA1962 azoR2 Fmn-Dependent Nadh-Azoreductase 2, Azor2 -2.37 PA1967   Hypothetical Protein 1.72 PA1972   Conserved Hypothetical Protein -2.35 PA1973 pqqF Pyrroloquinoline Quinone Biosynthesis Protein F -2.72 PA1974   Hypothetical Protein -2.59 PA1975   Hypothetical Protein -2.34 PA1976 ercS ErcS -2.75 PA1978 erbR Response Regulator ErbR -1.81 PA1980 eraR Response Regulator EraR -2.29 PA1982 exaA Quinoprotein Ethanol Dehydrogenase -1.91 PA1990 pqqH PqqH -2.55 PA1992 ercS ErcS -2.31 PA1993   Probable Major Facilitator Superfamily (Mfs) Transporter -2.07 PA1999 dchA Dehydrocarnitine Coa Transferase, DchA 3.51 PA2000 dchB Dehydrocarnitine Coa Transferase, DchB 3.02 PA2007 maiA Maleylacetoacetate Isomerase 1.77 PA2009 hmgA Homogentisate 1,2-Dioxygenase 2.68 PA2015 liuA Putative Isovaleryl-Coa Dehydrogenase 2.72 PA2016 liuR Regulator Of Liu Genes 2.42 PA2022   Probable Nucleotide Sugar Dehydrogenase -2.36 PA2023 galU Utp-Glucose-1-Phosphate Uridylyltransferase 2.73 PA2024   Probable Ring-Cleaving Dioxygenase -1.68 PA2032   Probable Transcriptional Regulator -2.02 PA2033   Hypothetical Protein 2.86 PA2035   Probable Decarboxylase -2.34 PA2036   Hypothetical Protein -4.71 PA2037   Hypothetical Protein -1.95 PA2042   Probable Transporter (Membrane Subunit) 2.75 PA2046   Hypothetical Protein -2.73 PA2048   Hypothetical Protein -1.64  156 PA2054 cynR Transcriptional Regulator CynR -2.42 PA2056   Probable Transcriptional Regulator -2.33 PA2057 sppR Tonb-Dependent Receptor, SppR -4.15 PA2058 sppA Abc Transporter Substrate-Binding Protein, SppA -2.99 PA2061 sppD Abc Transporter Atp-Binding Protein, SppD -4.10 PA2064 pcoB Copper Resistance Protein B Precursor -2.95 PA2065 pcoA Copper Resistance Protein A Precursor -1.72 PA2066   Hypothetical Protein 2.76 PA2069   Probable Carbamoyl Transferase 3.53 PA2070   Hypothetical Protein -3.43 PA2071 fusA2 Elongation Factor G 1.54 PA2078   (7S,10S)-Hydroperoxide Diol Synthase -5.34 PA2079   Probable Amino Acid Permease -2.19 PA2080 kynU Kynureninase KynU -2.12 PA2084   Probable Asparagine Synthetase -2.60 PA2093   Probable Sigma-70 Factor, Ecf Subfamily -2.40 PA2095   Hypothetical Protein -2.10 PA2097   Probable Flavin-Binding Monooxygenase -2.02 PA2100   Probable Transcriptional Regulator -2.83 PA2101   Conserved Hypothetical Protein -2.24 PA2102   Hypothetical Protein -2.39 PA2107   Hypothetical Protein -2.06 PA2115   Probable Transcriptional Regulator -1.52 PA2120   Hypothetical Protein -2.43 PA2121   Probable Transcriptional Regulator -1.65 PA2122   Hypothetical Protein -1.57 PA2124   Probable Dehydrogenase -3.79 PA2125   Probable Aldehyde Dehydrogenase -5.48 PA2126 cgrC CupA Gene Regulator C, CgrC -1.84 PA2129 cupA2 Chaperone Cupa2 -4.51 PA2130 cupA3 Usher Cupa3 -4.35 PA2134   Hypothetical Protein 2.53 PA2135   Probable Transporter -2.33 PA2136   Hypothetical Protein -1.83 PA2143   Hypothetical Protein 2.03 PA2144 glgP Glycogen Phosphorylase 2.58 PA2146   Conserved Hypothetical Protein 5.41 PA2149   Hypothetical Protein 1.51 PA2150   Conserved Hypothetical Protein 1.58 PA2151   Conserved Hypothetical Protein 2.01 PA2152   Probable Trehalose Synthase 1.60 PA2159   Conserved Hypothetical Protein 3.30 PA2160   Probable Glycosyl Hydrolase 2.89  157 PA2164   Probable Glycosyl Hydrolase 1.60 PA2165   Probable Glycogen Synthase 2.71 PA2166   Hypothetical Protein 3.34 PA2169   Hypothetical Protein 4.51 PA2170   Hypothetical Protein 3.03 PA2171   Hypothetical Protein 4.19 PA2172   Hypothetical Protein 1.93 PA2189   Hypothetical Protein 1.91 PA2193 hcnA Hydrogen Cyanide Synthase HcnA 2.14 PA2195 hcnC Hydrogen Cyanide Synthase HcnC 1.56 PA2206   Lysr-Type Transcriptional Regulator -1.65 PA2207   Hypothetical Protein -2.66 PA2209   Hypothetical Protein -2.03 PA2210   Probable Major Facilitator Superfamily (Mfs) Transporter -3.49 PA2211   Conserved Hypothetical Protein -2.24 PA2212   Conserved Hypothetical Protein -2.86 PA2214   Putative L-Lyxonate Transporter -5.61 PA2215 lyxD L-Lyxonate Dehydratase LyxD -2.48 PA2216   2-Keto-3-Deoxy-D-Arabinonate Dehydratase -3.30 PA2219 opdE Membrane Protein OpdE -3.50 PA2220   Probable Transcriptional Regulator -1.89 PA2227 vqsM Arac-Type Transcriptional Regulator VqsM -1.82 PA2228   Hypothetical Protein -2.83 PA2241 pslK PslK -2.24 PA2243 pslM Hypothetical Protein -4.14 PA2247 bkdA1 2-Oxoisovalerate Dehydrogenase (Alpha Subunit) -1.68 PA2248 bkdA2 2-Oxoisovalerate Dehydrogenase (Beta Subunit) -1.57 PA2249 bkdB Branched-Chain Alpha-Keto Acid Dehydrogenase (Lipoamide Component) -1.72 PA2250 lpdV Lipoamide Dehydrogenase-Val -1.51 PA2251   Hypothetical Protein -1.55 PA2252   Probable AgcS Sodium/Alanine/Glycine Symporter -2.23 PA2261   Probable 2-Ketogluconate Kinase -3.01 PA2262   Probable 2-Ketogluconate Transporter -1.70 PA2263   Probable 2-Hydroxyacid Dehydrogenase -2.87 PA2267   Probable Transcriptional Regulator -1.90 PA2268   Hypothetical Protein -1.96 PA2269   Conserved Hypothetical Protein -2.71 PA2271   Probable Acetyltransferase -1.62 PA2273 soxR SoxR -1.56 PA2274   Hypothetical Protein -1.83 PA2275   Probable Alcohol Dehydrogenase (Zn-Dependent) -2.79 PA2277 arsR ArsR Protein -1.59  158 PA2278 arsB ArsB Protein -3.86 PA2279 arsC ArsC Protein -1.62 PA2280   Oxidoreductase -2.38 PA2282   Hypothetical Protein -1.95 PA2283   Hypothetical Protein -3.35 PA2285   Hypothetical Protein -2.34 PA2290 gcd Glucose Dehydrogenase 1.96 PA2291   Probable Glucose-Sensitive Porin 4.16 PA2295   Probable Permease of ABC Transporter -1.77 PA2296   Hypothetical Protein -3.26 PA2300 chiC Chitinase 4.91 PA2310   Hypothetical Protein -2.61 PA2312   Probable Transcriptional Regulator -2.33 PA2313   Hypothetical Protein -2.93 PA2315   Hypothetical Protein -6.15 PA2316   Probable Transcriptional Regulator -1.99 PA2323   Probable Glyceraldehyde-3-Phosphate Dehydrogenase -1.60 PA2326   Hypothetical Protein -2.01 PA2327   Probable Permease of ABC Transporter -1.74 PA2328   Hypothetical Protein -1.60 PA2330   Hypothetical Protein -1.85 PA2332   Probable Transcriptional Regulator -1.67 PA2333   Probable Sulfatase -4.15 PA2335   Probable TonB-Dependent Receptor -4.82 PA2336   Hypothetical Protein -4.69 PA2337 mtlR Transcriptional Regulator MtlR -1.99 PA2338   Probable Binding Protein Component of ABC Maltose/Mannitol Transporter -1.75 PA2339   Probable Binding-Protein-Dependent Maltose/Mannitol Transport Protein -3.95 PA2340   Probable Binding-Protein-Dependent Maltose/Mannitol Transport Protein -2.73 PA2341   Probable ATP-Binding Component of ABC Maltose/Mannitol Transporter -3.04 PA2342 mtlD Mannitol Dehydrogenase -4.14 PA2344 mtlZ Fructokinase -3.42 PA2346   Conserved Hypothetical Protein -5.17 PA2354   Probable Transcriptional Regulator -1.9 PA2355   Probable Fmnh2-Dependent Monooxygenase -3.48 PA2365 hsiB3 HsiB3 3.76 PA2366 hsiC3 HsiC3 4.52 PA2367 hcp3 Hcp3 4.01 PA2369 hsiG3 HsiG3 1.57 PA2371 clpV3 ClpV3 1.73  159 PA2372   Hypothetical Protein 2.33 PA2373 vgrG3 VgrG3 1.80 PA2377   Hypothetical Protein -2.12 PA2384   Hypothetical Protein 3.89 PA2385 pvdQ 3-Oxo-C12-Homoserine Lactone Acylase PvdQ 1.96 PA2386 pvdA L-Ornithine N5-Oxygenase 4.97 PA2389 pvdR PvdR 1.73 PA2392 pvdP PvdP 1.91 PA2393   Putative Dipeptidase 3.64 PA2394 pvdN PvdN 3.73 PA2395 pvdO PvdO 3.34 PA2396 pvdF Pyoverdine Synthetase F 2.35 PA2397 pvdE Pyoverdine Biosynthesis Protein PvdE 2.97 PA2398 fpvA Ferripyoverdine Receptor 4.61 PA2399 pvdD Pyoverdine Synthetase D 2.00 PA2400 pvdJ PvdJ 4.57 PA2402   Probable Non-Ribosomal Peptide Synthetase 1.55 PA2403 fpvG FpvG 3.49 PA2404 fpvH FpvH 2.48 PA2405 fpvJ FpvJ 3.44 PA2411   Probable Thioesterase 2.99 PA2412   Conserved Hypothetical Protein 4.07 PA2413 pvdH L-2,4-Diaminobutyrate:2-Ketoglutarate 4-Aminotransferase, PvdH 2.19 PA2420   Probable Porin -3.37 PA2422   Hypothetical Protein -3.11 PA2425 pvdG PvdG 3.2 PA2426 pvdS Sigma Factor PvdS 5.35 PA2427   Hypothetical Protein 2.08 PA2432 bexR Bistable Expression Regulator, BexR -1.6 PA2433   Hypothetical Protein 2.06 PA2435   Probable Cation-Transporting P-Type ATPase -1.59 PA2440   Hypothetical Protein -2.72 PA2442 gcvT2 Glycine Cleavage System Protein T2 2.38 PA2443 sdaA L-Serine Dehydratase 2.16 PA2444 glyA2 Serine Hydroxymethyltransferase 3.68 PA2445 gcvP2 Glycine Cleavage System Protein P2 4.10 PA2446 gcvH2 Glycine Cleavage System Protein H2 4.64 PA2447   Probable Transcriptional Regulator -1.75 PA2459   Hypothetical Protein -4.69 PA2460   Hypothetical Protein -1.90 PA2461   Hypothetical Protein -2.33 PA2462   Hypothetical Protein -1.66  160 PA2465   Hypothetical Protein -2.69 PA2468 foxI Ecf Sigma Factor FoxI 1.84 PA2469   Probable Transcriptional Regulator -2.28 PA2471   Conserved Hypothetical Protein -2.37 PA2477   Probable Thiol:Disulfide Interchange Protein -2.54 PA2478   Probable Thiol:Disulfide Interchange Protein -2.25 PA2479   Probable Two-Component Response Regulator -2.45 PA2480   Probable Two-Component Sensor -2.28 PA2481   Hypothetical Protein -1.78 PA2482   Probable Cytochrome C -1.81 PA2483   Conserved Hypothetical Protein -2.75 PA2487   Hypothetical Protein -3.53 PA2488   Probable Transcriptional Regulator -2.4 PA2489   Probable Transcriptional Regulator -1.68 PA2490   Conserved Hypothetical Protein -2.80 PA2493 mexE Resistance-Nodulation-Cell Division (RND) Multidrug Efflux Membrane Fusion Protein MexE Precursor -1.58 PA2495 oprN Multidrug Efflux Outer Membrane Protein OprN Precursor -1.84 PA2497   Probable Transcriptional Regulator -2.83 PA2499   Probable Deaminase -3.45 PA2500   Probable Major Facilitator Superfamily (Mfs) Transporter -3.61 PA2502   Hypothetical Protein -1.61 PA2505 opdT Tyrosine Porin OpdT -2.84 PA2509 catB Muconate Cycloisomerase I -4.25 PA2510 catR Transcriptional Regulator CatR -2.80 PA2515 xylL Cis-1,2-Dihydroxycyclohexa-3,4-Diene Carboxylate Dehydrogenase -5.46 PA2518 xylX Toluate 1,2-Dioxygenase Alpha Subunit -4.51 PA2519 xylS Transcriptional Regulator XylS -3.23 PA2520 czcA RND Divalent Metal Cation Efflux Transporter CzcA -2.89 PA2521 czcB RND Divalent Metal Cation Efflux Membrane Fusion Protein CzcB  -4.92 PA2524 czcS CzcS -2.42 PA2534   Probable Transcriptional Regulator -2.12 PA2536   Probable Phosphatidate Cytidylyltransferase 2.21 PA2537   Probable Acyltransferase 1.68 PA2545 xthA Exodeoxyribonuclease III 1.63 PA2546   Probable Ring-Cleaving Dioxygenase -1.89 PA2548   Hypothetical Protein -3.43 PA2552   Probable Acyl-CoA Dehydrogenase 1.65 PA2555   Probable Amp-Binding Enzyme 1.86 PA2563   Probable Sulfate Transporter -1.89 PA2564   Hypothetical Protein 1.53  161 PA2565   Hypothetical Protein 2.59 PA2566   Conserved Hypothetical Protein 2.79 PA2569   Hypothetical Protein 2.07 PA2570 lecA LecA 5.79 PA2574 alkB1 Alkane-1-Monooxygenase -2.63 PA2576   Hypothetical Protein -2.15 PA2589   Hypothetical Protein -4.18 PA2592   Probable Periplasmic Spermidine/Putrescine-Binding Protein 2.28 PA2594   Conserved Hypothetical Protein -1.64 PA2596   Conserved Hypothetical Protein -3.70 PA2597   Hypothetical Protein -3.49 PA2598   Hypothetical Protein -2.82 PA2599   Conserved Hypothetical Protein -1.53 PA2600   Hypothetical Protein -1.78 PA2619 infA Initiation Factor 1.97 PA2629 purB Adenylosuccinate Lyase 1.85 PA2634 aceA Isocitrate Lyase AceA -1.75 PA2635   Hypothetical Protein -1.61 PA2650   Conserved Hypothetical Protein -2.24 PA2655   Hypothetical Protein -2.84 PA2656 carS Two-Component Sensor CarS -2.3 PA2657 carR Two-Component Response Regulator CarR -2.48 PA2658   Hypothetical Protein -3.22 PA2659   Hypothetical Protein -3.01 PA2660   Hypothetical Protein -1.71 PA2662   Conserved Hypothetical Protein -2.86 PA2663 ppyR Psl And Pyoverdine Operon Regulator, PpyR -2.08 PA2664 fhp Flavohemoprotein -1.61 PA2665 fhpR Transcriptional Activator of Flavohemoglobin, FhpR -2.17 PA2669   Hypothetical Protein -3.94 PA2670   Hypothetical Protein -2.28 PA2675   Probable Type Ii Secretion System Protein -2.24 PA2676   Probable Type Ii Secretion System Protein -2.55 PA2677   Probable Type Ii Secretion Protein -1.79 PA2678   Probable Permease of Abc-2 Transporter -2.99 PA2680   Probable Quinone Oxidoreductase -3.29 PA2681   Probable Transcriptional Regulator -3.11 PA2685 vgrG4 VgrG4 1.59 PA2689   Hypothetical Protein -2.39 PA2691   Conserved Hypothetical Protein -1.98 PA2693   Conserved Hypothetical Protein -2.18 PA2701   Probable Major Facilitator Superfamily (Mfs) Transporter -2.91 PA2711   Probable Periplasmic Spermidine/Putrescine-Binding Protein -1.88  162 PA2712   Hypothetical Protein -1.80 PA2719   Hypothetical Protein -1.63 PA2723   Hypothetical Protein -1.84 PA2724   Hypothetical Protein -2.05 PA2750   Hypothetical Protein -2.50 PA2758   Probable Transcriptional Regulator -2.94 PA2760 oprQ OprQ 1.71 PA2761   Hypothetical Protein 2.78 PA2762   Hypothetical Protein -2.33 PA2764   Hypothetical Protein -1.69 PA2772   Hypothetical Protein -1.51 PA2775 tsi4 Tsi4 1.58 PA2782 bamI Biofilm-Associated Metzincin Inhibitor, BamI -1.65 PA2783 mep72 Mep72 -3.11 PA2791   Hypothetical Protein -2.37 PA2792   Hypothetical Protein 1.63 PA2794   Pseudaminidase -1.52 PA2798   Probable Two-Component Response Regulator 1.51 PA2800 vacJ VacJ 1.52 PA2808 ptrA Pseudomonas Type Iii Repressor A -1.87 PA2809 copR Two-Component Response Regulator, CopR -1.60 PA2810 copS Two-Component Sensor, CopS -1.76 PA2814   Hypothetical Protein -2.36 PA2816   Hypothetical Protein -1.72 PA2818 arr Aminoglycoside Response Regulator -2.28 PA2819   Hypothetical Protein -2.84 PA2827   Conserved Hypothetical Protein -1.78 PA2833   Conserved Hypothetical Protein -2.91 PA2835   Probable Major Facilitator Superfamily (Mfs) Transporter -3.13 PA2839   Conserved Hypothetical Protein -2.63 PA2844   Conserved Hypothetical Protein -2.38 PA2848   Probable Transcriptional Regulator -2.65 PA2851 efp Translation Elongation Factor P 2.77 PA2852 earP EarP -1.67 PA2853 oprI Outer Membrane Lipoprotein OprI Precursor 1.68 PA2858   Conserved Hypothetical Protein -1.79 PA2861 ligT 2'-5' RNA Ligase -2.73 PA2874   Hypothetical Protein -1.63 PA2877   Probable Transcriptional Regulator -1.75 PA2878   Hypothetical Protein -3.12 PA2879   Probable Transcriptional Regulator -1.74 PA2880   Hypothetical Protein -2.25 PA2890 atuE Putative Isohexenylglutaconyl-CoA Hydratase -3.60  163 PA2891 atuF Geranyl-CoA Carboxylase, Alpha-Subunit (Biotin-Containing) -2.38 PA2892 atuG Gcase, Alpha-Subunit (Biotin-Containing) -2.31 PA2898   Hypothetical Protein -2.4 PA2903 cobJ Precorrin-3 Methylase CobJ -1.76 PA2907 cobL Precorrin-6Y-Dependent Methyltransferase CobL -1.84 PA2909   Hypothetical Protein -2.61 PA2919   Hypothetical Protein -1.57 PA2922   Probable Hydrolase -5.75 PA2923 hisJ Periplasmic Histidine-Binding Protein HisJ -3.39 PA2926 hisP Histidine Transport Protein HisP -3.16 PA2928   Hypothetical Protein -2.43 PA2929   Hypothetical Protein -2.88 PA2930   Probable Transcriptional Regulator -1.95 PA2931 cifR CifR -1.90 PA2935   Hypothetical Protein -2.62 PA2940   Probable Acyl-Coa Thiolase -2.52 PA2941   Hypothetical Protein -2.62 PA2950 pfm Proton Motive Force Protein, Pmf 1.84 PA2969 plsX Fatty Acid Biosynthesis Protein PlsX 2.14 PA2970 rpmF 50S Ribosomal Protein L32 3.5 PA2971   Conserved Hypothetical Protein 3.37 PA2982   Conserved Hypothetical Protein 1.93 PA2984   Hypothetical Protein -3.03 PA2999 nqrA Na+-Translocating NADH:Ubiquinone Oxidoreductase Subunit NrqA 1.67 PA3016   Hypothetical Protein -1.73 PA3017   Conserved Hypothetical Protein -1.92 PA3025   Probable FAD-Dependent Glycerol-3-Phosphate Dehydrogenase -2.54 PA3039   Probable Transporter -2.74 PA3041   Hypothetical Protein 1.81 PA3042   Hypothetical Protein 2.13 PA3045 rocA2 Two-Component Response Regulator, RocA2 -1.87 PA3047   Probable D-Alanyl-D-Alanine Carboxypeptidase 1.55 PA3051   Hypothetical Protein -1.62 PA3058 pelG PelG -2.24 PA3059 pelF PelF -2.50 PA3060 pelE PelE -3.81 PA3061 pelD PelD -2.92 PA3063 pelB PelB -4.29 PA3064 pelA PelA -3.84 PA3065   Hypothetical Protein -7.81 PA3066   Hypothetical Protein -8.49  164 PA3067   Probable Transcriptional Regulator -7.39 PA3068 gdhB NAD-Dependent Glutamate Dehydrogenase 1.66 PA3090   Hypothetical Protein -2.64 PA3092 fadH1 2,4-Dienoyl-Coa Reductase FadH1 -1.63 PA3101 xcpT General Secretion Pathway Protein G 1.67 PA3105 xcpQ General Secretion Pathway Protein D 1.64 PA3112 accD Acetyl-Coa Carboxylase Beta Subunit 1.66 PA3125   Hypothetical Protein -3.15 PA3132   Probable Hydrolase -3.18 PA3134 gltX Glutamyl-Trna Synthetase 1.55 PA3136   Probable Secretion Protein -1.50 PA3151 hisF2 Imidazoleglycerol-Phosphate Synthase, Cyclase Subunit -1.62 PA3152 hisH2 Glutamine Amidotransferase -2.01 PA3153 wzx O-Antigen Translocase -1.68 PA3161 himD Integration Host Factor Beta Subunit -1.62 PA3162 rpsA 30S Ribosomal Protein S1 2.61 PA3166 pheA Chorismate Mutase 1.82 PA3167 serC 3-Phosphoserine Aminotransferase 1.76 PA3168 gyrA Dna Gyrase Subunit A 1.74 PA3186 oprB Glucose/Carbohydrate Outer Membrane Porin Oprb Precursor 6.52 PA3187   Probable ATP-Binding Component of ABC Transporter 5.95 PA3188   Probable Permease of ABC Sugar Transporter 6.16 PA3189   Probable Permease of ABC Sugar Transporter 5.62 PA3190   Probable Binding Protein Component of ABC Sugar Transporter 7.12 PA3207   Hypothetical Protein -2.71 PA3215   Probable Transcriptional Regulator -1.72 PA3216   Hypothetical Protein -1.72 PA3219   Hypothetical Protein -2.55 PA3231   Hypothetical Protein 1.84 PA3236 betX BetX -2.59 PA3237   Hypothetical Protein -3.35 PA3241   Hypothetical Protein -1.58 PA3262   Probable Peptidyl-Prolyl Cis-Trans Isomerase, FkbP-Type 2.74 PA3274   Hypothetical Protein 2.63 PA3276   Hypothetical Protein 1.75 PA3279 oprP Phosphate-Specific Outer Membrane Porin OprP Precursor -2.62 PA3287   Conserved Hypothetical Protein -1.58 PA3291 tli1 Tli1 -2.18 PA3296 phoA Alkaline Phosphatase -2.71 PA3306   Hypothetical Protein -2.62 PA3307   Hypothetical Protein -4.54 PA3309   Conserved Hypothetical Protein 2.66  165 PA3312   Probable 3-Hydroxyisobutyrate Dehydrogenase -1.69 PA3316   Probable Permease of ABC Transporter -1.82 PA3320   Hypothetical Protein -5.21 PA3321   Probable Transcriptional Regulator -3.01 PA3324   Probable Short-Chain Dehydrogenase -2.64 PA3325   Conserved Hypothetical Protein -2.12 PA3326 clpP2 ClpP2 3.11 PA3358   Hypothetical Protein -2.89 PA3362   Hypothetical Protein -6.45 PA3363 amiR Aliphatic Amidase Regulator -6.53 PA3364 amiC Aliphatic Amidase Expression-Regulating Protein -6.28 PA3365   Probable Chaperone -6.37 PA3366 amiE Aliphatic Amidase -6.72 PA3369   Hypothetical Protein 1.65 PA3371   Hypothetical Protein 2.47 PA3372   Conserved Hypothetical Protein -1.72 PA3374   Conserved Hypothetical Protein -2.91 PA3378   Conserved Hypothetical Protein -3.75 PA3383   Binding Protein Component of ABC Phosphonate Transporter -1.58 PA3384 phnC Atp-Binding Component of ABC Phosphonate Transporter -2.64 PA3385 amrZ Alginate and Motility Regulator Z 1.68 PA3387 rhlG Beta-Ketoacyl Reductase -2.02 PA3389   Probable Ring-Cleaving Dioxygenase -1.96 PA3397 fprA FprA 3.37 PA3406 hasD Transport Protein HasD -1.67 PA3407 hasAp Heme Acquisition Protein HasAp 5.96 PA3408 hasR Heme Uptake Outer Membrane Receptor Hasr Precursor 1.51 PA3420   Probable Transcriptional Regulator -3.58 PA3421   Conserved Hypothetical Protein -2.29 PA3422   Hypothetical Protein -2.49 PA3424   Hypothetical Protein -2.88 PA3425   Hypothetical Protein -3.54 PA3428   Hypothetical Protein -1.60 PA3429   Probable Epoxide Hydrolase -1.63 PA3433   Probable Transcriptional Regulator -1.62 PA3436   Hypothetical Protein -3.41 PA3444   Conserved Hypothetical Protein -2.77 PA3456   Hypothetical Protein -1.89 PA3457   Hypothetical Protein -2.18 PA3459   Probable Glutamine Amidotransferase 2.19 PA3464   Hypothetical Protein -2.47 PA3476 rhlI Autoinducer Synthesis Protein RhlI 3.03 PA3477 rhlR Transcriptional Regulator RhlR 2.41  166 PA3478 rhlB Rhamnosyltransferase Chain B 3.1 PA3479 rhlA Rhamnosyltransferase Chain A 4.48 PA3486 vgrG4b Vgrg-4B 2.05 PA3487 tle5 Tle5 1.59 PA3492   Conserved Hypothetical Protein -1.79 PA3499   Hypothetical Protein -3.14 PA3504   Probable Aldehyde Dehydrogenase -3.02 PA3505   Hypothetical Protein -3.51 PA3507   Probable Short-Chain Dehydrogenase -2.74 PA3508   Probable Transcriptional Regulator -1.65 PA3509   Probable Hydrolase -4.04 PA3511   Probable Short-Chain Dehydrogenase -3.96 PA3512   Probable Permease of Abc Transporter -1.72 PA3513   Hypothetical Protein -2.1 PA3516   Probable Lyase -1.97 PA3517   Probable Lyase -1.58 PA3521 opmE Opme -4.1 PA3522 mexQ Mexq -2.78 PA3523 mexP Mexp -2.75 PA3530 bfd Bacterioferritin-Associated Ferredoxin Bfd 3.39 PA3531 bfrB Bacterioferritin -3.36 PA3534   Probable Oxidoreductase -1.89 PA3540 algD Gdp-Mannose 6-Dehydrogenase Algd -2.05 PA3541 alg8 Alginate Biosynthesis Protein Alg8 -2.84 PA3542 alg44 Alginate Biosynthesis Protein Alg44 -3.24 PA3543 algK Alginate Biosynthetic Protein AlgK Precursor -1.78 PA3544 algE Alginate Production Outer Membrane Protein AlgE Precursor -3.81 PA3545 algG Alginate-C5-Mannuronan-Epimerase AlgG -4.16 PA3548 algI Alginate O-Acetyltransferase AlgI -3.39 PA3550 algF Alginate O-Acetyltransferase AlgF -2.44 PA3551 algA Phosphomannose Isomerase -1.67 PA3552 arnB ArnB -3.66 PA3554 arnA ArnA -3.64 PA3559   Probable Nucleotide Sugar Dehydrogenase -2.84 PA3560 fruA Phosphotransferase System Transporter Fructose-Specific -3.59 PA3561 fruK 1-Phosphofructokinase -2.81 PA3562 fruI Phosphotransferase System Transporter Enzyme I, Frui -2.61 PA3564   Conserved Hypothetical Protein -2.23 PA3565   Probable Transcriptional Regulator -2.11 PA3569 mmsB 3-Hydroxyisobutyrate Dehydrogenase 1.85 PA3570 mmsA Methylmalonate-Semialdehyde Dehydrogenase 3.50 PA3573   Probable Major Facilitator Superfamily (Mfs) Transporter -1.63 PA3584 glpD Glycerol-3-Phosphate Dehydrogenase -1.65  167 PA3586   Probable Hydrolase -3.04 PA3590   Probable Hydroxyacyl-Coa Dehydrogenase -3.24 PA3595   Probable Major Facilitator Superfamily (Mfs) Transporter -2.31 PA3596   Probable Methylated-DNA-Protein-Cysteine Methyltransferase -2.12 PA3605   Hypothetical Protein -1.65 PA3607 potA Polyamine Transport Protein PotA -2.05 PA3608 potB Polyamine Transport Protein PotB -1.63 PA3611   Hypothetical Protein 1.77 PA3615   Hypothetical Protein -1.77 PA3635 eno Enolase 2.02 PA3636 kdsA 2-Dehydro-3-Deoxyphosphooctonate Aldolase 1.95 PA3637 pyrG Ctp Synthase 1.58 PA3640 dnaE Dna Polymerase Iii, Alpha Chain 1.76 PA3652 uppS Undecaprenyl Pyrophosphate Synthetase 1.61 PA3654 pyrH Uridylate Kinase 2.21 PA3656 rpsB 30S Ribosomal Protein S2 3.66 PA3661   Hypothetical Protein 2.34 PA3669   Hypothetical Protein -1.59 PA3681   Hypothetical Protein -2.05 PA3686 adk Adenylate Kinase 2.09 PA3691   Hypothetical Protein 4.96 PA3692 lptF Lipotoxon F, LptF 5.01 PA3700 lysS Lysyl-Trna Synthetase 2.28 PA3718   Probable Major Facilitator Superfamily (Mfs) Transporter -4.31 PA3719 armR Antirepressor For MexR, ArmR -5.23 PA3720   Hypothetical Protein -4.34 PA3721 nalC NalC -4.39 PA3726   Conserved Hypothetical Protein 2.32 PA3727   Hypothetical Protein 2.38 PA3731   Conserved Hypothetical Protein -2.61 PA3735 thrC Threonine Synthase 1.52 PA3740   Hypothetical Protein 1.75 PA3742 rplS 50S Ribosomal Protein L19 2.59 PA3743 trmD Trna (Guanine-N1)-Methyltransferase 3.21 PA3744 rimM 16S Rrna Processing Protein 2.94 PA3745 rpsP 30S Ribosomal Protein S16 2.78 PA3746 ffh Signal Recognition Particle Protein Ffh 2.21 PA3747   Conserved Hypothetical Protein 1.84 PA3749   Probable Major Facilitator Superfamily (Mfs) Transporter -3.83 PA3750   Hypothetical Protein -2.87 PA3752   Hypothetical Protein -2.25 PA3759   Probable Aminotransferase -2.52 PA3760   N-Acetyl-D-Glucosamine Phosphotransferase System -1.6  168 Transporter PA3761 nagE N-Acetyl-D-Glucosamine Phosphotransferase System Transporter -1.86 PA3765   Hypothetical Protein -2.89 PA3770 guaB Inosine-5'-Monophosphate Dehydrogenase 1.61 PA3772   Hypothetical Protein -1.87 PA3776   Probable Transcriptional Regulator -3.92 PA3799   Conserved Hypothetical Protein 2.22 PA3801   Conserved Hypothetical Protein 1.58 PA3802 hisS Histidyl-Trna Synthetase 1.94 PA3806   Conserved Hypothetical Protein 2.10 PA3807 ndk Nucleoside Diphosphate Kinase 3.09 PA3818   Extragenic Suppressor Protein Suhb 2.54 PA3820 secF Secretion Protein Secf 2.05 PA3821 secD Secretion Protein Secd 2.25 PA3822   Conserved Hypothetical Protein 2.30 PA3827 lptG Lipopolysaccharide Export System Permease Protein Lptg 1.6 PA3829   Hypothetical Protein -1.92 PA3830   Probable Transcriptional Regulator -2.46 PA3835   Hypothetical Protein -3.99 PA3851   Hypothetical Protein -1.5 PA3863 dauA Fad-Dependent Catabolic D-Arginine Dehydrogenase, DauA -1.83 PA3868   Hypothetical Protein -1.56 PA3870 moaA1 Molybdopterin Biosynthetic Protein A1 -2.77 PA3876 narK2 Nitrite Extrusion Protein 2 -2.34 PA3883   Probable Short-Chain Dehydrogenase -1.63 PA3884   Hypothetical Protein -3.15 PA3886   Hypothetical Protein -1.53 PA3893   Conserved Hypothetical Protein -2.88 PA3894   Probable Outer Membrane Protein Precursor -2.34 PA3899 fecI FecI 2.04 PA3903 prfC Peptide Chain Release Factor 3 2.38 PA3909 eddB Extracelullar Dna Degradation Protein, EddB -1.96 PA3910 eddA Extracelullar Dna Degradation Protein, EddA -2.53 PA3911   Conserved Hypothetical Protein -1.86 PA3914 moeA1 Molybdenum Cofactor Biosynthetic Protein A1 -4.32 PA3915 moaB1 Molybdopterin Biosynthetic Protein B1 -1.96 PA3926   Probable Major Facilitator Superfamily (Mfs) Transporter -2.27 PA3932   Probable Transcriptional Regulator -1.62 PA3937   Probable Atp-Binding Component Of Abc Taurine Transporter -3.20 PA3939   Hypothetical Protein -3.21 PA3941   Hypothetical Protein -1.98 PA3946 rocS1 Two-Component Sensor Rocs1 -2.49  169 PA3947 rocR RocR -1.51 PA3953   Conserved Hypothetical Protein -1.69 PA3955   Hypothetical Protein -1.51 PA3959   Hypothetical Protein -1.72 PA3960   Hypothetical Protein -1.53 PA3962   Hypothetical Protein 1.55 PA3964   Hypothetical Protein -2.55 PA3966   Hypothetical Protein 1.59 PA3971   Hypothetical Protein -2.59 PA3972   Probable Acyl-Coa Dehydrogenase -2.36 PA3973   Probable Transcriptional Regulator -1.60 PA3987 leuS Leucyl-Trna Synthetase 1.67 PA3991   Hypothetical Protein -4.46 PA3994   Probable Epoxide Hydrolase -4.25 PA4001 sltB1 Soluble Lytic Transglycosylase B 1.53 PA4003 pbpA Penicillin-Binding Protein 2 1.90 PA4004   Conserved Hypothetical Protein 1.50 PA4005   Conserved Hypothetical Protein 2.02 PA4006 nadD1 Nicotinate Mononucleotide Adenylyltransferase NadD1 1.76 PA4008   Probable Hydrolase -3.9 PA4009   Hypothetical Protein -2.59 PA4011   Hypothetical Protein -1.92 PA4021   Probable Transcriptional Regulator -1.80 PA4026   Probable Acetyltransferase -1.60 PA4031 ppa Inorganic Pyrophosphatase 2.68 PA4036   Probable Two-Component Sensor -2.29 PA4039   Hypothetical Protein -2.96 PA4040   Hypothetical Protein -2.10 PA4064   Probable ATP-Binding Component of ABC Transporter -1.55 PA4067 oprG Outer Membrane Protein OprG Precursor 2.95 PA4070   Probable Transcriptional Regulator -1.84 PA4074   Probable Transcriptional Regulator -2.29 PA4075   Hypothetical Protein -1.59 PA4081 cupB6 Fimbrial Subunit CupB6 -2.17 PA4082 cupB5 Adhesive Protein CupB5 -3.87 PA4084 cupB3 Usher CupB3 -2.78 PA4085 cupB2 Chaperone CupB2 -2.14 PA4087   Conserved Hypothetical Protein -3.27 PA4090   Hypothetical Protein 1.96 PA4091 hpaA 4-Hydroxyphenylacetate 3-Monooxygenase Large Chain 2.02 PA4093   Hypothetical Protein -1.92 PA4102 bfmS BfmS -1.57 PA4103   Hypothetical Protein -2.73  170 PA4104   Conserved Hypothetical Protein -2.61 PA4107 efhP EfhP -3.25 PA4111   Hypothetical Protein -2.63 PA4113   Probable Major Facilitator Superfamily (Mfs) Transporter -1.96 PA4126   Probable Major Facilitator Superfamily (Mfs) Transporter -2.11 PA4128   Conserved Hypothetical Protein -2.66 PA4136   Probable Major Facilitator Superfamily (Mfs) Transporter -3.08 PA4138 tyrS Tyrosyl-TrnA Synthetase -1.58 PA4141   Hypothetical Protein 8.49 PA4142   Probable Secretion Protein 3.42 PA4144   Probable Outer Membrane Protein Precursor -2.24 PA4146   Hypothetical Protein -2.08 PA4149   Conserved Hypothetical Protein -2.36 PA4158 fepC Ferric Enterobactin Transport Protein FepC -1.65 PA4160 fepD Ferric Enterobactin Transport Protein FepD -3.64 PA4161 fepG Ferric Enterobactin Transport Protein FepG -3.66 PA4162   Probable Short-Chain Dehydrogenase -2.78 PA4165   Probable Transcriptional Regulator -2.34 PA4166   Probable Acetyltransferase -1.58 PA4168 fpvB Second Ferric Pyoverdine Receptor FpvB 1.76 PA4171   Probable Protease 1.68 PA4174   Probable Transcriptional Regulator -1.66 PA4179   Probable Porin -3.60 PA4188   Conserved Hypothetical Protein -3.40 PA4189   Probable Aldehyde Dehydrogenase -4.15 PA4191   Probable Iron/Ascorbate Oxidoreductase -4.69 PA4194   Probable Permease of ABC Transporter -2.11 PA4195   Probable Binding Protein Component of ABC Transporter -2.94 PA4197 bfiS BfiS -2.06 PA4203 nmoR NmoR -2.38 PA4209 phzM Probable Phenazine-Specific Methyltransferase 3.50 PA4210 phzA1 Probable Phenazine Biosynthesis Protein 5.38 PA4212 phzC1 Phenazine Biosynthesis Protein PhzC 3.33 PA4216 phzG1 Probable Pyridoxamine 5'-Phosphate Oxidase 2.49 PA4217 phzS Flavin-Containing Monooxygenase 4.69 PA4227 pchR Transcriptional Regulator PchR 3.16 PA4237 rplQ 50S Ribosomal Protein L17 3.08 PA4238 rpoA Dna-Directed Rna Polymerase Alpha Chain 2.74 PA4239 rpsD 30S Ribosomal Protein S4 2.81 PA4240 rpsK 30S Ribosomal Protein S11 2.95 PA4241 rpsM 30S Ribosomal Protein S13 2.94 PA4242 rpmJ 50S Ribosomal Protein L36 1.98 PA4243 secY Secretion Protein Secy 3.02  171 PA4244 rplO 50S Ribosomal Protein L15 2.74 PA4245 rpmD 50S Ribosomal Protein L30 3.22 PA4246 rpsE 30S Ribosomal Protein S5 3.06 PA4247 rplR 50S Ribosomal Protein L18 3.47 PA4248 rplF 50S Ribosomal Protein L6 3.52 PA4249 rpsH 30S Ribosomal Protein S8 3.31 PA4250 rpsN 30S Ribosomal Protein S14 2.23 PA4251 rplE 50S Ribosomal Protein L5 2.72 PA4252 rplX 50S Ribosomal Protein L24 2.40 PA4253 rplN 50S Ribosomal Protein L14 2.06 PA4254 rpsQ 30S Ribosomal Protein S17 2.44 PA4255 rpmC 50S Ribosomal Protein L29 2.42 PA4256 rplP 50S Ribosomal Protein L16 2.36 PA4257 rpsC 30S Ribosomal Protein S3 2.39 PA4258 rplV 50S Ribosomal Protein L22 2.28 PA4259 rpsS 30S Ribosomal Protein S19 2.49 PA4260 rplB 50S Ribosomal Protein L2 2.62 PA4261 rplW 50S Ribosomal Protein L23 2.85 PA4262 rplD 50S Ribosomal Protein L4 2.77 PA4263 rplC 50S Ribosomal Protein L3 2.79 PA4264 rpsJ 30S Ribosomal Protein S10 2.68 PA4266 fusA1 Elongation Factor G 2.04 PA4267 rpsG 30S Ribosomal Protein S7 1.94 PA4268 rpsL 30S Ribosomal Protein S12 1.91 PA4276 secE Secretion Protein Sece 1.93 PA4279   Hypothetical Protein -1.74 PA4283 recD Exodeoxyribonuclease V Alpha Chain -1.65 PA4287   Hypothetical Protein -3.13 PA4288   Probable Transcriptional Regulator -1.78 PA4289   Probable Transporter -3.02 PA4292   Probable Phosphate Transporter 1.55 PA4294   Hypothetical Protein 2.45 PA4302 tadA Tada Atpase 2.84 PA4304 rcpA RcpA 2.95 PA4305 rcpC RcpC 2.62 PA4307 pctC Chemotactic Transducer PctcC 1.95 PA4309 pctA Chemotactic Transducer PctA -3.44 PA4317   Hypothetical Protein 2.60 PA4342   Probable Amidase -2.76 PA4343   Probable Major Facilitator Superfamily (Mfs) Transporter -2.86 PA4348   Conserved Hypothetical Protein 1.69 PA4349   Hypothetical Protein -2.40 PA4350 olsB OlsB -3.72  172 PA4353   Conserved Hypothetical Protein -1.91 PA4359   Conserved Hypothetical Protein 3.33 PA4363 iciA Inhibitor of Chromosome Initiation IciA -2.5 PA4370 icmP Insulin-Cleaving Metalloproteinase Outer Membrane Protein  2.96 PA4374 mexV RND Multidrug Efflux Membrane Fusion Protein MexV 1.74 PA4384   Hypothetical Protein 2.09 PA4385 groEL Groel Protein 2.05 PA4390   Hypothetical Protein 1.81 PA4402 argJ Glutamate N-Acetyltransferase 1.67 PA4403 secA Secretion Protein Seca 2.06 PA4406 lpxC Udp-3-O-Acyl-N-Acetylglucosamine Deacetylase 1.51 PA4407 ftsZ Cell Division Protein FtsZ 2.06 PA4408 ftsA Cell Division Protein FtsA 2.32 PA4409 ftsQ Cell Division Protein FtsQ 2.14 PA4410 ddlB D-Alanine-D-Alanine Ligase 2.06 PA4411 murC Udp-N-Acetylmuramate-Alanine Ligase 2.06 PA4413 ftsW Cell Division Protein FtsW 1.64 PA4414 murD Udp-N-Acetylmuramoylalanine-D-Glutamate Ligase 2.14 PA4415 mraY Phospho-N-Acetylmuramoyl-Pentapeptide-Transferase 1.94 PA4416 murF Udp-N-Acetylmuramoylalanyl-D-Glutamyl-2,6-Diaminopimelate-D-Alanyl-D-Alanyl Ligase 1.57 PA4418 ftsI Penicillin-Binding Protein 3 1.75 PA4419 ftsL Cell Division Protein Ftsl 2.21 PA4420   Conserved Hypothetical Protein 1.87 PA4421   Conserved Hypothetical Protein 1.75 PA4432 rpsI 30S Ribosomal Protein S9 3.56 PA4433 rplM 50S Ribosomal Protein L13 3.84 PA4437   Hypothetical Protein -1.59 PA4438   Conserved Hypothetical Protein 2.04 PA4449 hisG Atp-Phosphoribosyltransferase 1.6 PA4450 murA Udp-N-Acetylglucosamine 1-Carboxyvinyltransferase 1.7 PA4451   Conserved Hypothetical Protein 1.72 PA4457   Arabinose-5-Phosphate Isomerase Kdsd 1.82 PA4458   Conserved Hypothetical Protein 1.67 PA4459 lptC Lipopolysaccharide Export System Protein  1.54 PA4460 lptH LptH 1.68 PA4461 lptB Lipopolysaccharide Export System Atp-Binding Protein  1.65 PA4467   Hypothetical Protein 3.73 PA4468 sodM Superoxide Dismutase 5.59 PA4469   Hypothetical Protein 5.48 PA4470 fumC1 Fumarate Hydratase 5.98 PA4471   Hypothetical Protein 5.22 PA4480 mreC Rod Shape-Determining Protein MreC 1.97  173 PA4481 mreB Rod Shape-Determining Protein MreB 2.55 PA4482 gatC Glu-Trna(Gln) Amidotransferase Subunit C 1.53 PA4484 gatB Glu-Trna(Gln) Amidotransferase Subunit B 1.55 PA4487 magF MagF 1.63 PA4495   Hypothetical Protein 2.27 PA4496 dppA1 Probable Binding Protein Component of ABC Transporter 3.02 PA4507   Hypothetical Protein -2.41 PA4509   Hypothetical Protein -2.4 PA4514   Probable Outer Membrane Receptor for Iron Transport 2.38 PA4515   Conserved Hypothetical Protein 3.03 PA4516   Hypothetical Protein 1.96 PA4519 speC Ornithine Decarboxylase 2.35 PA4525 pilA Type 4 Fimbrial Precursor PilA 1.99 PA4527 pilC Type 4 Fimbrial Biogenesis Protein PilC (Putative Pseudogene) -1.57 PA4540   Hypothetical Protein -2.62 PA4541 lepA Large Extracellular Protease, LepA -1.79 PA4545 comL Competence Protein ComL 2.26 PA4559 lspA Prolipoprotein Signal Peptidase 1.52 PA4563 rpsT 30S Ribosomal Protein S20 3.98 PA4567 rpmA 50S Ribosomal Protein L27 1.75 PA4570   Hypothetical Protein 6.53 PA4571   Probable Cytochrome C 1.90 PA4575   Hypothetical Protein -2.08 PA4578   Hypothetical Protein 2.02 PA4585 rtcA Rna 3'-Terminal Phosphate Cyclase -2.14 PA4587 ccpR Cytochrome C551 Peroxidase Precursor 3.59 PA4593   Probable Permease of ABC Transporter -1.77 PA4597 oprJ Multidrug Efflux Outer Membrane Protein OprJ Precursor -3.39 PA4598 mexD RND Multidrug Efflux Transporter Mexd -2.44 PA4599 mexC RND Multidrug Efflux Membrane Fusion Protein Mexc Precursor -3.41 PA4602 glyA3 Serine Hydroxymethyltransferase 2.82 PA4610   Hypothetical Protein -2.45 PA4612   Conserved Hypothetical Protein -2.27 PA4621   Probable Oxidoreductase -2.10 PA4622   Probable Major Facilitator Superfamily (Mfs) Transporter -2.09 PA4624 cdrB Cyclic Diguanylate-Regulated Tps Partner B, CdrB 1.76 PA4625 cdrA Cyclic Diguanylate-Regulated Tps Partner A, CdrA 1.62 PA4629   Hypothetical Protein 1.55 PA4630   Hypothetical Protein -1.53 PA4636   Hypothetical Protein 1.73 PA4648 cupE1 Pilin Subunit CupE1 4.72 PA4649 cupE2 Pilin Subunit CupE2 3.14  174 PA4650 cupE3 Pilin Subunit CupE3 2.04 PA4651 cupE4 Pilin Assembly Chaperone CupE4 1.86 PA4654   Probable Major Facilitator Superfamily (Mfs) Transporter -2.21 PA4665 prfA Peptide Chain Release Factor 1 1.81 PA4669 ipk Isopentenyl Monophosphate Kinase 2.37 PA4670 prs Ribose-Phosphate Pyrophosphokinase 2.65 PA4671   Probable Ribosomal Protein L25 1.78 PA4672   Peptidyl-Trna Hydrolase 2.25 PA4675 chtA Chta 3.20 PA4685   Hypothetical Protein 1.9 PA4686   Hypothetical Protein 1.93 PA4708 phuT Heme-Transport Protein, PhuT 1.67 PA4709 phuS PhuS 2.57 PA4710 phuR Heme/Hemoglobin Uptake Outer Membrane Receptor PhuR 3.40 PA4723 dksA Suppressor Protein DksA 2.97 PA4738   Conserved Hypothetical Protein 4.07 PA4739   Conserved Hypothetical Protein 4.64 PA4741 rpsO 30S Ribosomal Protein S15 2.90 PA4742 truB Trna Pseudouridine 55 Synthase 2.40 PA4743 rbfA Ribosome-Binding Factor A 2.91 PA4744 infB Translation Initiation Factor If-2 2.52 PA4745 nusA N Utilization Substance Protein A 2.51 PA4746   Conserved Hypothetical Protein 2.98 PA4748 tpiA Triosephosphate Isomerase 1.89 PA4753   Conserved Hypothetical Protein 2.00 PA4765 omlA Outer Membrane Lipoprotein OmlA Precursor 2.29 PA4768 smpB SmpB Protein 2.43 PA4770 lldP L-Lactate Permease 1.50 PA4774   Hypothetical Protein -2.65 PA4779   Hypothetical Protein -1.79 PA4783   Conserved Hypothetical Protein -2.62 PA4784   Probable Transcriptional Regulator -1.52 PA4791   Hypothetical Protein -2.10 PA4795   Hypothetical Protein -1.86 PA4799   Hypothetical Protein -1.98 PA4802   Hypothetical Protein -2.6 PA4804  cupB3 Potra-Like Domain-Containing Usher, CupB3 -3.27 PA4805   Probable Class III Aminotransferase -2.16 PA4807 selB Selenocysteine-Specific Elongation Factor -1.52 PA4810 fdnI Nitrate-Inducible Formate Dehydrogenase, Gamma Subunit -1.63 PA4812 fdnG Formate Dehydrogenase-O, Major Subunit -1.58 PA4814 fadH2 2,4-Dienoyl-Coa Reductase FadH2 -4.95 PA4816   Hypothetical Protein -2.07  175 PA4817   Hypothetical Protein -1.86 PA4821   Probable Transporter -3.60 PA4822   Hypothetical Protein -1.72 PA4828   Conserved Hypothetical Protein -2.12 PA4830   Hypothetical Protein -3.14 PA4832   Probable Short-Chain Dehydrogenase -2.64 PA4843 gcbA GcbA 1.70 PA4844 ctpL CtpL -2.42 PA4846 aroQ1 3-Dehydroquinate Dehydratase 1.86 PA4848 accC Biotin Carboxylase 1.63 PA4850 prmA Ribosomal Protein L11 Methyltransferase 1.52 PA4853 fis Dna-Binding Protein Fis 2.04 PA4858   Conserved Hypothetical Protein -3.12 PA4864 ureD Urease Accessory Protein -2.06 PA4871   Hypothetical Protein -3.21 PA4876 osmE Osmotically Inducible Lipoprotein OsmE 3.49 PA4877   Hypothetical Protein 2.19 PA4880   Probable Bacterioferritin 1.88 PA4882   Hypothetical Protein -1.71 PA4885 irlR Two-Component Response Regulator -2.41 PA4886   Probable Two-Component Sensor -2.78 PA4896   Probable Sigma-70 Factor, Ecf Subfamily 2.73 PA4898 opdK Histidine Porin OpdK -2.86 PA4899   Probable Aldehyde Dehydrogenase -2.14 PA4900   Probable Major Facilitator Superfamily (Mfs) Transporter -4.06 PA4901 mdlC Benzoylformate Decarboxylase -4.47 PA4904 vanA Vanillate O-Demethylase Oxygenase Subunit -4.57 PA4905 vanB Vanillate O-Demethylase Oxidoreductase -2.99 PA4908   Hypothetical Protein -1.69 PA4921 choE Cholinesterase, ChoE -1.99 PA4925   Conserved Hypothetical Protein 1.66 PA4927   Conserved Hypothetical Protein -1.86 PA4928   Conserved Hypothetical Protein 1.8 PA4932 rplI 50S Ribosomal Protein L9 3.41 PA4933   Hypothetical Protein 3.48 PA4934 rpsR 30S Ribosomal Protein S18 4.34 PA4938 purA Adenylosuccinate Synthetase 1.69 PA4940   Conserved Hypothetical Protein 2.35 PA4941 hflC Protease Subunit HflC 1.73 PA4967 parE Topoisomerase Iv Subunit B 1.55 PA4968   Conserved Hypothetical Protein 1.72 PA4972   Hypothetical Protein 1.74 PA4976 aruH Arginine:Pyruvate Transaminas, AruH -1.57  176 PA4977 aruI 2-Ketoarginine Decarboxylase, AruI -2.32 PA4978   Hypothetical Protein -2.88 PA4981 lysP Lysine-Specific Permease -2.39 PA4982   Probable Two-Component Sensor -2.31 PA4985   Uncharacterized Protein -2.02 PA4986   Probable Oxidoreductase -1.51 PA4988 waaA 3-Deoxy-D-Manno-Octulosonic-Acid (Kdo) Transferase -2.00 PA4989   Probable Transcriptional Regulator -3.06 PA4994   Probable Acyl-Coa Dehydrogenase -2.1 PA4995   Probable Acyl-Coa Dehydrogenase -1.68 PA5005   Probable Carbamoyl Transferase 1.80 PA5010 waaG Udp-Glucose:(Heptosyl) Lps Alpha 1,3-Glucosyltransferase WaaG 2.10 PA5015 aceE Pyruvate Dehydrogenase 1.71 PA5016 aceF Dihydrolipoamide Acetyltransferase 1.60 PA5027   Hypothetical Protein 2.06 PA5029   Probable Transcriptional Regulator -1.87 PA5031   Probable Short Chain Dehydrogenase -3.45 PA5032   Probable Transcriptional Regulator -3.70 PA5034 hemE Uroporphyrinogen Decarboxylase 1.68 PA5041 pilP Type 4 Fimbrial Biogenesis Protein PilP 1.83 PA5042 pilO Type 4 Fimbrial Biogenesis Protein PilO 1.96 PA5043 pilN Type 4 Fimbrial Biogenesis Protein PilN 1.76 PA5045 ponA Penicillin-Binding Protein 1A 1.67 PA5046   Malic Enzyme 1.77 PA5049 rpmE 50S Ribosomal Protein L31 3.67 PA5053 hslV Heat Shock Protein HslV 2.33 PA5054 hslU Heat Shock Protein HslU 2.38 PA5058 phaC2 Poly(3-Hydroxyalkanoic Acid) Synthase 2 2.31 PA5059   Probable Transcriptional Regulator 2.00 PA5075   Probable Permease Of Abc Transporter 1.52 PA5078 opgG OpgG 1.70 PA5083 dguB DguB -2.30 PA5084 dguA DguA -3.82 PA5085 dguR DguR -1.73 PA5100 hutU Urocanase 2.09 PA5102   Hypothetical Protein -2.51 PA5103 puuR PuuR 1.66 PA5110 fbp Fructose-1,6-Bisphosphatase 2.08 PA5115   Conserved Hypothetical Protein -1.80 PA5118 thiI Thiazole Biosynthesis Protein ThiI 2.78 PA5128 secB Secretion Protein SecB 3.25 PA5129 grxC GrxC 2.36  177 PA5130   Conserved Hypothetical Protein 1.78 PA5131 pgm Phosphoglycerate Mutase 1.67 PA5132   Hypothetical Protein -2.80 PA5136   Hypothetical Protein 1.54 PA5145   Hypothetical Protein -1.90 PA5158   Probable Outer Membrane Protein Precursor -2.02 PA5161 rmlB Dtdp-D-Glucose 4,6-Dehydratase 2.55 PA5163 rmlA Glucose-1-Phosphate Thymidylyltransferase 2.33 PA5164 rmlC Dtdp-4-Dehydrorhamnose 3,5-Epimerase 2.58 PA5170 arcD Arginine/Ornithine Antiporter 2.56 PA5171 arcA Arginine Deiminase 2.94 PA5172 arcB Ornithine Carbamoyltransferase, Catabolic 2.99 PA5173 arcC Carbamate Kinase 2.44 PA5178   Conserved Hypothetical Protein 2.45 PA5179   Probable Transcriptional Regulator -1.55 PA5183   Hypothetical Protein -1.57 PA5186   Probable Iron-Containing Alcohol Dehydrogenase -2.73 PA5188   Probable 3-Hydroxyacyl-Coa Dehydrogenase -2.15 PA5189   Probable Transcriptional Regulator -2.21 PA5192 pckA Phosphoenolpyruvate Carboxykinase 1.78 PA5204 argA N-Acetylglutamate Synthase 1.78 PA5211   Conserved Hypothetical Protein -2.91 PA5217   Probable Binding Protein Component of ABC Iron Transporter 3.14 PA5218   Probable Transcriptional Regulator -1.87 PA5220   Hypothetical Protein 4.46 PA5230   Probable Permease of ABC Transporter 1.75 PA5231   Probable ATP-Binding/Permease Fusion ABC Transporter 1.70 PA5238   Probable O-Antigen Acetylase -1.57 PA5239 rho Transcription Termination Factor Rho 3.31 PA5264   Hypothetical Protein -1.95 PA5265   Hypothetical Protein -1.61 PA5267 hcpB Secreted Protein Hcp 2.29 PA5282   Probable Major Facilitator Superfamily (Mfs) Transporter -4.08 PA5286   Conserved Hypothetical Protein 1.71 PA5290   Conserved Hypothetical Protein -1.74 PA5294   Putative Multidrug Efflux Pump -4.03 PA5296 rep Atp-Dependent Dna Helicase Rep 1.78 PA5298   Xanthine Phosphoribosyltransferase 2.46 PA5303   Conserved Hypothetical Protein 1.64 PA5304 dadA D-Amino Acid Dehydrogenase, Small Subunit 1.54 PA5311   Probable Major Facilitator Superfamily (Mfs) Transporter -1.99 PA5315 rpmG 50S Ribosomal Protein L33 3.63 PA5316 rpmB 50S Ribosomal Protein L28 3.72  178 PA5321 dut Deoxyuridine 5'-Triphosphate Nucleotidohydrolase 1.71 PA5322 algC Phosphomannomutase AlgC 2.15 PA5323 argB Acetylglutamate Kinase 2.48 PA5324 sphR Sphingosine-Responsive Regulator, SphR -1.71 PA5369 pstS Periplasmic Phosphate-Binding Protein ABC Transporter, PstS 1.81 PA5379 sdaB L-Serine Dehydratase -2.28 PA5381   Hypothetical Protein -2.16 PA5382   Probable Transcriptional Regulator -2.51 PA5387 cdhC Cdhc, Carnitine Dehydrogenase-Related Gene C -3.19 PA5389 cdhR Cdhr, Transcriptional Regulator -2.05 PA5390   Probable Peptidic Bond Hydrolase -2.89 PA5398 dgcA Dgca, Dimethylglycine Catabolism -2.2 PA5399 dgcB Dgcb, Dimethylglycine Catabolism -2.93 PA5400   Probable Electron Transfer Flavoprotein Alpha Subunit -3.71 PA5401   Hypothetical Protein -3.24 PA5408   Hypothetical Protein -1.79 PA5409   Hypothetical Protein -1.82 PA5411 gbcB GbcB -1.61 PA5412   Hypothetical Protein -1.58 PA5421 fdhA Glutathione-Independent Formaldehyde Dehydrogenase -1.93 PA5427 adhA Alcohol Dehydrogenase 1.90 PA5431   Probable Transcriptional Regulator -3.26 PA5432   Probable Acetyltransferase -1.74 PA5433   Conserved Hypothetical Protein -2.11 PA5446   Hypothetical Protein -2.41 PA5465   Hypothetical Protein -2.14 PA5466   Hypothetical Protein -3.4 PA5468   Probable Citrate Transporter -2.11 PA5469   Conserved Hypothetical Protein -1.87 PA5471.1   Pa5471 Leader Peptide 1.72 PA5475   Hypothetical Protein 2.84 PA5479 gltP Proton-Glutamate Symporter 2.36 PA5480   Hypothetical Protein -1.98 PA5481   Hypothetical Protein 4.87 PA5482   Hypothetical Protein 4.05 PA5507   Hypothetical Protein 1.74 PA5515   Hypothetical Protein -1.52 PA5521   Probable Short-Chain Dehydrogenase -1.73 PA5522 pauA6 Glutamylpolyamine Synthetase -2.61 PA5523   Probable Aminotransferase -3.24 PA5524   Probable Short-Chain Dehydrogenase -2.85 PA5525   Probable Transcriptional Regulator -1.86 PA5531 tonB1 TonB1 2.43  179 PA5543   Hypothetical Protein -1.53 PA5548   Probable Major Facilitator Superfamily (Mfs) Transporter -2.00 PA5549 glmS Glucosamine-Fructose-6-Phosphate Aminotransferase 1.76 PA5550 glmR Glmr Transcriptional Regulator 2.16 PA5554 atpD ATP Synthase Beta Chain 1.95 PA5555 atpG ATP Synthase Gamma Chain 1.92 PA5556 atpA ATP Synthase Alpha Chain 1.9 PA5557 atpH ATP Synthase Delta Chain 2.27 PA5558 atpF ATP Synthase B Chain 1.96 PA5559 atpE ATP Synthase C Chain 1.59 PA5560 atpB ATP Synthase A Chain 2.23 PA5564 gidB Glucose Inhibited Division Protein B 2.26 PA5565 gidA Glucose-Inhibited Division Protein A 1.56 PA5568   Conserved Hypothetical Protein 2.65 PA5569 rnpA Ribonuclease P Protein Component 3.04 PA5570 rpmH 50S Ribosomal Protein L34 3.03 PA1291   Hypothetical protein -2.27 PA1321 cyoE Cytochrome o ubiquinol oxidase protein CyoE -1.59 PA1334   Probable oxidoreductase -3.30 PA1346   Hypothetical protein -6.48 PA1382   Probable type II secretion system protein -3.53 PA1383   Hypothetical protein -2.76 PA1384 galE UDP-glucose 4-epimerase -4.32 PA1385   Probable glycosyl transferase -2.57 PA1386   Probable ATP-binding component of ABC transporter -5.00 PA1387   Hypothetical protein -2.52 PA1388   Hypothetical protein -3.31 PA1390   Probable glycosyl transferase -3.18 PA1391   Probable glycosyl transferase -3.70 PA1393 cysC Adenosine 5'-phosphosulfate (APS) kinase -2.92 PA1418   Probable sodium:solute symport protein -2.93 PA1499   Conserved hypothetical protein -5.44 PA1503   Hypothetical protein -4.24 PA1507   Probable transporter -1.68 PA1519   Probable transporter -4.29 PA1525 alkB2 Alkane-1-monooxygenase 2 -2.41 PA1566 pauA3 Glutamylpolyamine synthetase -2.54 PA1600   Probable cytochrome c -5.88 PA1707 pcrH Regulatory protein PcrH -3.55 PA1708 popB Translocator protein PopB -3.70 PA1711 exsE ExsE -2.83 PA1739   Probable oxidoreductase -4.01  180 PA1875   Probable outer membrane protein precursor 4.30 PA1877   Probable secretion protein 3.42 PA1887   Hypothetical protein 2.57 PA1888   Hypothetical protein 2.05 PA1901 phzC2 Phenazine biosynthesis protein PhzC -3.09 PA1905 phzG2 Probable pyridoxamine 5'-phosphate oxidase -3.98 PA1914   Conserved hypothetical protein 4.73 PA1921   Hypothetical protein -3.99 PA1922   Probable tonB-dependent receptor -4.35 PA1923   Hypothetical protein -5.63 PA1924   Hypothetical protein -6.63 PA1925   Hypothetical protein -4.21 PA1927 metE 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase 3.70 PA1935   Hypothetical protein -2.55 PA1937   Conserved hypothetical protein 1.88 PA1977   Hypothetical protein -8.03 PA1979 eraS Sensor kinase, EraS -4.74 PA2013 liuC Putative 3-methylglutaconyl-CoA hydratase 1.82 PA2014 liuB Methylcrotonyl-coa carboxylase, beta-subunit 1.71 PA2073   Probable transporter (membrane subunit) -1.76 PA2074   Hypothetical protein -4.14 PA2089   Hypothetical protein -4.26 PA2096   Probable transcriptional regulator -4.17 PA2099   Probable short-chain dehydrogenase -4.56 PA2103 moeB Probable molybdopterin biosynthesis protein MoeB -1.93 PA2104   Probable cysteine synthase -1.98 PA2105   Probable acetyltransferase -1.9 PA2106   Hypothetical protein -2.36 PA2110   Hypothetical protein -1.61 PA2113 opdO Pyroglutamate porin OpdO 1.87 PA2114   Probable major facilitator superfamily (MFS) transporter 2.35 PA2147 katE Catalase HPII 2.48 PA2182   Hypothetical protein -1.79 PA2185 katN Non-heme catalase KatN -2.95 PA2186   Hypothetical protein -3.4 PA2188   Probable alcohol dehydrogenase (Zn-dependent) -3.07 PA2192   Conserved hypothetical protein -2.13 PA2204   Probable binding protein component of ABC transporter 2.38 PA2217   Probable aldehyde dehydrogenase -5.59 PA2218   Hypothetical protein -2.49 PA2221   Conserved hypothetical protein -2.66  181 PA2222   Hypothetical protein -1.52 PA2224   Hypothetical protein -2.05 PA2225   Hypothetical protein -2.42 PA2226 qsrO QsrO -1.76 PA2229   Conserved hypothetical protein -3.36 PA2260   Hypothetical protein -3.15 PA2294   Probable ATP-binding component of ABC transporter -6.17 PA2317   Probable oxidoreductase 1.68 PA2318   Hypothetical protein 1.93 PA2334   Probable transcriptional regulator -6.19 PA2343 mtlY Xylulose kinase -7.46 PA2381   Hypothetical protein -2.57 PA2423   Hypothetical protein 1.56 PA2429   Hypothetical protein -3.15 PA2434   Hypothetical protein -1.98 PA2437   Hypothetical protein -3.76 PA2438   Hypothetical protein -2.00 PA2439   Hypothetical protein -3.38 PA2441   Hypothetical protein -5.48 PA2470 gtdA Gentisate 1,2-dioxygenase -3.68 PA2507 catA Catechol 1,2-dioxygenase -6.31 PA2508 catC Muconolactone delta-isomerase -5.06 PA2511 antR AntR -2.71 PA2512 antA Anthranilate dioxygenase large subunit -5.30 PA2513 antB Anthranilate dioxygenase small subunit -5.11 PA2514 antC Anthranilate dioxygenase reductase -4.52 PA2516 xylZ Toluate 1,2-dioxygenase electron transfer component -5.88 PA2580   Conserved hypothetical protein -2.42 PA2588   Probable transcriptional regulator 2.15 PA2630   Conserved hypothetical protein 2.45 PA2636   Hypothetical protein -5.57 PA2672   Probable type II secretion system protein -4.53 PA2673   Probable type II secretion system protein -3.08 PA2682   Conserved hypothetical protein -2.76 PA2700 opdB Proline porin OpdB -1.94 PA2714   Probable molybdopterin oxidoreductase -3.42 PA2715   Probable ferredoxin -2.50 PA2736   Hypothetical protein -1.62 PA2754   Conserved hypothetical protein 1.92 PA2759   Hypothetical protein -1.76 PA2838   Probable transcriptional regulator -6.44 PA2847   Conserved hypothetical protein -5.53  182 PA2863 lipH Lipase modulator protein -2.14 PA2912   Probable ATP-binding component of ABC transporter -1.65 PA2913   Hypothetical protein -2.02 PA2914   Probable permease of ABC transporter -3.48 PA2934 cif CFTR inhibitory factor, Cif -1.62 PA2937   Hypothetical protein -3.67 PA2938   Probable transporter -4.54 PA2939   Probable aminopeptidase 4.54 PA3062 pelC Pelc -4.85 PA3137   Probable major facilitator superfamily (MFS) transporter -2.10 PA3142   Integrase -1.77 PA3160 wzz O-antigen chain length regulator -2.65 PA3181   2-keto-3-deoxy-6-phosphogluconate aldolase 2.08 PA3183 zwf Glucose-6-phosphate 1-dehydrogenase 3.74 PA3192 gltR Two-component response regulator GltR 1.58 PA3194 edd Phosphogluconate dehydratase 2.19 PA3195 gapA Glyceraldehyde 3-phosphate dehydrogenase 3.46 PA3266 capB Cold acclimation protein B 2.83 PA3281   Hypothetical protein -2.49 PA3282   Hypothetical protein -2.3 PA3315   Probable permease of ABC transporter -2.36 PA3319 plcN Non-hemolytic phospholipase C precursor -3.17 PA3323   Conserved hypothetical protein -2.56 PA3331   Cytochrome P450 1.69 PA3332   Conserved hypothetical protein 2.82 PA3334   Probable acyl carrier protein 2.00 PA3335   Hypothetical protein 1.71 PA3359   Hypothetical protein -4.85 PA3360   Probable secretion protein -5.66 PA3361 lecB Fucose-binding lectin PA-IIL 5.78 PA3390   Hypothetical protein -2.39 PA3393 nosD NosD protein -1.82 PA3415   Probable dihydrolipoamide acetyltransferase -2.23 PA3416   Probable pyruvate dehydrogenase E1 component, β chain -1.98 PA3417   Probable pyruvate dehydrogenase E1 component, α chain -2.44 PA3442   Probable ATP-binding component of ABC transporter -2.77 PA3450 lsfA 1-Cys peroxiredoxin LsfA 1.93 PA3467   Probable major facilitator superfamily (MFS) transporter -3.32 PA3497   Hypothetical protein -4.74 PA3498   Probable oxidoreductase -2.93 PA3500   Conserved hypothetical protein -3.26 PA3506   Probable decarboxylase -3.18  183 PA3520   Hypothetical protein 2.74 PA3546 algX Alginate biosynthesis protein AlgX -4.73 PA3588   Probable porin -6.87 PA3593   Probable acyl-coa dehydrogenase -6.03 PA3597   Probable amino acid permease -2.49 PA3655 tsf Elongation factor Ts 3.86 PA3662   Hypothetical protein -1.77 PA3724 lasB Elastase LasB 6.73 PA3769 guaA GMP synthase 2.62 PA3789   Hypothetical protein -1.74 PA3841 exoS Exoenzyme S -2.84 PA3843   Hypothetical protein -2.00 PA3901 fecA Fe(III) dicitrate transport protein 1.99 PA3906   Hypothetical protein 3.60 PA3907   Hypothetical protein 1.81 PA3908   Hypothetical protein 3.00 PA3935 tauD Taurine dioxygenase -3.01 PA3940   Probable DNA binding protein 4.02 PA3967   Hypothetical protein 2.28 PA4028   Hypothetical protein -6.42 PA4062   Hypothetical protein -2.68 PA4139   Hypothetical protein 4.11 PA4170   Hypothetical protein -2.33 PA4181   Hypothetical protein -2.00 PA4182   Hypothetical protein -2.00 PA4187   Probable major facilitator superfamily (MFS) transporter -5.84 PA4211 phzB1 Probable phenazine biosynthesis protein 6.31 PA4218 ampP AmpP 1.92 PA4220   Hypothetical protein 2.26 PA4221 fptA Fe(III)-pyochelin outer membrane receptor precursor 4.58 PA4223   Probable ATP-binding component of ABC transporter 1.76 PA4224 pchG Pyochelin biosynthetic protein 1.63 PA4225 pchF Pyochelin synthetase 2.01 PA4226 pchE Dihydroaeruginoic acid synthetase 2.10 PA4228 pchD Pyochelin biosynthesis protein 3.94 PA4229 pchC Pyochelin biosynthetic protein 2.22 PA4230 pchB Salicylate biosynthesis protein 5.09 PA4231 pchA Salicylate biosynthesis isochorismate synthase 4.11 PA4271 rplL 50S ribosomal protein L7 / L12 3.09 PA4272 rplJ 50S ribosomal protein L10 3.13 PA4273 rplA 50S ribosomal protein L1 1.83 PA4274 rplK 50S ribosomal protein L11 1.90  184 PA4277 tufB Elongation factor Tu 1.94 PA4300 tadC TadC 2.15 PA4303 tadZ TadZ 1.65 PA4306 flp Type IVb pilin, Flp 6.21 PA4355 pyeM PyeM -4.78 PA4364   Hypothetical protein -1.73 PA4365 lysE Lysine efflux permease -1.83 PA4443 cysD ATP sulfurylase small subunit 2.08 PA4549 fimT Type 4 fimbrial biogenesis protein FimT -3.99 PA4568 rplU 50S ribosomal protein L21 2.10 PA4586   Hypothetical protein -3.11 PA4590 pra Protein activator 3.78 PA4607   Hypothetical protein 1.80 PA4620   Hypothetical protein -1.88 PA4673   Conserved hypothetical protein 2.08 PA4714   Conserved hypothetical protein 2.38 PA4740 pnp Polyribonucleotide nucleotidyltransferase 2.65 PA4834   Putative nicotianamine synthase -1.88 PA4835   Hypothetical protein -2.15 PA4903   Probable major facilitator superfamily (MFS) transporter -7.58 PA4935 rpsF 30S ribosomal protein S6 4.34 PA4973 thiC Thiamin biosynthesis protein  1.57 PA4980   Probable enoyl-coA hydratase/isomerase -1.91 PA5001 ssg Cell surface-sugar biosynthetic glycosyltransferase, Ssg 1.66 PA5002 dnpA De-N-acetylase involved in persistence 1.53 PA5117 typA Regulatory protein TypA 2.89 PA5139   Hypothetical protein 1.98 PA5232   Conserved hypothetical protein 2.40 PA5274 rnk Nucleoside diphosphate kinase regulator 1.95 PA5354 glcE Glycolate oxidase subunit -1.55 PA5396   Hypothetical protein -1.83 PA5397   Hypothetical protein -1.96 PA5419 soxG Sarcosine oxidase gamma subunit -1.52 PA5426 purE Phosphoribosylaminoimidazole carboxylase, catalytic SU 2.18 PA5470   Probable peptide chain release factor -3.07 PA5537   Hypothetical protein -2.30 PA5538 amiA N-acetylmuramoyl-L-alanine amidase -2.82 PA5539   Hypothetical protein -2.86 PA0688 lapA Low-molecular-weight alkaline phosphatase A, LapA -5.12 PA0689 lapB Low-molecular-weight alkaline phosphatase B, LapB -2.01 PA0717   Hypothetical protein of bacteriophage Pf1 -2.43 PA0718   Hypothetical protein of bacteriophage Pf1 -2.44  185 PA0719   Hypothetical protein of bacteriophage Pf1 -2.45 PA0726   Hypothetical protein of bacteriophage Pf1 -4.14 PA0728   Probable bacteriophage integrase -3.28 PA0730   Probable transferase 2.68 PA0737   Hypothetical protein -3.20 PA0790   Hypothetical protein -6.54 PA0850   Hypothetical protein -1.61 PA0852 cbpD Chitin-binding protein CbpD precursor 5.64 PA0882   Hypothetical protein -4.24 PA0986   Conserved hypothetical protein 2.39 PA1151 imm2 Pyocin S2 immunity protein -2.24 PA1217   Probable 2-isopropylmalate synthase -1.68 PA1221   Hypothetical protein -2.36 PA1224   Probable NAD(P)H dehydrogenase -3.90 PA1251   Probable chemotaxis transducer -3.62 PA0187   Hypothetical protein -2.57 PA0188   Hypothetical protein -4.72 PA0229 pcaT Dicarboxylic acid transporter PcaT -2.10 PA0234   Hypothetical protein -2.2 PA0241   Probable major facilitator superfamily (MFS) transporter -5.31 PA0247 pobA P-hydroxybenzoate hydroxylase -3.59 PA0258   Hypothetical protein -2.49 PA0279   Probable transcriptional regulator -2.90 PA0283 sbp Sulfate-binding protein precursor 2.25 PA0284   Hypothetical protein 1.56 PA0349   Hypothetical protein -4.56 PA0417 chpE Probable chemotaxis protein -2.30 PA0434   Hypothetical protein -1.54 PA0435   Hypothetical protein -2.78 PA0439   Probable oxidoreductase -3.35 PA0476   Probable permease -3.72 PA0497   Hypothetical protein -3.57 PA0518 nirM Cytochrome c-551 precursor 3.53 PA0523 norC Nitric-oxide reductase subunit C 2.86 PA0524 norB Nitric-oxide reductase subunit B 2.41 PA0531   Probable glutamine amidotransferase -3.92 PA0578   Conserved hypothetical protein 3.90 PA0617   Probable bacteriophage protein 2.09 PA0634   Hypothetical protein 2.67 PA0654 speD S-adenosylmethionine decarboxylase proenzyme 2.19 PA0045   Hypothetical protein 4.14  186 PA0046   Hypothetical protein 3.91 PA0051 phzH Potential phenazine-modifying enzyme 2.82  Table A-5. Dysregulated genes in the DPA1463o mutant not dysregulated in PA14 wild-type surfing relative to swimming. RNA-Seq was performed on the surfing deficient DPA1463o mutant and PA14 WT surfing on SCFM with 0.4% mucin. The DPA1463o mutant log fold change was determined relative to wild-type surfing. PA14WT surfing was compared to a swim control. 653 genes were identified as uniquely dysregulated in the mutant relative to the wild-type under surfing conditions that were not found in wild-type surfing relative to swimming. Log fold-change cut-off of ± 1.5 and p-value < 0.05 was used. Gene annotations and descriptions come from www.pseudomonas.com (Winsor et al., 2016). Genes with no PAO1 gene ID were provided with their PA14 gene locus tag instead.  PA01 Gene ID/locus Gene Name Description  Log FC PA0043  Hypothetical Protein -1.86 PA0051 phzH Potential Phenazine-Modifying Enzyme 3.55 PA0057  Hypothetical Protein 2.22 PA0059 osmC Osmotically Inducible Protein OsmC 2.36 PA0060  Hypothetical Protein 1.78 PA0062  Lipoprotein 1.95 PA0071  Hypothetical Protein -2.08 PA0102  Carbonic Anhydrase -2.61 PA0103  Sulfate Transporter -2.50 PA0122  Hemolysin 1.64 PA0127  Lipoprotein -1.79 PA0143 nuh Nonspecific Ribonucleoside Hydrolase 1.80 PA0147  Oxidoreductase -1.61 PA0151  TonB-Dependent Receptor -1.74 PA0155 pcaR Transcriptional Regulator PcaR -1.75 PA0157  RND Efflux Membrane Fusion Protein 1.55 PA0173 cheB Chemotaxis-Specific Methylesterase 1.81 PA0174  Hypothetical Protein 2.16 PA0175  Chemotaxis Protein Methyltransferase 2.03 PA0178 cheA Two-Component Sensor -2.20 PA0197 tonB2 Hypothetical Protein 3.69 PA0201  Hypothetical Protein 1.56 PA0218  LysR Family Transcriptional Regulator -1.52 PA0226  CoA Transferase, Subunit A -4.27 PA0227  CoA Transferase Subunit B -4.03 PA0228 pcaF Beta-Ketoadipyl CoA Thiolase -3.85 PA0235 pcaK 4-Hydroxybenzoate Transporter PcaK -3.29 PA0236  IclR Family Transcriptional Regulator 1.72 PA0241  Mfs Transporter -2.79 PA0242  Hypothetical Protein -1.85 PA0247 pobA 4-Hydroxybenzoate 3-Monooxygenase -2.60  187 PA0263 hcpC Secreted Protein Hcp 3.31 PA0263 hcp2 Secreted Protein Hcp 3.39 PA0263 hcpB Secreted Protein Hcp 3.60 PA0264  Hypothetical Protein -1.89 PA0267  Hypothetical Protein 1.56 PA0277  Zn-Dependent Protease With Chaperone Function -1.53 PA0280 cysA Sulfate Transport Protein CysA 1.70 PA0281 cysW Sulfate Transport Protein CysW 2.25 PA0282 cysT Sulfate Transport Protein CysT 2.42 PA0283 sbp Sulfate-Binding Protein 3.26 PA0284  Hypothetical Protein 2.67 PA0289  Transcriptional Regulator -1.82 PA0296  Glutamine Synthetase -1.61 PA0297 spuA Glutamine Amidotransferase -1.81 PA0298 spuB Glutamine Synthetase -2.07 PA0299 spuC Aminotransferase -1.66 PA0320  Hypothetical Protein -5.78 PA0327  Transcriptional Regulator -3.71 PA0336 ygdP Dinucleoside Polyphosphate Hydrolase -1.87 PA0337 ptsP Phosphoenolpyruvate-Protein Phosphotransferase PtsP -1.65 PA0354  Hypothetical Protein 2.55 PA0355 pfpI Protease Pfpi 3.00 PA0359  Hypothetical Protein -1.77 PA0368  Hypothetical Protein -1.53 PA0377  Hypothetical Protein 1.71 PA0402 pyrB Aspartate Carbamoyltransferase -1.64 PA0403 pyrR Bifunctional Pyrimidine Regulatory Protein PyrR -1.80 PA0404 yqgF Holliday Junction Resolvase-Like Protein -2.11 PA0405  Hypothetical Protein -1.73 PA0433  Hypothetical Protein 1.79 PA0460  Hypothetical Protein 1.86 PA0469  Hypothetical Protein -1.83 PA0476  Permease -1.66 PA0489  Phosphoribosyl Transferase 1.63 PA0490  Hypothetical Protein 1.80 PA0496  Hydrolase -2.01 PA0532  Hypothetical Protein -2.25 PA0545  Hypothetical Protein -1.75 PA0553  Hypothetical Protein 1.70 PA0554  Hypothetical Protein 2.13 PA0567  Hypothetical Protein 2.01 PA0573  Hypothetical Protein -1.72 PA0578  Hypothetical Protein 1.80 PA0589 glpE Thiosulfate Sulfurtransferase -1.56 PA0593 pdxA 4-Hydroxythreonine-4-Phosphate Dehydrogenase 1.54  188 PA0602  ABC Transporter Substrate-Binding Protein -1.56 PA0612  Hypothetical Protein 1.81 PA0613  Hypothetical Protein 2.19 PA0632  Hypothetical Protein 3.36 PA0673  Hypothetical Protein -1.73 PA0677 hxcW HxcW -1.67 PA0679 hxcP HxcP -3.02 PA0693 exbB2 Transport Protein Exbb2 -1.85 PA0707 toxR Transcriptional Regulator ToxR 2.00 PA0713  Hypothetical Protein -1.56 PA0730  Transferase 1.82 PA0756  Two-Component Response Regulator 1.56 PA0761 nadB L-Aspartate Oxidase -1.76 PA0766 mucD Serine Protease MucD 1.73 PA0775  Hypothetical Protein 1.83 PA0781  Hypothetical Protein 2.70 PA0807  Hypothetical Protein 1.83 PA0841  Hypothetical Protein -2.09 PA0845  Hypothetical Protein -3.17 PA0852 cpbD Chitin-Binding Protein Cbpd 3.71 PA0875  Hypothetical Protein -1.62 PA0887 acsA Acetyl-Coa Synthetase -1.58 PA0895 argD BifunctionalN-Succinyldiaminopimelate-Aminotransferase/ 1.63 PA0907  Hypothetical Protein -1.58 PA0922  Hypothetical Protein -1.61 PA0938  Hypothetical Protein 2.14 PA0942  Transcriptional Regulator -2.15 PA0952  Hypothetical Protein -3.02 PA0979  Hypothetical Protein 1.60 PA0990  Hypothetical Protein 1.86 PA0996 pqsA PqsA 1.72 PA0997 pqsB PqsB 2.19 PA0998 pqsC PqsC 2.47 PA0999 pqsD 3-Oxoacyl-Acp Synthase 2.36 PA1000 pqsE Quinolone Signal Response Protein 2.36 PA1001 phnA Anthranilate Synthase Component I 2.93 PA1017 pauA Pimeloyl-CoA Synthetase -1.97 PA1029  Hypothetical Protein -2.71 PA1030  Hypothetical Protein -1.68 PA1068  Hsp90 Family Protein -2.00 PA1108  Mfs Family Transporter -1.96 PA1111  Hypothetical Protein 1.78 PA1122  Peptide Deformylase -1.54 PA1132  Hypothetical Protein 2.12  189 PA1134  Hypothetical Protein 2.00 PA1135 hchA Chaperone Protein HchA -2.25 PA1136  Transcriptional Regulator -2.92 PA1139  Hypothetical Protein -1.59 PA1160  Hypothetical Protein -1.54 PA1169  Lipoxygenase 1.71 PA1170  Hypothetical Protein 1.88 PA1178 oprH PhoP/Q And Low Mg2+ Inducible Outer Membrane Protein -2.67 PA1188  Hypothetical Protein -2.11 PA1190  Hypothetical Protein -2.11 PA1193  Hypothetical Protein 1.59 PA1209  Hypothetical Protein -1.51 PA1226  Transcriptional Regulator -1.75 PA1227  Hypothetical Protein -1.59 PA1242  Hypothetical Protein 2.54 PA1243  Sensor/Response Regulator Hybrid 2.39 PA1244  Hypothetical Protein -3.08 PA1245  Hypothetical Protein 2.75 PA1246 aprD Alkaline Protease Secretion Protein AprD 2.49 PA1247 aprE Alkaline Protease Secretion Protein AprE 2.70 PA1248 aprF Alkaline Protease Secretion Outer Membrane Protein AprF  3.03 PA1249 aprA Alkaline Metalloproteinase 5.43 PA1284  Acyl-CoA Dehydrogenase -1.81 PA1296  2-Hydroxyacid Dehydrogenase -1.67 PA1309  LysR Family Transcriptional Regulator -2.15 PA1315  Transcriptional Regulator -1.71 PA1317 cyoA Cytochrome O Ubiquinol Oxidase Subunit Ii 1.62 PA1323  Hypothetical Protein 2.67 PA1324  Hypothetical Protein 2.60 PA1332  Hypothetical Protein -1.76 PA1373 fabF2 3-Oxoacyl-Acp Synthase -2.12 PA1377  Acetyltransferase -1.59 PA14_00410  Dioxygenase -1.63 PA14_03166  Hypothetical Protein 1.64 PA14_03320  Hypothetical Protein 1.51 PA14_03330  Hypothetical Protein 1.58 PA14_03370  Hypothetical Protein 2.32 PA14_04830  Acetyltransferase -1.77 PA14_07460  Hypothetical Protein -1.54 PA14_13210  Hypothetical Protein 3.27 PA14_13630  Hypothetical Protein 2.03 PA14_13920  Hypothetical Protein 1.51 PA14_13950  Hypothetical Protein -1.55 PA14_14320  Hypothetical Protein 2.18 PA14_14420  Hypothetical Protein 1.52  190 PA14_14550  Hypothetical Protein 1.94 PA14_14560  Hypothetical Protein 6.67 PA14_15490  Hypothetical Protein 1.59 PA14_15570  Hypothetical Protein -2.12 PA14_16110  Hypothetical Protein 2.02 PA14_18070  Periplasmic Metal-Binding Protein -1.7 PA14_20060  Hypothetical Protein 2.98 PA14_21830  Hypothetical Protein 1.85 PA14_22080  Resolvase -2.67 PA14_22180  Hypothetical Protein 1.84 PA14_22210  Hypothetical Protein 1.86 PA14_22240  Hypothetical Protein 1.78 PA14_22270  Recombinase 1.72 PA14_22500  Protein-Disulfide Isomerase 1.76 PA14_23350 orfA Hypothetical Protein -2.32 PA14_23420  Zinc-Binding Dehydrogenase 1.56 PA14_23430  Heparinase 2.28 PA14_23440 orfL Group 1 Glycosyl Transferase 2.28 PA14_28240  Hypothetical Protein 1.51 PA14_28250  Secreted Acid Phosphatase 2.81 PA14_28360  Hypothetical Protein 1.81 PA14_28470  Hypothetical Protein -2.15 PA14_28520  Hypothetical Protein 2.89 PA14_28820  Hypothetical Protein 2.02 PA14_28830  Hypothetical Protein 2.33 PA14_28840  Helicase 1.98 PA14_29330  Hypothetical Protein 2.80 PA14_31060  Hypothetical Protein -1.53 PA14_31150  Hypothetical Protein -3.83 PA14_31430  Hypothetical Protein 3.04 PA14_33300  Hypothetical Protein 2.29 PA14_33310  Hypothetical Protein 2.39 PA14_33320  Hypothetical Protein 2.23 PA14_33330  Hypothetical Protein 1.64 PA14_33340  Helicase 2.32 PA14_33350  Hypothetical Protein 2.69 PA14_33970  Hypothetical Protein 3.72 PA14_33980  Hypothetical Protein 2.75 PA14_35740  Transposase 1.88 PA14_35760  Hypothetical Protein 2.22 PA14_35770  Hypothetical Protein 2.71 PA14_35780  Hypothetical Protein 3.10 PA14_35890  Diaminobutyrate-2-Oxoglutarate Aminotransferase 1.61 PA14_35920  Acetate Permease -1.72 PA14_36480  Hypothetical Protein 2.71  191 PA14_36790  Hypothetical Protein 2.89 PA14_36900  Hypothetical Protein 3.32 PA14_36940  Hypothetical Protein 1.82 PA14_39470  Hypothetical Protein 2.03 PA14_39480  Hypothetical Protein 2.40 PA14_39880 phzG2 Pyridoxamine 5'-Phosphate Oxidase 2.76 PA14_40740  Hypothetical Protein -2.77 PA14_46460  Hypothetical Protein 2.91 PA14_46510  Hypothetical Protein 2.13 PA14_46520  Hypothetical Protein 2.84 PA14_46530  Hypothetical Protein 5.38 PA14_46540  Hypothetical Protein 2.56 PA14_46550  Ribonuclease 2.85 PA14_49480  Hypothetical Protein 3.77 PA14_49990  Hypothetical Protein -2.16 PA14_51560  Acetyltransferase 1.55 PA14_51590  Hypothetical Protein -2.37 PA14_53450  Hypothetical Protein -1.92 PA14_53580  Hypothetical Protein 1.66 PA14_53590  Hypothetical Protein 2.83 PA14_53600  Hypothetical Protein 2.88 PA14_53610  Hypothetical Protein 2.87 PA14_54850  Hypothetical Protein 2.84 PA14_55080  Hypothetical Protein 2.00 PA14_55090  Hypothetical Protein 1.52 PA14_58730 pilA Type Iv Pilin Structural Subunit 2.31 PA14_59150  Single-Stranded DNA-Binding Protein 1.57 PA14_59240 pilL2 Type Iv B Pilus Protein -1.96 PA14_59340 pilT2 Type Iv B Pilus Protein 2.50 PA14_59350 pilV2 Type Iv B Pilus Protein -2.08 PA14_59390  Hypothetical Protein -1.72 PA14_59845  Hypothetical Protein -2.33 PA14_60030  Hypothetical Protein -1.5 PA14_60040  Hypothetical Protein -1.72 PA14_60050  Plasmid Stabilization Protein -1.92 PA14_60100 dtd Deoxycytidine Triphosphate Deaminase 2.77 PA14_60110  Hypothetical Protein 1.51 PA14_60120 dcd2 Deoxycytidine Deaminase 1.61 PA14_60140  XerD-Like Integrase 1.60 PA14_61110  Hypothetical Protein -1.67 PA14_64430  Hypothetical Protein 2.74 PA14_72370  Hypothetical Protein 2.49 PA1402  Hypothetical Protein -2.08 PA1405  Helicase -1.68 PA1413  LysR Family Transcriptional Regulator -1.98  192 PA1435  RND Efflux Membrane Fusion Protein -2.20 PA1436  RND Efflux Transporter -2.10 PA1437  Two-Component Response Regulator -1.88 PA1498 pykF Pyruvate Kinase -1.56 PA1500  Oxidoreductase -2.37 PA1501  Hydroxypyruvate Isomerase -3.70 PA1502 gcl Glyoxylate Carboligase -2.20 PA1505 moaA2 Molybdenum Cofactor Biosynthesis Protein A -1.54 PA1507  Transporter -1.52 PA1513  Hypothetical Protein -1.59 PA1514  Ureidoglycolate Hydrolase -3.83 PA1515 alc Allantoicase -1.68 PA1516  Hypothetical Protein -2.17 PA1517  Hypothetical Protein -2.00 PA1518  Transthyretin Family Protein -2.08 PA1519  Transporter -2.12 PA1525 alkB2 Alkane-1 Monooxygenase -1.69 PA1542  Hypothetical Protein -2.31 PA1545 pemB PemB -1.69 PA1569  Sugar Mfs Transporter -3.29 PA1572  Atp-Nad Kinase -2.65 PA1573  Hypothetical Protein -2.34 PA1597  Hypothetical Protein 2.65 PA1606  Hypothetical Protein 1.59 PA1607  Hypothetical Protein -1.66 PA1621  Hydrolase -1.55 PA1622  Hydrolase -1.56 PA1625  Hypothetical Protein 1.83 PA1627  GntR Family Transcriptional Regulator -2.07 PA1628  3-Hydroxyacyl-Coa Dehydrogenase -1.73 PA1629  Enoyl-CoA Hydratase -1.75 PA1656 hsiA2 Hsia2 1.70 PA1657 hsiB2 Hsib2 2.27 PA1658 hsiC2 Hsic2 2.60 PA1659  Hypothetical Protein 2.44 PA1660 hsiG2 Hsig2 2.86 PA1661 hsiH2 Hsih2 3.14 PA1663 sfa2 Sfa2 2.97 PA1664 lip2.2 Lip2.2 3.12 PA1665 fha2 Fha2 3.22 PA1668 dotU2 Dotu2 3.12 PA1736  Acetyl-CoA Acetyltransferase -1.91 PA1737  3-Hydroxyacyl-Coa Dehydrogenase -2.11 PA1755  Hypothetical Protein -2.26 PA1761  Hypothetical Protein -2.02  193 PA1769  Hypothetical Protein -1.84 PA1772 rraA Ribonuclease Activity Regulator Protein RraA -1.92 PA1779  Assimilatory Nitrate Reductase -1.77 PA1793 ppiB Peptidyl-Prolyl Cis-Trans Isomerase B -1.65 PA1825  Hypothetical Protein -1.51 PA1827  Short-Chain Dehydrogenase -1.74 PA1838 cysI Sulfite Reductase 1.53 PA1845  Hypothetical Protein -1.77 PA1847  Hypothetical Protein -1.53 PA1852  Hypothetical Protein -1.52 PA1864  Tetr Family Transcriptional Regulator 1.63 PA1865  Hypothetical Protein -1.66 PA1866  Hypothetical Protein -1.64 PA1869  Acyl Carrier Protein 2.73 PA1870  Hypothetical Protein 3.07 PA1871 lasA LasA Protease 2.64 PA1873  Cation Transporter 2.68 PA1877  Secretion Protein 1.65 PA1887  Hypothetical Protein 1.64 PA1888  Hypothetical Protein 1.73 PA1889  Hypothetical Protein 2.23 PA1899 phzA2 Phenazine Biosynthesis Protein 2.85 PA1900 phzB2 Phenazine Biosynthesis Protein 2.73 PA1913  Hypothetical Protein 3.94 PA1914  Hypothetical Protein 5.38 PA1921  Hypothetical Protein 4.07 PA1922  TonB-Dependent Receptor 4.01 PA1923 cobN Cobaltochelatase Subunit CobN 3.39 PA1925  Hypothetical Protein -2.97 PA1928 rimJ Ribosomal Protein Alanine Acetyltransferase -2.25 PA1929  Hypothetical Protein -1.83 PA1932  Hydroxylase Molybdopterin-Containing Subunit 2.02 PA1942  Hypothetical Protein -1.95 PA1968  Hypothetical Protein -1.91 PA1984  NAD+ Dependent Acetaldehyde Dehydrogenase -2.3 PA1991  Iron-Containing Alcohol Dehydrogenase -1.87 PA1992  Two-Component Sensor -2.17 PA1997  Acetoacetyl-CoA Synthetase -1.52 PA1998  LysR Family Transcriptional Regulator -2.16 PA2017  Hypothetical Protein -1.52 PA2019  Periplasmic Multidrug Efflux Lipoprotein -1.55 PA2021  Hypothetical Protein 2.67 PA2023 galU Utp-Glucose-1-Phosphate Uridylyltransferase 1.53 PA2024  Ring-Cleaving Dioxygenase -2.72 PA2035  Thiamine Pyrophosphate Protein -2.13  194 PA2041  Amino Acid Permease -1.62 PA2046  Hypothetical Protein 4.45 PA2062  Pyridoxal-Phosphate Dependent Protein 2.34 PA2066  Hypothetical Protein 2.78 PA2067  Hydrolase 2.68 PA2068  Mfs Transporter 2.35 PA2069  Carbamoyl Transferase 2.67 PA2086  Hydrolase 2.40 PA2090  Flavin-Dependent Oxidoreductase 2.91 PA2092  Mfs Transporter 2.50 PA2095  Hypothetical Protein 1.69 PA2096  AraC Family Transcriptional Regulator -1.65 PA2107  Hypothetical Protein 2.74 PA2108  Thiamine Pyrophosphate Protein 2.80 PA2109  Hypothetical Protein 1.85 PA2120  Hypothetical Protein -2.29 PA2134  Hypothetical Protein 2.32 PA2135  Transporter 2.51 PA2136  Hypothetical Protein 1.92 PA2140  Metallothionein 2.57 PA2141  Ompetence-Damaged Protein 2.88 PA2142  Short-Chain Dehydrogenase 3.09 PA2143  Hypothetical Protein 2.42 PA2144 glgP Glycogen Phosphorylase 2.43 PA2146  Hypothetical Protein -1.95 PA2147 katE Hydroperoxidase 3.21 PA2148  Hypothetical Protein 2.99 PA2149  Hypothetical Protein 2.38 PA2150  Ku Domain-Containing Protein 2.31 PA2151  Hypothetical Protein 2.80 PA2152  Trehalose Synthase 2.85 PA2153 glgB Glycogen Branching Protein 2.57 PA2154  Hypothetical Protein 4.06 PA2155  Cardiolipin Synthase 2 2.49 PA2156  Hypothetical Protein 3.53 PA2157  Hypothetical Protein 3.00 PA2158  Alcohol Dehydrogenase 2.59 PA2159  Hypothetical Protein 2.55 PA2160  Glycosyl Hydrolase 2.28 PA2161  Hypothetical Protein 3.23 PA2162  Maltooligosyl Trehalose Synthase 2.88 PA2163  4-Alpha-Glucanotransferase 3.00 PA2164  Glycosyl Hydrolase 2.89 PA2165 glgA Glycogen Synthase 2.59 PA2167  Hypothetical Protein 2.09  195 PA2168  Hypothetical Protein 1.76 PA2169  Hypothetical Protein 2.09 PA2171  Hypothetical Protein -1.76 PA2172  Hypothetical Protein -1.59 PA2173  Hypothetical Protein 2.17 PA2175  Hypothetical Protein 2.26 PA2176  Hypothetical Protein 2.32 PA2178  Hypothetical Protein -1.65 PA2179  Hypothetical Protein 2.93 PA2180  Hypothetical Protein 2.67 PA2181  Carboxylate-Amine Ligase -1.65 PA2187  Hypothetical Protein -2.13 PA2189  Hypothetical Protein 2.10 PA2204  ABC Transporter Substrate-Binding Protein 1.87 PA2210  Mfs Transporter -2.19 PA2212 pdxA 4-Hydroxythreonine-4-Phosphate Dehydrogenase -2.61 PA2235 pslE Hypothetical Protein -3.53 PA2236 pslF Hypothetical Protein -2.49 PA2244 pslN Hypothetical Protein 2.98 PA2245 pslO Hypothetical Protein 2.17 PA2261  2-Ketogluconate Kinase -2.09 PA2262  2-Ketogluconate Transporter -1.72 PA2263  2-Hydroxyacid Dehydrogenase -2.09 PA2281  AraC Family Transcriptional Regulator 1.60 PA2282  Hypothetical Protein -1.52 PA2291  Glucose-Sensitive Porin -1.90 PA2299  GntR Family Transcriptional Regulator 2.58 PA2300 chiC Chitinase 3.64 PA2309  ABC Transporter Substrate-Binding Protein 2.27 PA2311  Hypothetical Protein 1.94 PA2312  Xre Family Transcriptional Regulator 2.8 PA2338  Maltose/Mannitol ABC Transporter Substrate-Binding Protein -1.75 PA2345  Hypothetical Protein 1.58 PA2359 sfa3 Sfa3 1.56 PA2381  Hypothetical Protein -2.22 PA2382 lldA L-Lactate Dehydrogenase 1.79 PA2384  Hypothetical Protein 2.52 PA2385 pvdQ Penicillin Acylase-Related Protein 2.41 PA2386 pvdA L-Ornithine N5-Oxygenase 2.06 PA2392 pvdP Protein PvdP 3.43 PA2394 pvdN Protein PvdN 2.49 PA2397 pvdE Pyoverdine Biosynthesis Protein PvdE 2.26 PA2399 pvdD Pyoverdine Synthetase D 2.52 PA2400 pvdJ Protein PvdJ 2.54  196 PA2403  Hypothetical Protein 2.23 PA2404  Hypothetical Protein 2.10 PA2405  Hypothetical Protein 2.01 PA2406  Hypothetical Protein 2.52 PA2407  Adhesion Protein 2.06 PA2408  ABC Transporter ATP-Binding Protein 1.68 PA2409  ABC Transporter Permease 1.66 PA2410  Hypothetical Protein 1.87 PA2412  Hypothetical Protein 3.06 PA2413 pvdH Diaminobutyrate-2-Oxoglutarate Aminotransferase 2.42 PA2414 sndH L-Sorbosone Dehydrogenase 3.46 PA2415  Hypothetical Protein 3.53 PA2416 treA Trehalase 3.15 PA2424 pvdL Peptide Synthase 2.18 PA2425 pvdG Protein PvdG 3.13 PA2427  Hypothetical Protein 2.04 PA2432  Transcriptional Regulator -1.66 PA2437  Hypothetical Protein -1.72 PA2439  Hypothetical Protein -1.85 PA2440  Hypothetical Protein 3.79 PA2448  Hypothetical Protein 1.93 PA2467  Transmembrane Sensor -1.54 PA2469  Transcriptional Regulator -1.78 PA2481  Hypothetical Protein -1.95 PA2482  Cytochrome C -2.02 PA2483  Hypothetical Protein -1.57 PA2493 mexE Rnd Multidrug Efflux Membrane Fusion Protein MexE -1.70 PA2494 mexF Rnd Multidrug Efflux Transporter MexF -1.80 PA2495 oprN Multidrug Efflux Outer Membrane Protein OprN Precursor -1.73 PA2507 catA Catechol 1,2-Dioxygenase -6.22 PA2508 catC Muconolactone Delta-Isomerase -6.54 PA2509 catB Muconate Cycloisomerase I -2.23 PA2510 catR Transcriptional Regulator CatR -1.67 PA2511  Transcriptional Regulator -3.12 PA2512 antA Anthranilate Dioxygenase Large Subunit -2.39 PA2513 antB Anthranilate Dioxygenase Small Subunit -7.09 PA2514 antC Anthranilate Dioxygenase Reductase -7.8 PA2518 xylX Toluate 1,2-Dioxygenase Subunit Alpha -2.2 PA2519 xylS Transcriptional Regulator XylA -3.32 PA2531  Aminotransferase -1.69 PA2536  Phosphatidate Cytidylyltransferase 1.57 PA2540  Hypothetical Protein 1.79 PA2551  LysR Family Transcriptional Regulator -1.96 PA2569  Hypothetical Protein 3.29 PA2570 pa1L Pa-I Galactophilic Lectin -1.98  197 PA2582  Osmoprotectant Transporter Activator Protein -1.94 PA2587 pqsH Fad-Dependent Monooxygenase 1.70 PA2601  Lysr Family Transcriptional Regulator -2.15 PA2602  Hypothetical Protein -2.32 PA2603  Thiosulfate Sulfurtransferase -1.86 PA2604  Hypothetical Protein -1.66 PA2631  Acetyl Transferase -1.96 PA2634  Isocitrate Lyase -2.60 PA2656  Two-Component Sensor -1.91 PA2657  Two-Component Response Regulator -2.30 PA2658  Hypothetical Protein -3.25 PA2659  Hypothetical Protein -2.99 PA2675  Type Ii Secretion System Protein -2.03 PA2681  LysR Family Transcriptional Regulator -1.73 PA2682  Hypothetical Protein -1.76 PA2688 pfeA Outer Membrane Receptor PfeA -2.32 PA2693  Hypothetical Protein -1.77 PA2694  Thioredoxin 1.86 PA2696  Transcriptional Regulator -1.74 PA2708  Hypothetical Protein 2.04 PA2711  Periplasmic Spermidine/Putrescine-Binding Protein -3.57 PA2737  Transcriptional Regulator -1.64 PA2738 ihfA Integration Host Factor Subunit Alpha -1.65 PA2751  Hypothetical Protein 1.53 PA2754  Hypothetical Protein 2.54 PA2758  LysR Family Transcriptional Regulator -1.77 PA2759  Hypothetical Protein -2.90 PA2762  Hypothetical Protein -3.67 PA2764  Hypothetical Protein -1.63 PA2773  Hypothetical Protein 1.96 PA2776  Hypothetical Protein -1.82 PA2782  Hypothetical Protein -1.88 PA2786  Hypothetical Protein 2.03 PA2814  Hypothetical Protein -2.14 PA2833  Hypothetical Protein -1.67 PA2846  LysR Family Transcriptional Regulator -1.75 PA2886  Hypothetical Protein 1.57 PA2893  Long-Chain-Acyl-CoA Synthetase -1.71 PA2895 sbrR SbrR 1.69 PA2896 sbrI SbrI 1.55 PA2927  Hypothetical Protein 1.82 PA2934  Hydrolase 1.92 PA2937  Hypothetical Protein -2.46 PA2938  Transporter -1.66 PA2939  Aminopeptidase 2.41  198 PA2985  Hypothetical Protein -1.5 PA3041  Hypothetical Protein 1.64 PA3042  Hypothetical Protein 1.69 PA3049 rmf Ribosome Modulation Factor -1.90 PA3053  Hydrolytic Enzyme -1.73 PA3069  Lipoprotein 2.08 PA3126 ibpA Heat-Shock Protein IbpA -1.93 PA3146 orfM NAD Dependent Epimerase/Dehydratase 2.75 PA3161 ihfB Integration Host Factor Subunit Beta -2.96 PA3191  Two-Component Sensor 1.62 PA3195 gapA Glyceraldehyde-3-Phosphate Dehydrogenase 2.02 PA3205  Hypothetical Protein -1.91 PA3215  AraC Family Transcriptional Regulator -1.75 PA3222  Permease -1.5 PA3229  Hypothetical Protein -2.5 PA3230  Hypothetical Protein 1.66 PA3231  Hypothetical Protein 1.56 PA3234 actP Acetate Permease -1.67 PA3235  Hypothetical Protein -2.16 PA3236  Glycine Betaine-Binding Protein -3.18 PA3273  Hypothetical Protein 2.57 PA3274  Hypothetical Protein 2.47 PA3276  Hypothetical Protein 1.51 PA3293  Hypothetical Protein 2.45 PA3294  Hypothetical Protein 2.94 PA3306 alkB Hypothetical Protein -1.57 PA3307  Hypothetical Protein -1.99 PA3321  LysR Family Transcriptional Activator -2.90 PA3323  Hypothetical Protein -1.91 PA3324  Short Chain Dehydrogenase -1.51 PA3338  Hypothetical Protein -1.72 PA3369  Hypothetical Protein 1.94 PA3370  Hypothetical Protein 2.20 PA3371  Hypothetical Protein 1.75 PA3384 phnC ABC Phosphonate Transporter ATP-Binding Protein 1.56 PA3389  Ring-Cleaving Dioxygenase -1.52 PA3390  Hypothetical Protein -2.31 PA3397 fpr Ferredoxin-NADP+ Reductase 1.54 PA3406 hasD Transport Protein HasD -1.53 PA3417  Pyruvate Dehydrogenase E1 Component Subunit α -3.36 PA3420  Transcriptional Regulator -2.07 PA3422  Hypothetical Protein -1.79 PA3424  Hypothetical Protein -4.02 PA3425  Hypothetical Protein -5.08 PA3427  Oxidoreductase -2.89  199 PA3441  Molybdopterin-Binding Protein 4.36 PA3442 ssuB Aliphatic Sulfonates Transport ATP-Binding Subunit 4.11 PA3444 ssuD Alkanesulfonate Monooxygenase 4.39 PA3446  NAD(P)H-Dependent FMN Reductase 3.71 PA3449  Hypothetical Protein 4.47 PA3450 lsfA 1-Cys Peroxiredoxin LsfA 4.02 PA3459  Asparagine Synthetase 2.29 PA3460  Gnat Family Acetyltransferase 2.14 PA3461  Hypothetical Protein 1.99 PA3467  Mfs Transporter -2.01 PA3473  Hypothetical Protein -1.85 PA3478 rhlB Rhamnosyltransferase Chain B 2.35 PA3479 rhlA Rhamnosyltransferase Chain A 2.23 PA3529  Peroxidase -1.90 PA3531 bfrB Bacterioferritin -2.67 PA3554  Bifunctional UDP-Glucuronic Acid Decarboxylase -1.63 PA3566  Hypothetical Protein -1.61 PA3567  Oxidoreductase -1.62 PA3577  Hypothetical Protein -1.55 PA3584 glpD Glycerol-3-Phosphate Dehydrogenase -2.61 PA3588  Porin -1.53 PA3598  Hypothetical Protein 2.29 PA3600 rpmJ 50S Ribosomal Protein L36 -5.11 PA3601 rpmE2 50S Ribosomal Protein L31 -4.56 PA3617 recA Recombinase A -1.62 PA3623  Hypothetical Protein -1.74 PA3677  Efflux Transmembrane Protein -1.61 PA3687 ppc Phosphoenolpyruvate Carboxylase -1.56 PA3689 cadR Transcriptional Regulator CadR -2.57 PA3691  Lipoprotein 2.60 PA3692 ompA Outer Membrane Protein, OmpA 2.52 PA3709  MFS Transporter 1.51 PA3720  Hypothetical Protein -2.21 PA3721  Transcriptional Regulator -1.81 PA3729  Hypothetical Protein 1.63 PA3730  Hypothetical Protein -2.04 PA3731  Hypothetical Protein -3.77 PA3732  Hypothetical Protein -3.02 PA3741  Hypothetical Protein 1.92 PA3752  Hypothetical Protein -2.14 PA3753  Hypothetical Protein -1.96 PA3754  Hypothetical Protein -2.13 PA3765  Hypothetical Protein -1.75 PA3769 guaA GMP Synthase 1.63 PA3795  Oxidoreductase 1.59  200 PA3813  Scaffold Protein -1.62 PA3814 iscS Cysteine Desulfurase -1.84 PA3835  Hypothetical Protein -1.71 PA3865  Amino Acid ABC Transporter -2.48 PA3888  ABC Transporter Permease 2.51 PA3889  ABC Transporter Substrate-Binding Protein 1.59 PA3890  Abc Transporter Permease 2.05 PA3891  Abc Transporter ATP-Binding Protein 1.88 PA3895  LysR Family Transcriptional Regulator -1.99 PA3901 fecA Fe(III) Dicitrate Transport Protein FecA -2.11 PA3904  Hypothetical Protein 1.71 PA3905  Hypothetical Protein 2.06 PA3906  Hypothetical Protein 2.89 PA3907  Hypothetical Protein 3.20 PA3908  Hypothetical Protein 2.82 PA3931  Hypothetical Protein 3.17 PA3937  Taurine ABC Transporter ATP-Binding Protein 2.34 PA3938  Taurine ABC Transporter Periplasmic Protein 3.83 PA3952  Hypothetical Protein 1.78 PA3962  Hypothetical Protein 1.83 PA3969  Hypothetical Protein -1.61 PA4026  Acetyltransferase -1.83 PA4063  Hypothetical Protein -3.31 PA4065  Permease -2.61 PA4066  Hypothetical Protein -1.89 PA4070  DNA-Binding Transcriptional Activator Fear -2.59 PA4078  Nonribosomal Peptide Synthetase 1.56 PA4082 cupB5 Adhesive Protein CupB5 -1.67 PA4094  AraC Family Transcriptional Regulator -1.69 PA4111  Hypothetical Protein -1.72 PA4127 hpcG 2-Oxo-Hepta-3-Ene-1,7-Dioic Acid Hydratase -1.74 PA4139  Hypothetical Protein -1.93 PA4140  Hypothetical Protein -1.67 PA4141  Hypothetical Protein 1.87 PA4142  Secretion Protein 2.14 PA4143  Toxin Transporter 1.93 PA4144  Outer Membrane Protein 1.99 PA4148  Short-Chain Dehydrogenase -1.8 PA4149  Hypothetical Protein -1.59 PA4150  Dehydrogenase E1 Component -1.76 PA4152  Branched-Chain Alpha-Keto Acid Dehydrogenase Subunit E2 -2.07 PA4153 adh 2,3-Butanediol Dehydrogenase -1.67 PA4154  Sh3 Domain-Containing Protein 1.90 PA4159 fepB Iron-Enterobactin Transporter Periplasmic Binding Protein -1.74  201 PA4163  Amidase -1.70 PA4164  Hypothetical Protein -1.64 PA4171  Protease 1.80 PA4172  Exonuclease Iii 2.93 PA4175 prpL PvdS-Regulated Endoprotease, Lysyl Class 1.62 PA4185  GntR Family Transcriptional Regulator -1.87 PA4204  Hypothetical Protein 1.89 PA4222  ABC Transporter ATP-Binding Protein 1.75 PA4223  ABC Transporter ATP-Binding Protein 1.74 PA4224 pchG Pyochelin Biosynthetic Protein PchG 2.33 PA4225 pchF Pyochelin Synthetase 1.99 PA4226 pchE Dihydroaeruginoic Acid Synthetase 1.86 PA4229 pchC Pyochelin Biosynthetic Protein PchC 1.60 PA4230 pchB Isochorismate-Pyruvate Lyase 2.19 PA4231 pchA Salicylate Biosynthesis Isochorismate Synthase 1.94 PA4304  Type Ii Secretion System Protein 1.64 PA4305  Pilus Assembly Protein -2.12 PA4306  Hypothetical Protein -3.59 PA4309 pctA Chemotactic Transducer PctA -2.15 PA4324  Hypothetical Protein -2.11 PA4344  Hydrolase 1.63 PA4345  Hypothetical Protein 1.83 PA4349  Hypothetical Protein -1.56 PA4352  Hypothetical Protein -1.53 PA4354  Hypothetical Protein -1.97 PA4357  Hypothetical Protein -1.95 PA4359 feoA Ferrous Iron Transport Protein A -2.52 PA4384  Hypothetical Protein 2.33 PA4390  Hypothetical Protein 2.65 PA4394  Hypothetical Protein 2.44 PA4442 cysN Bifunctional Sulfate Adenylyltransferase Subunit 1 1.74 PA4443 cysD Sulfate Adenylyltransferase Subunit 2 2.12 PA4463  Hypothetical Protein -1.61 PA4500 dppA3 Dipeptide ABC Transporter Substrate-Binding Protein Dppa3 -1.57 PA4521  Hypothetical Protein -1.51 PA4541  Large Exoprotein -2.07 PA4552 pilW Type 4 Fimbrial Biogenesis Protein PilW 2.11 PA4553 pilX Type 4 Fimbrial Biogenesis Protein PilX 1.88 PA4554 pilY1 Type 4 Fimbrial Biogenesis Protein PilY1 1.74 PA4556 pilE Type 4 Fimbrial Biogenesis Protein PilE 1.92 PA4598 mexD Multidrug Efflux RND Transporter MexD -1.91 PA4611  Hypothetical Protein -1.73 PA4616  C4-Dicarboxylate-Binding Protein -1.66 PA4618  Hypothetical Protein -1.74  202 PA4624  Hypothetical Protein -2.11 PA4625  Hypothetical Protein -1.77 PA4630  Hypothetical Protein -3.36 PA4697  Hypothetical Protein -1.58 PA4738  Hypothetical Protein 2.24 PA4739  Hypothetical Protein 1.90 PA4763 recN DNA Repair Protein RecN -1.96 PA4791  Hypothetical Protein -1.64 PA4799  Adenylate Kinase 1.63 PA4803  Methyltransferase -1.90 PA4810 fdnI Nitrate-Inducible Formate Dehydrogenase Subunit Gamma -1.79 PA4811 fdnH Nitrate-Inducible Formate Dehydrogenase Subunit Beta -2.07 PA4812 fdnG Formate Dehydrogenase-O, Major Subunit -1.69 PA4825 mgtA Mg(2+) Transport ATPase, P-Type 2 -1.71 PA4834  Hypothetical Protein -4.18 PA4835  Hypothetical Protein -3.48 PA4836  Hypothetical Protein -5.30 PA4837  Outer Membrane Protein -5.04 PA4838  Hypothetical Protein -3.12 PA4876 osmE Dna-Binding Transcriptional Activator OsmE 2.59 PA4877  Hypothetical Protein 2.13 PA4879  Hypothetical Protein 1.76 PA4880  Bacterioferritin 2.22 PA4881  Hypothetical Protein -1.93 PA4885 irlR Two-Component Response Regulator -1.63 PA4899  Aldehyde Dehydrogenase -1.74 PA4900  Mfs Transporter -2.43 PA4904 vanA Vanillate O-Demethylase Oxygenase -2.05 PA4916  Hypothetical Protein 1.57 PA4917  Hypothetical Protein 1.79 PA4921  Hypothetical Protein -1.95 PA4929  Hypothetical Protein -2.38 PA4980  Enoyl-CoA Hydratase/Isomerase -1.96 PA4984  TetR Family Transcriptional Regulator -1.82 PA4989  Transcriptional Regulator -1.90 PA4995  Acyl-CoA Dehydrogenase -1.71 PA5020  Acyl-CoA Dehydrogenase -2.41 PA5023  Hypothetical Protein -1.87 PA5055  Hypothetical Protein -1.70 PA5058 phaC2 Poly(3-Hydroxyalkanoic Acid) Synthase 2 1.64 PA5059  TetR Family Transcriptional Regulator 1.89 PA5085  LysR Family Transcriptional Regulator -2.13 PA5099  Cytosine/Purines Uracil Thiamine Allantoin Permease 1.55 PA5109  Hypothetical Protein 1.99 PA5111 gloA3 Lactoylglutathione Lyase 1.85  203 PA5131 pgm Phosphoglyceromutase 1.54 PA5161 rmlB DtdP-D-Glucose 4,6-Dehydratase 1.66 PA5162 rmlD DtdP-4-Dehydrorhamnose Reductase 1.59 PA5163 rmlA Glucose-1-Phosphate Thymidylyltransferase 1.60 PA5164 rmlC Dtdp-4-Dehydrorhamnose 3,5-Epimerase 1.70 PA5177  Hydrolase -1.64 PA5179  LysR Family Transcriptional Regulator -1.60 PA5206 argE Acetylornithine Deacetylase -1.63 PA5209  Hypothetical Protein 1.57 PA5218  LysR Family Transcriptional Regulator -1.54 PA5228  5-Formyltetrahydrofolate Cyclo-Ligase -2.63 PA5230  ABC Transporter Permease 1.57 PA5231  ABC Transporter ATP-Binding Protein/Permease 1.80 PA5266 vgrG14 VgrG14 2.31 PA5269  Hypothetical Protein -1.52 PA5284  Fimbrial Protein -2.53 PA5291  Choline Transporter 1.73 PA5297 poxB Pyruvate Dehydrogenase (Cytochrome) 1.57 PA5308 lrp Leucine-Responsive Regulatory Protein -1.92 PA5312  Aldehyde Dehydrogenase -1.55 PA5313  Omega Amino Acid-Pyruvate Transaminase -1.73 PA5314  Hypothetical Protein -1.86 PA5323 argB Acetylglutamate Kinase 1.64 PA5324 sphR Sphingosine-Responsive Regulator, SphR -1.89 PA5325 sphA SphA -3.23 PA5326 sphD SphD 1.60 PA5327 sphC SphC -2.92 PA5328 sphB SphB -2.97 PA5332 crc Catabolite Repression Control Protein -1.52 PA5379 sdaB L-Serine Dehydratase -2.89 PA5381  Hypothetical Protein -1.75 PA5383  Hypothetical Protein 4.15 PA5388  Hypothetical Protein -1.51 PA5389  AraC Family Transcriptional Regulator -1.77 PA5396  Hypothetical Protein 1.59 PA5397  Hypothetical Protein -3.78 PA5398  FMN Oxidoreductase -3.34 PA5399  Ferredoxin -3.49 PA5400  Electron Transfer Flavoprotein Alpha Subunit -3.08 PA5401  Hypothetical Protein -2.33 PA5408  Hypothetical Protein -1.93 PA5409  Hypothetical Protein -1.94 PA5410  Ring Hydroxylating Dioxygenase, Alpha-Subunit -2.89 PA5411  Ferredoxin -2.22 PA5415 glyA1 Serine Hydroxymethyltransferase -2.65  204 PA5416 soxB Sarcosine Oxidase Beta Subunit -2.84 PA5417 soxD Sarcosine Oxidase Delta Subunit -2.69 PA5418 soxA Sarcosine Oxidase Alpha Subunit -2.69 PA5419 soxG Sarcosine Oxidase Gamma Subunit -2.71 PA5420 purU2 Formyltetrahydrofolate Deformylase -2.49 PA5421 fdhA Glutathione-Independent Formaldehyde Dehydrogenase -3.21 PA5425 purK Phosphoribosylaminoimidazole Carboxylase ATPase Subunit 1.77 PA5432  GnaT Family Acetyltransferase -2.05 PA5433  GnaT Family Acetyltransferase -2.27 PA5435  Pyruvate Carboxylase Subunit B -2.27 PA5436  Pyruvate Carboxylase Subunit A -2.19 PA5446  Hypothetical Protein -3.02 PA5465  Hypothetical Protein -1.83 PA5481  Hypothetical Protein 2.58 PA5484  Two-Component Sensor 2.00 PA5514  Beta-Lactamase 2.11 PA5518  Potassium Efflux Transporter -1.58 PA5523  Aminotransferase -1.8 PA5524  Short-Chain Dehydrogenase -1.79 PA5526  Lipoprotein 1.61 PA5534  Hypothetical Protein -5.97 PA5535  Hypothetical Protein -5.84 PA5536  DksA/TraR Family C4-Type Zinc Finger Protein -5.04 PA5538 amiA N-Acetylmuramoyl-L-Alanine Amidase 3.16 PA5539  GTP Cyclohydrolase -3.36 PA5540  Hypothetical Protein -3.85 PA5541 pyrQ Dihydroorotase 5.40  Table A-6. Influence of mucin on MIC. Liquid MIC results done in SCFM with 0.4% mucin and without mucin grown at 37°C overnight with an inoculum size of 2-7 x 105cells (n=3-5). Antibiotic MIC (µg/ml) + mucin - mucin Gentamicin 4 1 Tobramycin 2 2 Amikacin 16 4 Imipenem 0.325 0.625 Meropenem 0.125 0.125 Ceftazidime 31.25 31.25 Aztreonam 4 8 Piperacillin 4 4 Erythromycin 500 250 Clarithromycin 2000 2000 Polymyxin B 16 16 Colistin 16 2  205 Norfloxacin 16 16 Ciprofloxacin 1 0.5 Trimethoprim 128 128 Tetracycline 64 256 Chloramphenicol 32 16  Table A-7. Resistome mutant susceptibility under surfing conditions - raw data. Average zone of inhibition measurements for resistome mutants tested for five selected antibiotics. Mutants of up-regulated resistome genes were tested against 10 µg/disk of antibiotic and down-regulated against 100 µg/disk. Statistical significance relative to wild-type was determined using two-way ANOVA. (n=3 resistome mutants; n=6 wild-type) * p<0.5, ** p<0.01, *** p< 10-3, **** p<10-4. Standard deviations range from 0 to 2.5mm. Mutant Zone of Inhibition (mm) Imipenem Tetracycline Polymyxin B Tobramycin Norfloxacin 10 µg/disk antibiotic concentration Wild-type 5.7 5.0 5.6 3.3 1.0 ΔrecG 7.3 8.7* 9.7** 12.5**** 7.3**** ΔddaH 9.0* 0*** 5.3 3.0 2.3 ΔPA5130 10** 0*** 0**** 0* 6.5**** cycH 4.5 4.3 11**** 9.7**** 0 100 µg/disk antibiotic concentration Wild-type 12.3 6.7 8 12 14.7 ΔarmR 0**** 0**** 1**** 6.3**** 0**** ΔPA3576 12.0 3.0* 6.0 8.3* 10.7* ΔPA1428 12.7 7.7 8.0 7.0*** 0.0**** ΔPA2047 12.3 7.0 5.7 7.3** 9.7*** ΔPA1553 9.0 7.3 7.7 8.0* 10.5* ΔatpB* 9.7 4.0 8.0 8.3* 9.7*** ΔPA4292 7.7** 5.3 17**** 5**** 10** ΔclpS 8.3* 6.3 15**** 6.7*** 8.3**** ΔnuoB 10.7 0**** 18**** 6.3**** 10.3** ΔPA3721 10 2** 14.5**** 0**** 10** ΔPA4429 11 8.7 20**** 0**** 7.7**** ΔetfA 12.3 9 15**** 7.3** 10** ΔnuoG 9.7 6.5 6 0**** 7.3**** ΔPA4781 12.3 10 10.3 6.7*** 9**** ΔserA 14.7 8.7 12.7** 7.3** 11* ΔccmF 14 8.7 13.3*** 7.3** 8.7**** ΔPA3667 15.7 0.0**** 7.7 10.0 12.0 ΔPA1513 10.7 0.0**** 7.3 10.7 14.0 ΔpchF 9.7 5.0 7.3 1.1**** 13.3 Δrph 13.0 9.0 6.0 5.0**** 15.0 ΔPA2566 11.0 4.0 7.0 9.3 9.7*** ΔgidA 9.7 7.3 9.0 10.5 10.0**  206 ΔmutS 16.0 9.3 4.3* 6.3**** 11.5 ΔthiG 6.3**** 6.7 7.0 8.7 10.3** ΔnuoF 11.0 5.3 6.7 7.0*** 14.7 ΔpckA 9.7 4.8 7.0 6.3**** 12.0 ΔPA2571 12.7 6.7 7.0 7.3** 11.7 ΔPA4766 13.7 6.0 6.5 7.0*** 13.7 ΔPA1348 16.7** 5.5 8.3 10.7 10.7* ΔbraB 11.0 6.7 5.7 10.0 10.7* ΔhtpX 12.7 5.7 8.3 11.0 9.5** ΔspeA 11.3 4.5 5.3 10.3 12.0 ΔadhA 12.3 7.0 6.7 10.3 13.3 Table A-8. RT-qPCR results confirmed the dysregulation of resistome genes shown in RNA-Seq. The relative fold-change of expression of select resistome genes under surfing conditions (SCFM + 0.4% mucin) relative to swimming (SCFM 0.3% agar) from both the RNA-Seq experiment and RT-qPCR of cells collected from the centre and edge of a surfing colony relative to swimming cells. (FC cut-off of RNA-Seq is ± 1.5).   Gene Expression (FC) RT-qPCR RNA-Seq Gene Centre Edge Centre Edge recG 16.4 8.1 1.9 2.1 PA5130 2.2 1.5 NC 2.4 ddaH 2.8 3.2 4.9 2.4 PA1428 -2.2 -3.3 -3.4 NC PA2047 3.1 -1.4 NC -2.1 thiG -2.5 1.8 -2.9 NC PA3667 -1.2 -3.4 -1.7 -2.5 PA3576 2.3 -4.4 NC -2.9 atpB -1.5 1.1 -2.1 NC PA4292 -2.3 -2.4 -6.7 NC nuoB -1.0 -1.2 -2.8 NC PA3721 -2.0 2.1 -5.3 -2.7 clpS 5.0 -3.8 NC -2.3 armR -4.4 2.9 -3.2 -5.1 cycH 5.7 1.4 NC 2.2     

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