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Physiological contribution of the Pseudomonas aeruginosa OprD family of porins Tamber, Sandeep 2006

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PHYSIOLOGICAL CONTRIBUTION OF T H E P S E U D O M O N A S  AERUGINOSA  O P R D F A M I L Y O F PORINS BY SANDEEP T A M B E R  B.Sc (Honours Microbiology), University of Alberta, 1998 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS OF T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y  in THE  F A C U L T Y OF G R A D U A T E STUDIES  (Microbiology & Immunology)  THE  U N I V E R S I T Y OF BRITISH C O L U M B I A  May, 2006 © Sandeep Tamber, 2006  To circumvent the permeability barrier of its outer membrane, Pseudomonas aeruginosa has evolved a series of specific porins to facilitate nutrient uptake. These channels have substrate-specific binding sites that selectively permit the passage of related classes of molecules. In this study, the identification of a novel 19 member family of porins is reported. The members of this family share a considerable degree of protein sequence conservation (46% to 57%) and fall into one of two phylogenetically distinct clusters; one bearing high similarity to the basic amino acid specific porin OprD and the other being most similar to the phenylacetic acid uptake porin, PhaK, of P. putida.  The physiological contribution of this family was investigated by predicting  substrates for of each homologue based on the genomic context of their genes. These predictions were then tested by performing growth curves with porin deficient mutants in minimal media containing the proposed substrate as the sole carbon source.  The  following substrates were identified for 7 of the 18 novel homologues: OpdB - proline, OpdC - histidine, OpdP - glycine-glutamate, OpdT - tyrosine, OpdH - cz's-aconitate, OpdK - vanillate and OpdO - pyroglutamate. Functional overlap was observed between the basic amino acid specific porin, OprD, and the glycine-glutamate specific porin, OpdP, with respect to arginine transport. Thus, members of this family had diverged to take up unique substrates but had also retained some redundancy which may allow them to compensate for one another in the event of a gene loss or mutation. To gain insights regarding the evolution of this family, one homologue, OpdH, was characterized in detail. This porin was specifically induced by citrate, isocitrate and cw-aconitate through the action of the PA0756-757 two-component regulatory system and was involved in the uptake of the latter two compounds. The channel properties of  ii  OpdH differed significantly from those of OprD. OpdH demonstrated an average single channel conductance of 0.7 nS in 1 M KC1. The channel was cation selective and did not harbour a tricarboxylate specific binding site.  The structural and evolutionary  implications of the differences between the two porins are discussed.  iii  TABLE OF CONTENTS ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST OF T A B L E S  viii  LIST OF FIGURES  ix  LIST OF ABBREVIATIONS  xi  ACKNOWLEDGEMENTS  xii  S T A T E M E N T OF AUTHORSHIP  xiii  1 INTRODUCTION  2  1.1 P S E U D O M O N A S A E R U G I N O S A  2  1.2 ROOTS OF P S E U D O M O N A S A E R U G I N O S A VERSATILITY  2  1.3 T H E P S E U D O M O N A S A E R U G I N O S A OUTER MEMBRANE  7  1.3.1 Outer membrane structure 1.3.2 Barrier function of the outer membrane 1.3.3 Uptake pathways through the outer membrane 1.3.3.1 Hydrophobic pathway 1.3.3.2 Self-promoted uptake of polycations 1.3.3.3 Porin pathway 1.4 M E T H O D S USED TO STUDY PORIN FUNCTION  1.4.1 Studies with intact cells 1.4.2 Model membrane systems 1.4.3 Structural studies 1.4.3.1 Structure and functional mechanism of general porins 1.4.3.2 Structural features unique to specific porins and their functional implications 1.5 P S E U D O M O N A S A E R U G I N O S A PORINS  1.5.1 General porins of P. aeruginosa 1.5.1.1 OprF... 1.5.2 Specific porins of P. aeruginosa 1.5.2.1 OprB  7 9 11 11 11 12 14  14 15 17 17 21 22  22 22 25 25  1.5.2.2 OprPand OprO 1.5.2.3 OprD 1.5.2.4 OprD homologues  26 27 29  1.6 AIMS OF THIS STUDY  30  1.7 REFERENCES  32  2 R O L E O F T H E O P R D F A M I L Y O F PORINS IN N U T R I E N T U P T A K E  42  2.1 INTRODUCTION  42  2.2 M A T E R I A L S AND M E T H O D S  44  2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6  Bacterial strains, plasmids, primers, and media Genetic manipulations Sequence analysis Reporter gene assays Growth assays Semi-quantitative (SQ)-PCR  44 47 48 48 48 49  2.3 RESULTS  2.3.1 2.3.2 2.3.3 2.3.4 2.3.5  50  A novel family of specific porins in P. aeruginosa Involvement of OprD in arginine uptake Involvement of OpdK in vanillate uptake Growth phenotypes of the other OprD homologues Induction of oprD homologues by their substrates  50 52 59 63 84  2.4 DISCUSSION  86  2.5 REFERENCES  92  3 FUNCTIONAL COMPENSATION WITHIN T H EOPRD FAMILY  96  3.1 INTRODUCTION  96  3.2 MATERIALS AND M E T H O D S  98  3.2.1 Bacterial strains and growth conditions 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7  98  Primers and reagents Construction of mutants Quantitative and semi-quantitative P C R Outer membrane isolation and protein electrophoresis Radioactive arginine uptake assays Minimum inhibitory concentration (MIC) determinations  99 100 101 101 102 102  v  3.3 RESULTS  103  3.3.1 Expression profiles of eight OprD homologues 3.3.2 Arginine-mediated induction of OpdP... 3.3.3 Involvement of OpdP in the compensatory uptake of arginine  ..103 105 106  3.4 DISCUSSION  108  3.5 REFERENCES  113  4 FUNCTIONAL CHARACTERIZATION OF OPDH  116  4.1 INTRODUCTION  116  4.2 MATERIALS AND M E T H O D S  117  4.2.1 Bacterial strains, plasmids, primers, and media 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8  117  Genetic manipulations Outer membrane isolation, protein electrophoresis and sequencing Reporter Gene Assays Semi-quantitative PCR (SQ-PCR) Biofilm Formation Assay OpdH purification Planar Bilayer Analysis  118 120 121 121 ..122 123 124  4.3 RESULTS  124  4.3.1 4.3.2 4.3.3 4.3.4 4.4.5 4.3.6 4.3.7  124 126 128 128 132 134 136  Genomic context of opdH Construction of an opdH knock-out and transcriptional fusion Role of OpdH in tricarboxylate uptake Tricarboxylate specific induction of OpdH Regulation of opdH by PA0756-PA0757 Involvement of OpdH in biofilm formation Channel function of OpdH  4.4 DISCUSSION  140  4.5 R E F E R E N C E S .  146  5 DISCUSSION  151  5.1 IDENTIFICATION OF A NOVEL FAMILY OF SPECIFIC PORINS IN P S E U D O M O N A S AERUGINOSA  151  vi  5.2 M E M B E R S OF T H E O P R D FAMILY EXHIBIT DISTINCT Y E T COMPLEMENTARY FUNCTIONS....  152  5.3 COMPARISON OF T H E CHANNEL PROPERTIES OF O P R D AND O P D H  154  5.4 RE-EVALUATION OF O P D H AS A SPECIFIC PORIN  155  5.5 BASIS OF T H E PHYLOGENETIC RELATIONSHIPS BETWEEN O P R D FAMILY MEMBERS  156  5.6 DIFFERENTIAL REGULATION OF O P R D FAMILY MEMBERS  157  5.7 CONTRIBUTION OF T H E O P R D FAMILY JO P S E U D O M O N A S A E R U G I N O S A PHYSIOLOGY  158  5.8 REFERENCES  161  vii  LIST OF T A B L E S C H A P T E R 1 INTRODUCTION TABLE  2  1.1 Transporter classes in P. aeruginosa  5  C H A P T E R 2 R O L E OF T H E O P R D FAMILY OF PORINS IN NUTRIENT UPTAKE  42  2.1 OprD homologues in P. aeruginosa and other bacteria T A B L E 2.2 Bacterial strains and plasmids used in this study T A B L E 2.3 Oligonucleotide sequences used in this study T A B L E 2.4 Amino acid composition of the second surface loop of the OprD homologues T A B L E 2.5 Amino acid composition of the third surface loop of the OprD homologues T A B L E 2.6 Doubling times of P. aeruginosa PAO, P A K , an oprD mutant and an opdK mutant grown in BM2 + either arginine or vanillate T A B L E 2.7 Catechol-2,3-dioxygenase (CDO) activities of an opdK::xylE-Gm transcriptional fusion and an oprD::xylE-Gm transcriptional fusion grown in various aromatic compounds T A B L E 2.8 Predicted substrates of the P. aeruginosa OprD homologues  44 45 46 56 56  C H A P T E R 3 FUNCTIONAL COMPENSATION WITHIN T H E O P R D FAMILY  96  TABLE  58  T  r  61 90  3.1 Substrate specificities of 8 members of the P. aeruginosa OprD family 3.2 Bacterial strains and plasmids used in this study T A B L E 3.3 Primers used in this investigation T A B L E 3.4 Major inhibitory concentrations (MIC) of imipenem and meropenem for P. aeruginosa strains lacking OprD, OpdP, or both porins  98 99 100  C H A P T E R 4 FUNCTIONAL CHARACTERIZATION OF O P D H  116  4.1 Bacterial strains and plasmids used in this study T A B L E 4.2 D N A primers used in this investigation T A B L E 4.3 Properties of E. coli and Pseudomonas porins  118 119 138  TABLE TABLE  108  TABLE  viii  LIST OF FIGURES CHAPTER 1 INTRODUCTION FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE  1.1 1.2 1.3 1.4 1.5 1.6  Gram-negative bacterial cell wall Planar bilayer apparatus Three dimensional porin structures of E. coli Charge segregation within the eyelet of the E. coli OmpF general pore Structures of the substrates taken up by OprD Expression of OprD and OprD homologues in diverse carbon sources  CHAPTER 2 ROLE OF THE OPRD FAMILY OF PORINS IN NUTRIENT UPTAKE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE FIGURE  2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8  Phylogenetic analysis of the OprD family in P. aeruginosa and P. putida Protein sequence alignment of the P. aeruginosa OprD family of porins Physical parameters of the P. aeruginosa OprD porin family Correlation of optical density with colony forming units in P. aeruginosa Genomic context of opdK Genomic contexts of the P. aeruginosa OprD porin family Growth phenotypes of P. aeruginosa OprD homologue mutants Expression of OprD homologues in response to their putative substrates  CHAPTER 3 FUNCTIONAL COMPENSATION WITHIN THE OPRD FAMILY  2  8 17 18 20 28 30 42  51 53 57 59 60 64 83 85 96  3.1 Expression profile of eight OprD homologues 104 F I G U R E 3.2 Outer membrane protein profiles of wild-type P. aeruginosa and an oprD deficient mutant 105 F I G U R E 3.3 Transcription of opdP in wild-type P. aeruginosa and an oprD deficient mutant ...106 F I G U R E 3.4 Growth and uptake of arginine by an oprDlopdP deficient mutant 107 FIGURE  CHAPTER 4 FUNCTIONAL CHARACTERIZATION OF O P D H  116  4.1 Genomic context of opdH 125 4.2 Outer membrane profiles of wild-type P. aeruginosa, an opdH mutant, and an opdH complemented strain 127 F I G U R E 4.3 Growth phenotype of an opdH deficient mutant 129 F I G U R E 4.4 Expression of OpdH in citrate 130 F I G U R E 4.5 Expression of OpdH in the Kreb's Cycle intermediates 131 F I G U R E 4.6 Effect of metal ions on OpdH expression 132 F I G U R E 4.7 Regulation of opdH and PA0754 by the PA0756-757 two-component regulatory system 134 F I G U R E 4.8 Involvement of opdH in biofilm formation ...135 F I G U R E 4.9 Selective solubilization of OpdH 136 F I G U R E 4.10 Purification of OpdH 137 F I G U R E 4.11 Single channel conductance of OpdH 139 F I G U R E 4.12 Macroscopic conductance experiment of OpdH 140 FIGURE FIGURE  FIGURE  4.13 Alignrnent of OprD and OpdH  142  C H A P T E R 5 DISCUSSION FIGURE  151  5.1 Specific porins of P. aeruginosa  158  x  LIST OF ABBREVIATIONS  A ABC AIDS Ap ATP bp Cb cDNA CDO CoA Da DTT DNA DNase EDTA Gm HCI HIV hr L LPS MIC min mRNA Octyl-POE PA PBS PCR psi RPM S SDS SDS-PAGE SQ-PCR TCA TAE Tc TM Tris TTT  Absorbance A T P binding cassette transporter Acquired immune deficiency syndrome Ampicillin Adenosine triphosphate Base pair Carbanicillin Complementary deoxyribonucleic acid Catechol-2,3 -dioxygenase Coenzyme A Dalton Dithiotheritol Deoxyribonucleic acid Deoxyribonuclease Ethylene diamine tetra acetic acid Gentamicin Hydrochloric acid Human immunodeficiency virus Hour Loop Lipopolysaccharide Minimal inhibitory concentration Minute Messenger ribonucleic acid Octyl-polyoxyethylene Pseudomonas aeruginosa locus of identity Phosphate buffered saline Polymerase chain reaction Pounds per square inch Revolutions per minute Sieman Sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis Semi-quantitative polymerase chain reaction Tricarboxylic acid Tris acetate ethylene diamine tetra acetic acid Tetracycline Transmembrane Tris(hydroxymethyl) aminomethane Tripartite tricarboxylate transporter  ACKNOWLEDGMENTS  When I moved to Vancouver to start my life as a graduate student, I had two main objectives; the first was to become a better scientist, the second was to learn how to snowboard.  If I have achieved the first goal, it is largely due to the efforts of my  supervisor, Dr. Bob Hancock.  His wisdom and enthusiasm regarding all aspects of  science, as well as his willingness to let me learn from my mistakes is very much appreciated. I would also like to thank the members of my supervisory committee, Drs. Tom Beatty, Bill Mohn, and Natalie Strynadka for their valuable insights and suggestions; and also the members of the Hancock lab past and present for their advice and camaraderie, particularly: Dr. Martina Ochs, who first conceived the idea for this project, Manjeet Bains and Susan Farmer, for superb technical advice and Barbara Sherman, for her excellent organizational skills. For attaining the second objective and so much more, I have to thank the many wonderful friends that I met while in Vancouver. I would also like to give a big 'ol shout out to my Edmonton crew, and my family, particularly my parents.  Their love and  support has been invaluable to me over the last six years.  xii  STATEMENT OF AUTHORSHIP  Large sections of Chapter 1 have been published in:  Tamber, S . and R. E . W. Hancock.  2003.  On the mechanism of solute uptake in  Pseudomonas. Front Biosci 8:S472-S483.  Tamber, S. and R.E.W. Hancock.  2 0 0 4 . The Outer Membranes of Pseudomonads. In J. L . Ramos (ed.), Pseudomonas, V o l I. Kluwer Academic. New York, N Y  Hancock, R. E . W. and S. Tamber. 2 0 0 4 . Porins of the Outer Membrane of Pseudomonas aeruginosa, p. 6 1 - 7 7 . In R. Benz (ed.), Bacterial and Eukaryotic Porins. Wiley-VCH, Weinheim. Chapter 2 was largely published as:  Tamber, S., M. M. Ochs, and R. E . W. Hancock.  2 0 0 6 . Role of the novel OprD family of porins in nutrient uptake in Pseudomonas aeruginosa. J Bacteriol 188:45-54.  Chapter 3 is being prepared for publication as:  Tamber, S. and R. E . W. Hancock.  2 0 0 6 . Involvement of two related porins, OprD and OpdP, in the uptake of arginine by Pseudomonas aeruginosa.  Chapter 4 is being prepared for publication as:  Tamber, S. and R. E . W. Hancock. 2 0 0 6 . Characterization of OpdH: A Pseudomonas aeruginosa porin involved in the uptake of tricarboxylates. In all instances where previously published materials or materials submitted for publication are reproduced in this thesis, they represent the original research and writing of the author. A footnote at the beginning of each chapter clearly indicates the contribution of each author. Dr. R.E.W. Hancock has co-authored all of the publications listed above and his reading of the thesis will serve to verify this statement of authorship.  1 INTRODUCTION  2  1.1 P S E U D O M O N A S A E R U G I N O S A  2  1.2 ROOTS OY P S E U D O M O N A S A E R U G I N O S A VERSATILITY  2  1.3 T H E  7  P S E U D O M O N A S A E R U G I N O S A OUTER MEMBRANE  1.3.1 Outer membrane structure 1.3.2 Barrier function of the outer membrane 1.3.3 Uptake pathways through the outer membrane 1.3.3.1 Hydrophobic pathway 1.3.3.2 Self-promoted uptake of polycations 1.3.3.3 Porin pathway 1.4 M E T H O D S USED TO STUDY PORIN FUNCTION  1.4.1 Studies with intact cells 1.4.2 Model membrane systems 1.4.3 Structural studies 1.4.3.1 Structure and functional mechanism of general porins 1.4.3.2 Structural features unique to specific porins and their functional implications  7 9 11 11 11 12 14  14 15 17 17 21  1.5 P S E U D O M O N A S A E R U G I N O S A PORINS  22  1.5.1 General porins of P. aeruginosa 1.5.1.1 OprF 1.5.2 Specific porins of P. aeruginosa 1.5.2.1 OprB 1.5.2.2 OprP and OprO 1.5.2.3 OprD 1.5.2.4 OprD homologues  22 22 25 25 26 27 29  1.6 AIMS OF THIS STUDY  30  1.7 REFERENCES  32  1 * Components of this chapter have been published in Tamber, S. and R. E. W. Hancock. 2003. Front Biosci 8:S472-S483., Tamber, S. and R.E.W. Hancock. 2004. In J.L. Ramos (ed.) Pseudomonas, and Hancock, R. E. W. and S. Tamber. 2004. In R. Benz (ed.), Bacterial and Eukaryotic Porins.  1 INTRODUCTION 1.1 P S E U D O M O N A S  AERUGINOSA  Pseudomonas aeruginosa is a ubiquitous free-living bacterium belonging to the yproteobacterial group that is commonly found in terrestrial and aquatic environments throughout the world. This organism is noted for its ability to adapt to new surroundings and has been isolated from numerous man-made environments such as hot-tubs, saline solutions, medical instruments, sink faucets, dilute disinfectant solutions, various pharmaceuticals, and cosmetics (5, 22, 84, 87, 108). In addition, it has the ability to colonize and infect a variety of eukaryotic hosts including slime molds (Dictostylium ducreyii), worms {Ceanorhabitias elegans), fruit flies (Drosophilia melanogaster), plants (Arabidopsis  thaliana),  and immuno-compromised mice and humans (57, 81).  Clinically, P. aeruginosa has been isolated from various human tissues including; the dermis, eyes, ears, the urinary tract, the respiratory tract, the gastrointestinal tract, and the reproductive organs (56).  Indeed, its ability to thrive in the hospital setting and infect  patients undergoing treatments for burns, HIV/AIDS, cancer, cystic fibrosis, or other underlying conditions, has made P. aeruginosa the third most prevalent cause of nosocomial infections (69). Once these infections are established they cause significant damage to the host due to the array of toxins and virulence factors produced by this organism (56, 83).  Eradication of P. aeruginosa infections is difficult owing to its  intrinsic resistance to many classes of antibiotics (90). Thus, these infections are often chronic or recurring, and eventually fatal. 1.2 ROOTS OY P S E U D O M O N A S A E R U G I N O S A VERSATILITY  2  Gene duplication is a rapid means of generating functional diversity, whereby one copy of the gene retains its original function while the other one, through natural selection, adopts a new role (73). This mechanism seems to have been favoured by P. aeruginosa, as approximately 15% of its genes have at least one paralogue in the genome; nearly twice the amount found in organisms with comparably sized genomes. A large proportion of these gene families, as is discussed below, are involved in metabolism, transport, or regulatory activities (98). In addition to their distinct cellular activities, many paralogues, especially those that have recently evolved, may still share some functional redundancy with their parent gene.  The presence of these multiple  copies of related genes is advantageous in the event of a gene loss or mutation, as it ensures that the function of the compromised gene will not be completely lost. This fitness effect was recently demonstrated in yeast strains, wherein strains harbouring multiple copies of genes were able to out-compete haploid strains (25). The primary factor contributing to P. aeruginosa's success in adaptation to diverse niches is its metabolic and energetic versatility. This organism has the ability to respire both aerobically and anaerobically (in the presence of nitrate) and its nutritional requirements are minimal because it can use a large variety of organic compounds such as sugars, amino acids, alcohols, amines, small aromatic compounds, fatty acids, dicarboxylic acids and other organic acids as energy sources (97). These compounds are initially processed via specific metabolic pathways into intermediates that ultimately are funneled into the tricarboxylate acid (TCA) cycle (13). This convergence permits the use of a broad range of compounds without investing a considerable amount of cellular resources.  3  Approximately 10% of the genes encoded by the P. aeruginosa genome are involved in metabolism (98). These genes include those involved in central metabolic pathways such as the T C A cycle, the Entner-Doudoroff pathway, the pentose phosphate pathway, and gluconeogenesis. However, a far larger number are purportedly involved in the initial catabolic steps of many compounds.  For example, there are at least 25  probable acyl-CoA dehydrogenases and 16 probable enoyl-CoA hydratases that may be involved in the oxidation of fatty acids into acetyl-CoA and propionyl-CoA (24). The genome contains 221 probable oxidoreductase genes (including dehydrogenases, oxygenases, dioxygenases) which are likely to be involved in the initial conversion of small aromatic and other stable compounds into less stable, higher energy intermediates. In addition there are multiple copies of aldolases, amidases, hydratases, and transferases. Although many of these genes have only been identified based on homology and have yet to be studied in P. aeruginosa; their presence in the genome underlies the enormous catabolic potential of this organism. The metabolic capacity of P. aeruginosa is matched by its transport capabilities. Approximately 516 genes in this organism are involved in the transport of small molecules (Table 1.1).  A T P binding cassette (ABC) transport systems comprise the  largest family of cytoplasmic membrane transporters in P. aeruginosa, with 101 putative members. This family of transporters consists of high affinity uptake systems and efflux pumps that use A T P hydrolysis to drive transport.  Five A B C uptake systems in P.  aeruginosa have been experimentally characterized; PstABC (phosphate), GltBFGK (glucose), SpuDEFGH (spermidine), BraCDEFG (branched chain amino acids), and AotJMPQ (arginine, ornithine) (1, 38, 54, 66, 68).  There is strong bioinformatic  4  evidence implicating 26 other systems in nutrient uptake. Many of these systems are proposed to take up amino acids, peptides, and other nitrogenous compounds such as taurine and polyamines (86). TABLE  1.1 Transporter classes in P. aeruginosa  Transporter Class Approximate Number of Members Cytoplasmic membrane transporters 428 A B C transporters 132 Ion channels 19 Phosphotransferase systems 6 Secondary transporters 273 Unclassified transporters 4 Outer membrane transporters 88 General porins 3 Specific porins 28 Gated porins 32 Efflux channel tunnels 18 Type II secretion pores 4 Type III secretion pores 3 Total 516 numbers based on those determined in references (86) and (30)  a  a  Secondary transporters couple the energy derived from an ion or compound moving down its concentration gradient to the uphill movement of another ion or compound. There are a large number of secondary transport protein families that differ mainly with respect to substrate, coupling molecule, and/or direction of transport (75). The 273 secondary transporters of P. aeruginosa belong to 45 distinct families. Three uptake proteins of this class, the arginine/ornithine antiporter, ArcD, and the branched amino acid transporters, BraB and BraZ, have been characterized functionally in P. aeruginosa (39, 40, 105). O f the remaining transporters, there is strong bioinformatic evidence implicating 92 in solute uptake. The putative substrates for the majority of  5  these permeases include amino acids, small aromatic molecules, Kreb's cycle intermediates, di- and tricarboxylic acids, nucleosides, and inorganic ions (86). The outer membrane also boasts a large number of transport proteins.  This  membrane houses three major classes of import proteins. General porins are involved in the uptake of a variety of small compounds. Specific porins have specific binding sites which enable the passage of structurally related classes of molecules. Gated porins are large channels that utilize energy input from the TonB protein to open and permit bulky substrates such as iron-siderophore complexes and vitamin B12 to pass through (48). While the P. aeruginosa outer membrane has a paucity of general porins, it is noted for having a large number of specific porins that take up a variety of compounds including carbohydrates, amino acids, and inorganic anions (30).  A striking feature of the P.  aeruginosa outer membrane is the presence of three large families of paralogous transporters; the 18 member OprM family of efflux and type I secretion channel-tunnels, the TonB-dependant family of 32 gated porins, and the OprD family of specific porins which has 19 members.  As the majority of these homologues were identified upon the  sequencing of the P. aeruginosa genome, they have yet to be characterized functionally (98). The coordination of P. aeruginosa's solute uptake and metabolic systems is carried out by a correspondingly large number of regulatory loci.  One of every nine  genes in the genome encodes a probable transcriptional regulator. Quite a few of these regulatory proteins have demonstrated roles in metabolism and transport (15, 67, 88). Many more have been implicated in metabolism based on their homology to other known regulators (98), and/or their expression profiles in different Pseudomonas strains or  6  strains that were grown in varying environments (23). The use of global gene expression technologies and bioinformatics approaches has offered a large-scale overview of how P. aeruginosa can orchestrate its metabolism to suit particular environments. However, to fully understand the versatility of this organism, it will be necessary to determine the substrate specificities of its metabolic enzymes and transporters, how these systems are interrelated, and how they respond to environmental cues. 1.3 T H E P S E U D O M O N A S A E R U G I N O S A OUTER MEMBRANE  1.3.1 Outer membrane structure The asymmetric nature of the Gram-negative outer membrane makes it unusual among biological membranes and imparts this structure with many of its unique features (Figure 1.1). The inner leaflet is comprised of phospholipids similar in composition to those  of the  cytoplasmic membrane,  predominating species.  where  phophatidyl ethanolamine  is the  The outer leaflet contains few, i f any, of these same  phospholipids but is primarily composed of the glycolipid, lipopolysaccharide (LPS) (32). LPS is a complex molecule having a tripartite structure consisting of the endotoxic Lipid A component, the rough core oligosaccharide, and the antigenic Opolysaccharide. Lipid A comprises the majority of the hydrophobic external surface of the outer membrane and is highly conserved among Pseudomonas species.  This  molecule consists of a phosphorylated P-l 6-glucosamine disaccharide backbone that is ?  anchored to the outer membrane by 6 or 7 fatty acyl chains (45).  The core  oligosaccharide is covalently linked to the Lipid A molecule. The components of the core are common to all Pseudomonas species examined to date and include the unique  7  octose 2-keto-3-deoxyoctulosonic acid (KDO), glucose, galactosamine, rhamnose, L glycero-D-manno-heptose, as well as alanine and phosphate (32).  1.1 Gram-negative bacterial cell wall. The cell wall consists of the cytoplasmic membrane (CM), periplasm (PP) and outer membrane (OM). The outer leaflet of the outer membrane is primarily made up of LPS which is comprised of the Lipid A moiety (1), core oligosaccharide (2), and O-antigen (3). The two major porin types are also shown; general porins discriminate between solutes based on their physicochemical properties, and specific porins, such as the maltodextrin-specific LamB porin illustrated above, facilitate the diffusion of related classes of molecules by virtue of their substrate specific binding sites (inset). Once across the outer membrane solutes enter the cell through cytoplasmic membrane transport systems. FIGURE  Indeed, the Pseudomonas core region is rich in phosphate molecules, containing approximately twice the amount found in the Enterobacteriaceae  (64). It has been  suggested on immunological grounds that there are 4 core structures in P. aeruginosa but this may represent differential substitution or connectivity of the component sugars (78, 107). Capping off approximately 10% of the LPS molecules is the O-polysaccharide (32).  Because of its location on the cell surface, the O-polysaccharide is the major  antigen recognized by the immune system.  There has been considerable selective  8  pressure to generate variability in this structure as it is the most diverse region of the LPS molecule and varies with respect to sugar composition, linkage, sequence and branch length. Currently, there are 20 known O-serotypes of P. aeruginosa (78). A typical Pseudomonas O-polysaccharide chain is made up of 0 to 50 repeating units of 3 to 5 sugars consisting of such sugars of glucosamine, glucose, rhamnose, fucosamine and often amino hexuronic acids such as quinavosamine, 2-imidazolinomannuronic acid, and 2,3-diacetamido-2,3-dideoxyhexuronic acid (59).  The variability in the number of  oligosaccharide repeats of the O-antigen gives rise to the characteristic ladder-like banding pattern observed when LPS is resolved on acrylamide gels. Embedded within the phospholipid and LPS matrix of the P. aeruginosa outer membrane is a collection of approximately 160 outer membrane proteins. These proteins cover approximately 60% of the outer membrane surface (9) and carry out the important roles of this organelle including the selective transport of molecules into and out of the cell; interaction with the immune system, immune cells, molecules and surfaces in the environment; an ability to exclude and resist enzyme attack; cell structure and stability; and the anchoring of structures like pili and flagella. Most of these proteins form (3-barrel structures in the outer membrane (49). Many of the remaining are putative lipoproteins, some of which associate with the inner monolayer of the outer membrane and anchor it to the peptidoglycan (89).  1.3.2 Barrier function of the outer membrane Gram-negative bacteria tend to have a higher intrinsic resistance to many classes of antibiotics, dyes, detergents, and toxins than their Gram-positive counterparts (27, 65). This resistance is contingent on the permeability barrier presented by the outer  9  membrane, which works in conjunction with secondary resistance mechanisms such as efflux and inactivating enzymes. The lower rate of entry into the cell ensures that the secondary resistance mechanisms work efficiently, and are not overwhelmed by a high concentration of toxins (27). The basis of the outer membrane permeability defect lies in the unique properties of its component LPS and protein molecules. The presence of phosphate residues on the Lipid A moiety and in the core region imparts a strong negative charge to the cell surface, repelling many hydrophobic, anionic and neutral polar compounds. In addition, the LPS chains are stabilized in the membrane through the ionic cross-bridging action of the divalent cations, M g  2 +  and C a . The fluidity of this tight network of LPS molecules is 2+  much less than that of the cytoplasmic membrane, and thus, further restricts the diffusion of hydrophobic compounds (96). The extent of the outer membrane barrier function towards hydrophobic molecules is dependant on the length, as well as the stability of the LPS chains. Studies on a series of mutants defective in the synthesis of the core region demonstrated that these strains increasingly permitted the passage of hydrophobic antibiotics as the length of the LPS chains decreased (65). Also, treating the outer membrane with polycations or chelators, such as E D T A , destabilizes it by disrupting the electrostatic interactions bridging adjacent LPS molecules and therefore, makes it more permeable to large molecules, including hydrophobic antibiotics (see Section 1.3.3.2, (34)). The uptake of hydrophilic compounds through the outer membrane occurs primarily through water-filled channels called porins. These channels represent the major class of outer membrane proteins, usually present at a copy number of 10 (9). Despite 5  10  these high numbers, porins only offer transit to a limited subset of molecules. The exclusion limit of most porins, defined by the narrowest region of the pore, is 600 Da, approximately the size of a di- or tri-saccharide molecule (49). Further selectivity is achieved via electrostatic attraction to or repulsion of solute molecules by the amino acids lining the mouth of the pore (92). Some porins also may have substrate-specific binding sites that only facilitate the diffusion of a particular structural class of compounds.  1.3.3 Uptake pathways through the outer membrane 1.3.3.1 Hydrophobic pathway Hydrophobic compounds enter Gram-negative cells simply by diffusing through the outer membrane (62). Once through, many toxic compounds are recognized and excreted from the cell by active efflux systems; whereas others are retained and metabolized. Plesiat and Nikaido (80) reported that the outer membrane permeation rate of hydrophobic compounds is 100 fold less than through the cytoplasmic membrane. However, compared to the doubling times of organisms such as P. aeruginosa that can use aliphatic hydrocarbons as energy sources, the rate of influx of these compounds is relatively rapid and thus sufficient to support growth.  1.3.3.2 Self-promoted uptake of polycations Polycationic compounds, and possibly organic cations such as Tris, can promote their own uptake by disrupting the structural integrity of the outer membrane.  These  agents bind to LPS with high affinities and competitively displace the M g  2+  2 +  and C a  ions  that bridge adjacent LPS chains (64). The displacement of the divalent cations by these more bulky permeabilizing agents distorts the structure of the outer membrane barrier and  11  permits the passage of these and other molecules, normally excluded from Gram-negative bacteria (34, 53). Outer membrane permeabilization by divalent cation chelators, such as E D T A is believed to occur by a similar mechanism; with the exception that the M g Ca  2 +  and  are removed rather than displaced. Several  Gram-negative  enteric  and  pseudomonad  species  have evolved  mechanisms to resist the outer membrane damaging effects of polycations and chelators. When grown in the presence of these agents, or in environments where the divalent cation concentration is limiting, these bacteria modify their LPS by adding 4-amino-4deoxyarabinose residues to the Lipid A disaccharide (20, 58, 100). This modification presumably renders the outer membrane more stable in environments with low concentrations of divalent cations by decreasing the net negative charge of LPS. 1.3.3.3 Porin pathway The permeability of the outer membrane towards hydrophilic compounds is defined by its porins. As discussed in section 2, these channels contribute to the barrier function of the outer membrane; however, they also provide small polar compounds with their main route of entry into the cell. For example, at least 90% of the uptake of the cephalosporin antibiotic cephloridine occurs through porin channels (118). In addition, clinical isolates of antibiotic resistant Gram-negative pathogens are frequently deficient in one or more porin species, highlighting the important role of these proteins in antibiotic permeability (14, 82, 95). Porins can either be general or specific. The distinction arises from the types of molecules taken up.  A variety of structurally unrelated molecules can traverse general  porins providing that they are of the correct size, charge, and polarity. Specific porins, in  12  contrast have stereo-specific binding sites that facilitate the diffusion of structurally related classes of molecules. However, it should be noted that specific porins also permit the non-specific passage of structurally unrelated compounds, as long as they are smaller than the exclusion limit of the pore (18, 41). Transport through porins occurs by the process of simple diffusion wherein the uptake kinetics rely on the concentration gradient of the solute in question. In nutrientrich environments, this gradient is steep and the rapid influx of molecules is favored. As the nutrient supply decreases, the removal of solute from the periplasm via the high affinity  transporters  of the cytoplasmic membrane becomes more important in  maintaining the concentration differential and the subsequent high rates of uptake across the outer membrane. In environments where nutrients are limited (i.e. in the micromolar range), simple diffusion is no longer sufficient to saturate the transport systems of the cytoplasmic membrane, and thus, the permeability of the outer membrane is rate limiting for growth (37, 65). It is in these conditions that specific porins are necessary for growth. Relative to the rates observed with general porins, the rate of diffusion through specific porins is accelerated at low concentrations of solute. This facilitation occurs via the substrate-specific binding sites of the porin, which are believed to orient their respective substrates in energetically favorable positions as they traverse the channel (21, 93, 114). As the binding sites of specific porins are saturable, the increased rates of uptake are only observed in nutrient limited conditions. Altering the pore properties of a porin can greatly alter the permeability of the outer membrane towards polar compounds. For example, mutants expressing a porin engineered to have a greater channel diameter are more sensitive to various hydrophilic  13  antibiotics (42). As well, there have been several investigations wherein the charge or substrate selectivity of a porin has been altered by changing the nature of the amino acids lining the pore mouth, eyelet, or comprising the substrate binding site (6, 99, 103). More telling, however, is the comparison of the porin profiles of E. coli and P. aeruginosa. The outer membrane of E. coli is rich in general porins that permit the passage of a variety of structurally unrelated compounds and has a few specific porins that are primarily involved in the uptake of large, bulky substrates (i.e. maltodextrins, nucleosides) (63). P. aeruginosa, in contrast, possesses one major general porin, OprF, and a large number of specific porins. The channels formed by OprF are inefficient, and therefore, most polar compounds are thought to primarily enter the cell via specific porins (30). This difference in porin composition is associated with a greatly reduced outer membrane permeability of P. aeruginosa, which, depending on the compound, is 10 to 500 times lower than that of E. coli (27, 116). 1.4 M E T H O D S USED TO STUDY PORIN FUNCTION  1.4.1 Studies with intact cells Studies of outer membrane permeability in intact cells are commonly used to investigate the physiological roles of porins. The methods described below are especially useful for the comparative analysis of uptake by isogenic strains that either lack one or more porin species, or harbour mutated porin channels.  However, many of these  experiments are indirect, and consequently any functional assignments made must be confirmed by more specific alternative methods. Transport through the outer membrane is assessed by following the uptake of hydrophilic compounds into the cell. Compounds must be labelled in order to follow their transport directly. However, the transport  14  process can be inferred by following the growth of cells on unlabelled compounds (116). Methods also exist to determine the uptake of antibiotics (117), and substrates of periplasmic enzymes (116). The use of any of these methods requires that the outer membrane permeability is the limiting step in these processes. This condition is achieved in nutrient poor environments, where the enzymes of the periplasm and the transporters of the inner membrane are relatively unsaturated. 1.4.2 Model membrane systems A variety of model membrane systems have been developed to complement the in vivo approaches discussed above. These methods investigate the diffusion properties of purified porins that have been reconstituted into liposomes or planar bilayers and produce insights regarding porin architecture and mechanism of action. Proteoliposome based approaches were among the first to be used to demonstrate the channel activity of the first named porin (61).  That particular assay involved the encapsulation of radiolabeled  sugars within the liposome and the subsequent detection of their efflux through the porin. Another type of assay measures the rate of substrate uptake into proteoliposomes containing hydrolytic enzymes (28). A third method, the liposome swelling assay, takes advantage of the reduced light scattering properties of swollen vesicles (55). Basically, the proteoliposomes are placed in isotonic buffer solutions containing various solutes. Solutes that can enter the vesicle via its porins will be accompanied by water in order to maintain the osmotic equilibrium; the influx of water causes the liposomes to swell which leads to a corresponding decrease in absorbance. Although these assays are conceptually simple and have been used extensively to identify porin activity and estimate their channel sizes, they are limited to the investigation of radiolabeled compounds, substrates  15  of hydrolytic enzymes, and uncharged compounds. In addition, these assays suffer from a number of drawbacks that often complicate data interpretation (28). Planar or black lipid bilayer systems are used to study the movement of charged compounds through porins (28).  The basic planar bilayer apparatus consists of a  rectangular Teflon cell that is divided into two chambers by a Teflon wall that has a small hole in its centre. A n electrode is placed in each of the chambers; one of the electrodes is connected to a voltage source, the other to a current amplifier (Figure 1.2). Channel activity is monitored on an oscillator and recorded using a chart recorder. The chambers are filled with a buffered salt solution and a bilayer is formed by painting a solution of lipids dissolved in decane on either side of the hole. As the solvent evaporates the lipid film thins out and forms a bilayer that is optically black and impermeable to ions. Porins, when added to one of the chambers, spontaneously insert into the membrane rendering it permeable to ions. This permeability is monitored as step-wise increases in electrical conductance, with each conductance step corresponding to a single channel insertion event. A wealth of information can be gleaned from these studies, including an estimate of the channel size (measured in Siemans or Ohms" ), as well as its ion selectivity, and 1  heterogeneity. The presence of substrate specific binding sites in specific porins can also be determined using this methodology.  16  B.  A. Voltmeter  Teflon cell  Current amplifier  Oscilloscope  1.2 Planar bilayer apparatus. A . Dual chambered Teflon cell. Porins spontaneously insert into the membrane bilayer which separates the two compartments. As they do, ions of the bathing solution pass through the porin causing an increase in conductance. B. Single channel experiment. The increase in conductance is monitored on an oscilloscope. Each step-wise increase corresponds to one channel insertion event. C. Macroscopic inhibiton experiment. The increase in conductance is monitored using an electrometer. The conductance increases exponentially as the channels insert into the bilayer. Once this increase plateaus, concentrated solutions of substrate are added to the Teflon cell (arrows). Should the substrate bind to the channel, a decrease in conductance will be observed. FIGURE  1.4.3 Structural studies 1.4.3.1 Structure and functional mechanism of general porins The  crystal structures of several general porins from diverse bacterial species  have been solved (Figure 1.3). These data, in conjunction with those obtained by genetic and biochemical means have revealed the probable mechanism by which these channels function. Despite the considerable sequence divergence among them, the structures of the general porins solved to date share a related three-dimensional fold (16, 52).  17  Three dimensional porin structures of E. coli. A . The general OmpF porin. B. The maltodextrins-specific LamB porin. C. The nucleoside-specific porin Tsx. The top panel depicts the side view of the channels and the bottom panel depicts the top view of the pores. Beta stransds are labeled in blue and the alpha helices in red. FIGURE 1.3  The quintessential OmpF porin molecule is composed of three 16-stranded P-sheet cylinders or barrels (one of which is shown in Figure 1.3 A). The membrane-spanning P strands are amphipathic; the hydrophobic amino acid side chains are oriented towards the membrane while the polar amino acid side chains line the channel interior producing a hydrophilic environment. The terminal residues of the P-strands are predominantly aromatic. These aromatic amino acids form a ring around the exterior, which is believed to anchor and stabilize the porin within the membrane. At the periplasmic face, the Pstrands are connected by seven short turns ranging from 1 to 12 amino acids in length and at the extracellular surface by eight hydrophilic loops ranging from 2 to 46 amino acids in length (49). These loops are the most variable structures among porin molecules and confer on the channels many of their unique properties.  Some loops (e.g. L2) are  18  involved in stabilizing the porin trimer by folding away from the channel and interacting with LPS, or other porin monomers (77). Others fold over the mouth of the channel and partially cover the entrance.  The longest loop, L3, folds into the channel interior, and  there forms part of the pore eyelet, the narrowest region of the channel. Examination of the general porin interior reveals the mechanism by which these seemingly simple channels permit the rapid diffusion of selected molecules. A vertical cross-section of the pore resembles an hourglass with three main regions; the mouth, the eyelet, and the exit. The mouth acts as a coarse filter, its opening is obscured by the extracellular loops that fold over it and thereby prevent the entry of many large solutes. A large proportion of the amino acids in the vicinity of the mouth are charged, which contributes to the charge selectivity of the channel (92). The mouth opens up to a large chamber, the primary function of which is thought to present the eyelet with a high concentration of pre-screened solutes, thus ensuring a high rate of transport. The amino acids within this chamber exhibit charge segregation whereby positive residues line one side of the channel and negative residues, mostly from L 3 , line the other (Figure 1.4). This unusual distribution of amino acids creates a transverse electric field that increases in strength as the channel narrows and approaches the eyelet (44).  In addition to  preventing the entry of non-polar molecules, this local electric field is thought to orient charged compounds as they traverse the channel such that random tumbling is reduced and high rates of diffusion are maintained.  At the eyelet, solutes undergo their final  selection before entering the cell. The diameter of the pore is narrowed to a minimum of 7 A by the extracellular loop, L3.  19  1.4 Charge segregation within the eyelet of the E. coli OmpF general pore. Oxygen atoms are labeled red, and nitrogen atoms are labeled blue. FIGURE  This loop also contributes a number of acidic amino acids to the region which, in conjunction with the basic residues of the channel wall, increase the local field strength to approximately 20 times as that of the mouth (92). This strong potential causes the amino acid side chains to extend fully, constricting the pore further and giving it a rigid structure. Molecules that are larger than the pore diameter, or do not form the adequate charge-charge interactions, are prevented from crossing the channel any further.  This  selectivity can be changed by altering the amino acid composition of the eyelet. For example, the ion selectivity can be modified by replacing a charged residue with one that is oppositely charged (6, 99). Similarly, substitution of the longer charged amino acids with shorter neutral ones allows larger molecules to traverse the channel (46).  The  diffusion of molecules through the exit region is relatively unhindered. This area is wider and contains fewer charged residues than the external mouth. Also, the periplasmic turns are almost flush with the inner leaflet of the outer membrane. Together, these features are believed to ensure that solutes, once past the eyelet, can enter the cell with the greatest of ease. 20  1.4.3.2 Structural features unique to specific porins and their functional implications The crystal structures of three specific porins have been solved to date; the E. coli maltodextriris-specific porin, LamB, the S. enterica serovar Typhimurium sucrosespecific porin, ScrY, and the E. coli nucleoside-specific porin, Tsx (21, 93, 114). Overall, the tertiary structure of these channels resembles that of the general porins; however, there are several differences that impact on the functional mechanism of specific porins. In contrast to general porins, both the LamB and ScrY trimers consist of 18stranded p-barrels. The channel entrance is restricted by three extracellular loops, L I , L3, and L6; however, L3 alone constricts the pore eyelet to a minimum of 5 A for LamB and 8.5 A for ScrY. The loop regions of the two sugar-selective porins contain a large number of aromatic amino acids (16 per monomer for LamB, 10 for ScrY, compared with 2 for OmpF), over half of which are located in the novel 9  th  extracellular loop. It is  believed that these aromatic residues may comprise the initial, low affinity adsorption sites that direct sugar molecules into the channel mouth. This hypothesis is supported by the observation that deletions in L9 of LamB impair maltose binding and import (47). Once in the channel, the disaccharide molecules are oriented by 6 contiguous aromatic amino acids, termed the greasy slide, that stretch from the external chamber down to the periplasmic exit (inset in Figure 1.1). As its name implies, the greasy slide forms the path down which the pyranose rings travel. The hydroxyl moieties of the substrate sugars are stabilized by the large number of ionizable residues that line the channel wall (104).  21  The proposed functional mechanism of nucleoside transport through Tsx is similar to that of the sugar-specific porins, however, its structure is markedly different (114). Tsx is a monomeric protein composed of a 12-stranded P-barrel (Figure 1.3). The barrel is not obstructed by any extracellular loops but the 6 (3-strands of its front side fold inward toward the membrane such that the barrel adopts the shape of a flattened cylinder and the pore adopts a keyhole like conformation. Rather than possessing an eyelet, the Tsx  channel is narrow almost throughout its entire length; the width of the narrower  portion of the keyhole is 3-5 A and that of the wider portion is 7-8 A . The loops and channel interior contain a number of aromatic amino acids that are involved in forming stacking interactions and van der Waals contacts with the base and sugar moieties of the nucleoside. As with disaccharide transport in LamB and ScrY, there is a binding site in the Tsx loop region that is proposed to be a weak affinity site that directs nucleosides into the pore. Once inside the channel, nucleosides are believed to be oriented such that the base is located in the narrow region of the keyhole and the sugar residues in the wider part. Transport of the solutes may occur by successive binding to two additional sites that collectively make up a 5-membered greasy slide (114). The hydroxyl groups of the nucleoside are thought to be stabilized through the formation of hydrogen bonds with the charged amino acids found throughout the porin. Thus, instead of segregated charges, specific porins have binding sites that facilitate diffusion through the continual formation and disruption of contacts within the channel interior. 1.5 P S E U D O M O N A S A E R U G I N O S A PORINS  1.5.1 General porins of P. aeruginosa  1.5.1.1 OprF  22  OprF is the major outer membrane protein of P. aeruginosa, usually present in excess of 2 x 10 copies per cell (30). The structure of OprF is unlike that of the general 5  porins of other Gram-negative bacteria.  Crosslinking studies indicate that it is an  oligomer, possibly a trimer that is associated with both LPS and peptidoglycan (3). The protein consists of three domains; the N-terminus, a hinge region, and the C-terminus. A three dimensional model of the N-terminus of OprF (the first 160 amino acids) was constructed based on the structure of the homologous protein, OmpA from E. coli and indicated that it is an eight stranded P-barrel (11). The proposed hinge from amino acids 161 to 209 joins the C- and N-termini. This region contains multiple proline-alanine repeats and has two disulfide bonds (30). It has been proposed that analogous to the Cterminus of OmpA, the corresponding region of OprF (amino acids 210 to 326) may be a globular domain that lies in the periplasm because it is involved in peptidoglycan binding. However, there is considerable evidence suggesting that this region, or a portion of it, is surface-exposed.  First, bioinformatic evidence strongly suggests that the C-  terminus contains two membrane spanning P-strands (29). Second, this region contains a cleavage site accessible to extracellular proteases and is highly antigenic (17, 43, 60, 106). Indeed, the C-terminus of OprF is regarded as a promising candidate for use in a P. aeruginosa vaccine (35). Planar bilayer methods have shown that P. aeruginosa OprF is a non-specific, weakly cation-selective channel with one of two channel sizes. The channels can be small (0.36 ns) or relatively large (2-5 ns) (8). The large channel size was confirmed by intact cell studies in which P. aeruginosa, normally unable to grow on di- and trisaccharides was provided with a raffinose metabolism system.  Comparisons of the growth and  23  plasmolysis rates between the resulting strain and an isogenic oprF deficient mutant demonstrated that OprF was involved in the uptake of large sugars (8). Despite this data, the existence of the large channel was in apparent contradiction with the measured low permeability of the Pseudomonas outer membrane, and was a controversial matter for many years.  The controversy was resolved by the demonstration that only a small  proportion of OprF channels (approximately 400 out of 200,000) (11) form the large size channels and that outer membrane permeability of smaller molecules (~ 200 Da, the size of most P. aeruginosa substrates) is largely managed by lower abundance specific porins such as OprD and OprB (30). Interestingly, the full length OprF protein is required for large pore formation. Mutants with C-terminal truncations in OprF only form the smaller sized pores (11), suggesting that OprF adopts a different conformation for the larger channel size that probably involves the translocation of the C-terminal half from the periplasm to cell surface. It has been proposed that the alternative disulfide bonding of the four cysteine residues of the hinge region may also be involved in adopting the two separate channel forms (7). In addition to its porin function, OprF performs a number of other important roles related to cell structure and pathogenesis.  P. aeruginosa mutants deficient in OprF  synthesis have an almost spherical appearance, are shorter than wild-type cells and do not grow in low osmolarity medium (109) suggesting OprF is involved in the maintenance of cell shape. Analysis of OprF mutants with truncated C-termini showed that this region may be involved in the above structural roles, as well as binding to peptidoglycan (85). As mentioned previously, OprF is a potent antigen and antibodies to the porin have been  24  isolated from the lungs of chronically infected patients (115). As well, OprF is involved in adhesion to cultured lung epithelial cells and to other P. aeruginosa cells in the form of anaerobic biofilms (4, 36). OprF has also been shown to bind to the human cytokine, interferon gamma (110).  As a result of this binding, the expression of a number of  virulence factors is induced through the RhIIR quorum sensing system. It is not clear whether OprF plays other roles in infection. Clinical isolates that lack OprF and are resistant to multiple antibiotics have been isolated (12). However, the role of OprF in antibiotic uptake has not been conclusively demonstrated to date.  1.5.2 Specific porins of P. aeruginosa  1.5.2.1 OprB OprB was first identified as a glucose uptake porin based on its induction when P. aeruginosa was grown in glucose containing media. It is co-regulated with a periplasmic glucose binding protein of an A B C transporter and therefore, forms part of the high affinity glucose uptake system in P. aeruginosa.  Subsequent work with interposon  mutants expanded the role of this porin to include the uptake of other simple carbohydrates such as mannitol, fructose and glycerol (112). Circular dichroism spectroscopy of P. aeruginosa OprB indicates that the betasheet content of this protein is 40%, which is in agreement with the beta-sheet content of other porins (111). Also, modelling of OprB to the structure of the homologous LamB maltodextrins specific porin of E. coli, suggests that like LamB, OprB monomers have 18 transmembrane P-strands. In addition, OprB has a group of aromatic residues that cluster into a motif resembling the greasy slide of LamB (30, 93).  25  OprB is positively regulated in the presence of its substrates glucose, fructose, glycerol, and mannitol (2). In glucose, this induction is mediated via a two component regulatory system (91). Salicylate, a compound released by plants upon infection, and citric acid cycle intermediates such as succinate repress the expression of OprB. The effects of pH and temperature on OprB expression were also examined by Adewoye et al. (2). They found that OprB is expressed optimally at pH 7 and that expression increases with temperature up to 42°C. P. aeruginosa has one OprB paralogue, OpbA (PA2291) that is 96% identical to OprB at the amino acid level. The opbA gene lies directly downstream of a glucose dehydrogenase gene, which is involved in the low affinity glucose uptake pathway (26). Thus it may be possible that P. aeruginosa uses two porins for glucose uptake: OprB at low external glucose concentrations and OpbA at higher glucose concentrations.  1.5.2.2 OprP and OprO OprP is the anion-selective, phosphate uptake porin of P. aeruginosa. Unlike OprB, which is induced by its substrate, OprP was first identified based on its induction in phosphate-limiting conditions (33). This regulation is mediated by the PhoB regulator which also controls the expression of a periplasmic phosphate binding protein (29). Studies with a transposon mutant lacking oprP confirmed that in P. aeruginosa, OprP is involved in high affinity phosphate uptake (99). Studies of OprP in a black lipid bilayer model membrane system showed that when phosphate is thought to be bound to the inside of the channel, the conductance of chloride ions through it decreases (10). In an attempt to identify the phosphate-binding site in OprP, the lysines in the N-terminal half of the protein were systematically changed  26  to glutamates by site-directed mutagenesis. Three lysine residues were found to be important in phosphate binding. In the KI21 mutant, phosphate binding, as measured by CI" conductance, was greatly reduced. In the K74 and K126 mutants, phosphate binding was reduced slightly (99). A topology model of OprP, generated by molecular modeling and insertion mutagenesis (10), predicts that both K121 and K126 are in loop 3 and K74 is found in the fourth beta-strand. It is proposed that K121 forms the major phosphate binding site of OprP, while both K74 and K126 are secondary binding sites. The oprO gene was first identified as an open reading frame immediately upstream of oprP. The two proteins are homologous, sharing 76% identity. Like OprP, OprO is an anion-selective channel, but preferentially takes up pyrophosphate as opposed to phosphate. OprO expression requires that the cells be in stationary phase in addition to being in phosphate limiting conditions (94). 1.5.2.3 OprD Interest in OprD began when it was noticed that several clinical P. aeruginosa isolates resistant to the carbapenem antibiotic imipenem, were missing this outer membrane protein (82).  Subsequent work using OprD deficient mutants and model  membrane systems demonstrated that the channel was specific for carbapenem antibiotics (101). Later work showed that the structural analogues arginine, lysine and dipeptides containing either residue could competitively inhibit the passage of imipenem, suggesting that these amino acids were the physiological substrates of OprD (Figure 1.5, (102)). However, OprD can also act as a general porin as it permits the non-specific passage of small sugars and their derivatives such as gluconate (41).  27  OprD shares 15% amino acid sequence similarity with OmpF of E. coli. Alignment of these two proteins suggested that the OprD monomer consists of a 16stranded P-barrel. This model was tested and refined by constructing OprD variants with 4 to 8 amino acid deletions in the putative loop regions (42, 70). Further analysis of the deletion mutants has shown that both loops 2 and 3 are involved in substrate binding (70). Loop 7 may also have a role in substrate binding since the OprD proteins of clinical isolates resistant to meropenem have several amino acid substitutions and a two amino acid deletion in this region (19). Mutants with deletions in loops 5, 7, and 8 have larger channels and permit the passage of multiple antibiotics (42). Therefore, it is likely that these regions fold to constrict the channel opening. In contrast to all of the other porins studied to date, the C-terminal amino acid of OprD is a leucine rather than an aromatic residue, but how this may contribute to the overall tertiary structure or folding remains to be determined. OH  0  o  Histidine  Imipenem  Lysine  0 Meropenem  O NH Arginine  FIGURE  OH OH OH OH Gluconate  1.5 Structures of the substrates taken up by OprD.  28  Uptake through OprD is a highly controlled process as evidenced by the complex regulation of this protein. It is induced by its substrate arginine via the ArgR regulator and the amino acids alanine and glutamate independently of ArgR (71). In the presence of succinate, OprD expression is repressed, presumably by the Crc catabolite repression control protein (72).  Although carbapenem resistant clinical isolates often lack or  express lower levels of OprD, it is unclear how this regulation is brought about (51). Two regulatory systems that may be involved have been described. The first involves the MexT regulator which represses OprD levels concomitantly with the induction of the mexEF-oprN efflux operon (50). The second mechanism exerts its effects indirectly by decreasing OprD transcription in response to micromolar concentrations of zinc, which is commonly released from urinary catheters (76). Aromatic compounds such as salicylate and benzoate also downregulate OprD, however the regulator(s) involved remain(s) to be identified (72). 1.5.2.4 OprD homologues Prior to the release of the P. aeruginosa genome sequence, two OprD homologues, OprE (113) and OprQ (74), were identified in this organism. Sixteen other homologues have since been discovered, giving the OprD family in P. aeruginosa 19 members. The discovery of this family considerably extended our view of the capacity and complexity of outer membrane permeability. It is now believed that the novel OprD homologues likely give P. aeruginosa a competitive edge by enabling the selective uptake of many different metabolites in a variety of environmental conditions. Interestingly, the supposed OprD proteins from P. aeruginosa strains grown in different conditions often demonstrated different migration patterns on SDS-PAGE gels (Figure  29  1.6).  Although the molecular weight of OprD is quite variable among different P.  aeruginosa strains and isolates (79), my research indicates that those aberrantly migrating proteins were most likely OprD homologues, suggesting that these different porins are differentially regulated. Indeed, the continued study of this family will lead to insights concerning the molecular evolution of these porins as well as their contribution to outer membrane permeability. oprD' 1  2  3  4  5  6  7  FIGURE 1.6 Expression of OprD and OprD homologues in diverse carbon sources.  Outer membranes were prepared from stationary phase cultures of P. aeruginosa and 20 pg protein were resolved on an 11% SDS-PAGE gel. Lane 1 - wild-type P. aeruginosa grown in arginine. A l l other outer membranes were isolated from an oprD' mutant; lane 2 - glycine-glutamate, lane 3 - tyrosine, lane 4 - vanillate, lane 5 - cw-aconitate, lane 6 pyroglutamate, lane 7 - glucose. The locations of OprB, OprD, OprE, OprF, OprG, and the lipoprotein OprL are shown (31).  1.6 AIMS OF THIS STUDY  The primary objective of this study was to investigate the contribution of the OprD family to the physiology of P. aeruginosa.  It was hypothesized that the  maintenance of OprD and its 18 homologues in the genome of organism served a dual purpose. First, as a consequence of their divergence from OprD both in sequence and in  30  function, these porins specialized in the diffusion of specific compounds, enabling P. aeruginosa to take up a variety of compounds without compromising the integrity of the outer membrane. Secondly, as the members of this family had still retained considerable amino acid sequence similarity (46-57% similar to OprD); it was believed that some homologues may continue to share overlapping activity allowing them to compensate for a loss or mutation of a related protein. 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Science 309:774-7. Wylie, J. L., C. Bernegger-Egli, J. D. O'Neil, and E. A. Worobec. 1993. Biophysical characterization of OprB, a glucose-inducible porin of Pseudomonas aeruginosa. J Bioenerg Biomembr 25:547-56. Wylie, J. L., and E. A. Worobec. 1995. The OprB porin plays a central role in carbohydrate uptake in Pseudomonas aeruginosa. J Bacteriol 177:3021-6. Yamano, Y., T. Nishikawa, and Y. Komatsu. 1993. Cloning and nucleotide sequence of anaerobically induced porin protein E l (OprE) of Pseudomonas aeruginosa P A O l . Mol Microbiol 8:993-1004. Ye, J., and B. van den Berg. 2004. Crystal structure of the bacterial nucleoside transporter Tsx. Embo J 23:3187-95. Yoon, S. S., R. F. Hennigan, G. M. Hilliard, U. A. Ochsner, K. Parvatiyar, M. C. Kamani, H. L. Allen, T. R. DeKievit, P. R. Gardner, U. Schwab, J. J. Rowe, B. H. Iglewski, T. R. McDermott, R. P. Mason, D. J. Wozniak, R. E. W. Hancock, M. R. Parsek, T. L. Noah, R. C. Boucher, and D. J. Hassett. 2002. 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Antimicrob Agents Chemother 12:368-72.  40  2 ROLE OF T H EOPRD FAMILY OF PORINS IN NUTRIENT UPTAKE  42  2.1 INTRODUCTION  42  2.2 M A T E R I A L S AND M E T H O D S  44  2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6  Bacterial strains, plasmids, primers, and media Genetic manipulations Sequence analysis Reporter gene assays Growth assays Semi-quantitative (SQ)-PCR  44 47 48 48 48 49  ;  2.3 RESULTS  2.3.1 2.3.2 2.3.3 2.3.4 2.3.5  50  A novel family of specific porins in P. aeruginosa Involvement of OprD in arginine uptake Involvement of OpdK in vanillate uptake Growth phenotypes of the other OprD homologues Induction of oprD homologues by their substrates  50 52 59 63 84  ;  2.4 DISCUSSION  86  2.5 R E F E R E N C E S  92  ;  41  *This chapter was largely published as T a m b e r , S., M . M . O c h s , a n d R. E . W . H a n c o c k . 2006. Role of the novel OprD family of porins in nutrient uptake in Pseudomonas aeruginosa. J Bacteriol 188:45-54. Martina M . Ochs constructed the opdK::xylE-Gm transcriptional fusion and performed CDO assays with this strain using the last 12 compounds listed in Table 2.5. R.E.W. Hancock edited this manuscript. r  2 ROLE OF T H EOPRD FAMILY OF PORINS IN NUTRIENT  UPTAKE  2.1 INTRODUCTION  Pseudomonas aeruginosa is an extremely versatile organism that grows in many diverse habitats such as terrestrial, marine, and fresh water environments. It is also capable of forming intimate associations with plants and animals.  Consistent with this ecological diversity, P.  aeruginosa can utilize a wide variety of compounds such as carboxylates, small aromatic compounds, and amino acids and amino acid derivatives as carbon sources (33). P. aeruginosa is noted for having 10 to 500 fold lower outer membrane permeability to various compounds in comparison with Escherichia coli, a feature that is central to its high intrinsic resistance to antimicrobials (7). While this decreased permeability permits the organism to resist attack by antibiotics produced by competitors in the environment, it creates potential problems for substrate uptake at low concentrations. In general, hydrophilic molecules traverse the outer membrane barrier via water-filled channels called porins. There are two major classes of porins, general and specific, differing primarily with respect to substrate selectivity. Many Gram-negative species, with the exception of the pseudomonads, possess general porins in high copy numbers that provide an aqueous environment through which small, structurally unrelated, hydrophilic molecules can diffuse. The kinetics of diffusion through general porins is largely driven by the physicochemical properties (i.e. size, shape, charge, polarity) and concentration gradient of the molecule in question (see Section 1.4.3.1). Specific porins, in contrast, are thought to possess stereo-specific substrate binding sites that serve to facilitate the diffusion of structurally related classes of molecules by orienting them into energetically favourable positions as they cross the narrowest regions of the channel (31,  42  42). This accelerated uptake is necessary in nutrient deficient conditions when uptake through the outer membrane becomes rate-limiting for growth and simple diffusion through general porins becomes inefficient.  The presence of binding sites in specific porins does not preclude  the entry of structurally unrelated compounds however, and thus these channels may also display some characteristics common to general porins (12). Specific porins are often induced to higher copy number by their substrates. Unlike other Gram-negative organisms, such as Escherichia coli, which possess a large number of general porins and a few specific ones, Pseudomonas sp. and other closely related soil organisms are unique in that they almost exclusively use specific porins for uptake through the outer membrane.  The major general porin of P. aeruginosa, OprF, for example, forms an  inefficient uptake pathway, whereby the majority of the OprF channels are too small to allow sufficient passage of molecules (7, 8). The specific porins of P. aeruginosa characterized to date include the glucose specific porin OprB (outer membrane protein B), the phosphate and polyphosphate specific porins, OprP and OprO, and the basic amino acid specific porin, OprD, which also takes up the structurally related antibiotics, imipenem and meropenem (37, 38). In addition, OprD is the prototype of a large paralogous family of porins (34). There are 18 OprD homologues in P. aeruginosa with primary amino acid sequences that are 46 to 57% similar to that of OprD. Members of this family are also found in other Pseudomonas species and closely related soil bacteria, such as Acinetobacter strain ADP1, as well as more divergent bacterial species, such as Escherichia coli (4), Shigella flexneri (14), and Yersinia pestis (26) (Table 2.1). A few OprD homologues have been implicated in nutrient uptake based on their placement in operons containing genes for the metabolism and transport of certain growth substrates.  For  43  example, the gene encoding the putative vanillate uptake porin VanP, in Acinetobacter,  lies in  the middle of the vanillate degradation operon (17). The benF and phaK putative porin genes of Pseudomonas putida (18) lie in the middle of operons responsible for the degradation of benzoate and phenylacetic acid, respectively. In addition, a number of OprD homologues have been observed during the investigation of global gene expression patterns of Pseudomonas grown under specific conditions (2, 5, 16, 39, 40). In this study, we hypothesize that in P. aeruginosa,  the OprD family members are expressed under specific conditions and are  responsible for the uptake of a variety of metabolites. The substrate selectivities of 7 of the 18 novel OprD homologues are described and discussed in the context of the phylogenetic framework of this family. T A B L E 2.1 O p r D homologues i n  Organism  P. aeruginosa a n d other proteobacteria Protobacterial group  OprD homologues P. aeruginosa P A O l 19 Y P. putida KT2440 21 T P.fluorescens PfO-1 14 y P. syringae pv tomato strain DC3000 10 y Azotobacter vinelandii 29 y Acinetobacter strain ADP1 4 y Burkholderia cepacia 3 P B. pseudomallei 1 P Xanthomonas campestris 1 y Bradyrhizobium japonicum U S D A 110 a 1 Shigella flexerni 1 y Yersinia species 3 y Escherichia coli 1 y Salmonella species 1 y percent similarity at the level of the primary amino acid sequence based on the scoring matrix (11)  Similarity to OprD 46-59% 44-63% 47-74% 45-74% 43-54% 40-50% 46-48% 49% 46% 33% 36% 34% 37% 38% B L O S U M 62 a  2.2 MATERIALS AND M E T H O D S  2.2.1 B a c t e r i a l strains, plasmids, p r i m e r s , a n d media  44  A l l strains and plasmids used in this study are listed in Table 2.2; the sequences of the D N A primers used are listed in Table 2.3. Strains were maintained on Luria Bertani agar. Antibiotics for either selection or maintenance were supplied at the following concentrations: ampicillin, 100 pg/mL for E. coli; carbenicillin, 300 pg/mL for P. aeruginosa, gentamicin, 10 pg/mL for P. aeruginosa, and tetracycline, 100 pg/mL for P. aeruginosa. TABLE  P. aeruginosa strains H103 H846 H859 PAOl PAK oprD oprE oprQ opdB opdC opdD opdF opdG opdH opdl opdJ opdK opdL opdN opdO opdP opdQ opdR opdT E. coli strains DH5a S17-1 Plasmids pCR2.1 pEXIOOT pX1918GT  2.2 Bacterial strains and plasmids used in this study Description wild-type strain HI03 oprD::xylE-Gm HI03 opdK::xylE-Gm wild-type strain wild-type strain P A O oprD: :miniTn5-Tc • P A K oprE::mmiTn5-Tc P A K oprQ::mimTn5-Tc P A O opdB::mmiTn5-Tc P A K opdC: :miniTn5-Tc P A K o/?dD::miniTn5-Tc P A K op<iF::miniTn5-Tc P A K opdG: :miniTn5-Tc P A K opdH: :miniTn5-Tc P A K o/?d/::miniTn5-Tc P A K opdJ::mimTn5-Tc P A K oj!?^::miniTn5-Tc P A K opJI::miniTn5-Tc P A K o/?<#V::miniTn5-Tc P A K opJO::miniTn5-Tc P A K opdP: :miniTn5-Tc P A K o/?dg::miniTn5-Tc P A K opc//?::miniTn5-Tc P A K opdT::mmiTn5-Tc  Reference or source (22) This study Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp. Pathogenesis Corp Pathogenesis Corp Pathogenesis Corp Pathogenesis Corp Pathogenesis Corp  T  r  r  T  r  T  r  r  r  r r  r  T  r  r  r  r  r  r  r  T  General cloning strain Mobilizing strain  (6) (25)  T A cloning vector, A p , K m suicide vector containing sacB gene, A p source of xylE-Gm cassette, A p , G m r  r  r  r  r  r  Invitrogen (32) (32)  45  TABLE  Primer opdK gene F opdK gene R PA4897 real-time F PA4897 real-time R pPA4899 real-time F pPA4899 real-time R pPA4900 real-time F pPA4900 real-time R pmdlC real-time F pmdlC real-time R mdlC real-time F mdlC real-time R PA4902 real-time F PA4902 real-time R PA4903 real-time F PA4903 real-time R PA4911 real-time F PA4911 real-time R PA4915 real-time F PA4915 real-time R oprD real-time F oprD real-time R opdB real-time F opdB real-time R opdC real-time F opdC real-time R opdJ real-time F opdJ real-time R opdP real-time F opdP real-time R opdT real-time F opdT real-time R opdD real-time F opdD real-time R opdH real-time F opdH real-time R opdK real-time F opdK real-time R opdO real-time F opdO real-time R rpsL real-time F rpsL real-time R  2.3 Oligonucleotide sequences used in this study  Sequence (5'- 3') GGCCATCGAGGAATTCACCACC ATCGTATTCGAAGCAGAACCTCACCGACCGACCGCTAT AGCTGGCCTACTTCAACAACACC ATCTGGCCCGGATCGTAATAG TTTCAGGGACGACCTAGCC TCACCACCGACCACTGGATCA GATCGCCTGCATCCACCTG GGTCGGTATAACGGGAATCGC TAATAGTTGCGCAAGACCAGGTC AATCCGTCGTAAAACCGCAGTC CCAACGCCTGGTACTCCCATAG GCTCGTGGCTCCATTTCACC ATCTCAACCTGATCCGCACCTTC GCGTAGCTCACCGATGGCTG CTCGCCCGGTTGCTTTGC TCGGCGTCCAGCTCATGAT TGCCGATCCACGAGTTCCTC GTCAGCCGGTGCTTGATGTAGA GGACCTTTTCAACAACCGGAT CGCACGGTACTTGTGCAGG TCAACATCTACCGCACAAACGA AAAGTGTGCGCCATCCAGAGTG CGACTTCGTCGGAGGTGACT GCCCGGACCGTTGAAATC ACGAGATCGACGACAACTGGTC CGAAATGCAGGCTGTAGGTGTT TCGACTTCATCGCGCAGAA CCTGGTACTGGATCTTCCACGA GCGGGAAAACATGAAGGACAG TGTACTTGAGAACGGTGCCCTG GAAAGAAACGACTGTGGACC GATGAAACCTTCCTTGCTCC CCAGGCGGAGTTTTTTGC GCCCACTCCTCCGAATAGGA CATCGGTACAACCGAACAACG CGGATCTTGGTCTTTTCCAGG TCTACCTTGGCCTGCAGAAGGT TGAAGTTGCCGTTGAACATGTC CCGTACCTGGTCAACTTCATCC CCATTTCACCCCGAGGTTCTT TGCGTAAGGTATGCCGTGTA CAGCAGTACGCTGTGCTCTT  A l l other manipulations were done with strains grown in B M 2 minimal media (62 m M potassium phosphate buffer (pH 7), 0.5 m M M g S 0 , 10 p M FeSC^) supplemented with specific carbon 4  sources.  A l l chemicals used were obtained from either Sigma (St. Louis, MO) or Fisher  (Hampton, NH) with the exception of glycine-glutamate, which was obtained from Bachem (Torrence, C A ) and the diterpenoid compounds which were a kind gift from Dr. W.W. Mohn (Vancouver, UBC). 2.2.2 G e n e t i c m a n i p u l a t i o n s  Routine molecular biology techniques were carried out according to standard protocols. Enzymes and cloning kits were provided by Invitrogen (Carlsbad, C A ) . A n opdK transcriptional fusion was constructed by amplifying the gene from P. aeruginosa strain HI03 using Taq polymerase in I X reaction buffer containing 2 m M MgCb, 5% D M S O , 0.4 m M dNTPs and 1 u.M of each primer. After an initial denaturation step at 94°C for 2 minutes, the amplification was incubated for 25 cycles at 94°C for 1 minute, 65°C for 1 minute, and 72°C for 1.5 minutes followed by a final extension step at 72°C for 5 minutes.  The resulting amplicon was then  cloned into pCR2.1 using the T A cloning kit and transformed into chemically competent E. coli DH5a. The opdK gene was excised from pCR2.1 using EcoRl and blunt ends were created by using the large fragment of Klenow D N A polymerase. This fragment was cloned into the Smal site of pEXlOOT. A 400 base-pair fragment of opdK was then excised by digestion with Pstl and a xylE-Gm cassette from pX1918GT was cloned into this site. The orientation of the xylE-Gm r  cassette was confirmed by restriction analysis.  T  This plasmid was transformed into electro-  competent E. coli SI7-1 and then mobilized into P. aeruginosa HI03 by biparental mating followed by successive selection on gentamicin and 5% sucrose.  The replacement of native  opdK with opdK::xylE-Grd in the resulting strain was confirmed by Southern blotting.  47  2.2.3 Sequence analysis Sequences of the P. aeruginosa OprD homologues and neighbouring genes were obtained from the most recent versions of the Pseudomonas genome data base at www.pseudomonas.com. The corresponding amino acid sequences were aligned using the basic local alignment sequence tool (1) hosted by the NCBI.  Genes were considered homologous i f they were at least 30%  similar over the length of the entire protein. Protein similarity scores were determined using the BLOSUM62 scoring matrix (11). SignalP was used to predict the signal peptides of the OprD homologues (3). The protein sequences without the signal peptides were multiply aligned using ClustalX (36) and manually edited using GeneDoc (http://www.psc.edu/biomed/genedoc).  Phylogenetic trees were made in  ClustalX and viewed with TreeView (24).  2.2.4 Reporter gene assays The catechol-2,3-dioxygenase (XylE) activity of the opdK transcriptional fusion was assayed as described (32).  Briefly, cells from overnight cultures were collected by  centrifugation, resuspended in 50 m M potassium phosphate buffer (pH 7.5) + 10% acetone, and disrupted by sonication.  Unbroken cells and other debris were removed by centrifugation.  Aliquots of the cell lysates were added to 50 m M potassium phosphate buffer (pH 7.5) containing 0.3 m M catechol and the increase in absorbance at 375 nm was monitored.  The  change in absorbance was then used to calculate the amount of 2-hydroxymuconic semialdehyde produced using a molar extinction coefficient of 44 000. The protein content of the cell extracts was determined by a modified Lowry method (30).  2.2.5 Growth assays  48  The Pseudomonas strains were grown overnight in B M 2 media supplemented with the specific carbon sources as indicated in the text. The overnight cultures were sub-cultured to a starting OD600 of 0.01 into pre-warmed fresh BM2 containing the specific carbon sources. These cultures were incubated in a 37°C water bath with an orbital shaker shaking at 200 R P M . To ensure the cultures were receiving sufficient aeration, the 10 mL of growth cultures were grown in 125 mL flasks. Aliquots of the growth cultures were taken at specified time intervals and the turbidity at 600 nm was determined using a spectrophotometer. The number of colony forming units was determined by serially diluting the aliquots to 10" to 10" in Luria-Bertani broth and 4  6  then plating onto Luria-Bertani agar plates. 2.2.6 S e m i - q u a n t i t a t i v e (SQ)-PCR  mRNA was isolated from exponential phase P. aeruginosa P A O l cells grown in B M 2 media plus 10 m M glucose or the specified carbon source using an RNeasy mini R N A isolation kit (Qiagen, Mississauga, ON). Contaminating genomic D N A was removed using a DNA-free kit (Ambion, Austin, TX).  The R N A was quantified by determining its absorbance at 260 nm.  R N A quality was assessed by comparing its absorbance at 260 nm with its absorbance at 280 nm and by examining its appearance on a 2% agarose-TAE gel. R N A aliquots were stored at -80°C. Reverse transcription was carried out by combining 4 pg of total R N A with 750 ng of the random decamer (NS) and incubated at 70°C for 10 minutes followed by a ten minute 5  incubation at 25°C. A mixture containing I X reaction buffer, 10 u M DTT, 0.5 u M dNTPS, 500 units/mL of Superase LN (Ambion), and 10 000 units/mL of Superscript RT II (Invitrogen) was . then added to the R N A and incubated for an hour at 37°C and two hours at 42°C. The R N A was destroyed by the addition of 170 m M NaOH and incubation at 65°C for 15 min. The mixture was neutralized by the addition of 170 m M HCI.  49  One microliter of the resulting c D N A was used as the template for SQ-PCR using primers designed to the internal regions of the OprD homologous genes, as well as the control gene, rpsL.  The utility of all primer pairs was checked by using genomic D N A template  controls. Amplification reactions were carried out using Taq polymerase (Invitrogen) in I X reaction buffer containing 2 m M MgCb, 5% DMSO, 0.4 m M dNTPs, and 40 n M of forward and reverse primers. The reactions were cycled for 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C. A l l reactions were amplified for 25 rounds with the exception of oprD and the ribosomal subunit control gene rpsL which were amplified for 20 cycles. The reactions using the opdB primers were cycled 35 times and the opdT reaction was cycled 40 times. The amplicons (ranging from 85 to 100 base pairs in length) were resolved by electrophoresis on 2% agaroseT A E gels stained with 50 pg/mL ethidium bromide. The intensities of the resulting bands on the agarose gels were quantified using ImageJ (http://rsb.info.nih.gov/ij/). 2.3 RESULTS  2.3.1 A novel family of specific porins in P. aeruginosa The genome of P. aeruginosa encodes 18 proteins that share considerable amino acid similarity (ranging from 46-57%) with the basic amino acid specific porin, OprD. Included in this family are the previously identified porins, OprE, (41), OprE3 (which has been renamed to OprQ) (23), and OprD3 (now known as OpdT) (34).  The 15 hitherto unknown OprD  homologues were named with the prefix Opd (for outer membrane protein D family). Phylogenetic analysis of this family (Figure 2.1) showed that it is comprised of two distinct groups. The smaller group, the OprD sub-family consisted of eight members in P. aeruginosa including, OprD, OprQ, OpdT and 5 additional OprD homologues. The other group, the OpdK sub-family, contained the anaerobically induced porin OprE plus 10 uncharacterized OprD  50  homologues. Members of the OpdK subfamily of porins demonstrated a higher degree of amino acid similarity to the phenylacetate uptake porin, PhaK, of P. putida than they do to OprD (5769% similar to PhaK vs. 47-51% similar to OprD). — PA0988 OprD  fj  • PP1206 OprD PP0268 OprQ  PA27GO OprQ . PP2754  . PA2606 OpdT - PP3630 _ PP0046  . PA2420 OpdJ  OprD sub-family  . PA01S2OpdC PA01S9 Opdl . PP5250  - PA2700OpdB PP0883  . PA4601 OpdP - PA3S88 OpdR . PP1419  • PA0765 OpdH PP3764  PA4179 OpdN - PP3271 PhaK  • PA4137 OpdL - PA211JOpdO . PA3018 OpdO PA102SOpdD  OpdK sub-family  PP2058  PA0231 OprE PP0234 OprE PP4465 . PP3390 - PP3656  . PA2213 0pdG PA4S98 OpdK  f=  PP2517 PA0240 OpdF - PP1383 PPI173 . PP3168 BenF PP3939  F I G U R E 2.1 P h y l o g e n e t i c a n a l y s i s o f t h e O p r D f a m i l y i n P. aeruginosa a n d P. putida. Phylogenetic trees were constructed using the neighbour-joining distance matrix method in ClustalX. A l l bootstrap values were over 700 (out of 1000 trials) with the exception of the branches indicated by thinner lines. In P. aeruginosa (bold-face type), OprD, OprE, OprQ, and OpdT (originally described as OprD3, (34)) had been previously identified. The remaining homologues were given names beginning with Opd (for outer membrane protein, OprD-like). The P. putida OprD homologues were labelled according to their ORF (PP) numbers including the previously named OprD, OprQ, OprE, BenF, and PhaK porins (indicated with regular type).  51  Eleven of the 19 porins in P. aeruginosa have clear orthologues in P. putida and are likely to share the same function in the two organisms. Six proteins in P. aeruginosa, (OpdJ, Opdl, OpdR, OpdO, OpdD, and OpdQ) did not have any orthologues in P. putida and thus might have arisen from a post-speciation duplication event and would be proposed to exhibit functions unique to P. aeruginosa. A protein sequence alignment of the OprD homologues (Figure 2.2) indicated that this family of porins might share similar secondary structures. When the OprD topology model was applied to the alignment, it showed that the highest degree of conservation lay in the regions that corresponded to the 16 putative trans-membrane (3-strands and periplasmic turns of OprD (13, 20). Interestingly, the N - and C-terminal (3-strands (TM1-TM4 and TM13-TM16) were more similar in sequence than the P-strands in the central region of the porins (TM5-TM12). The lowest degree of sequence conservation lay in the blocks corresponding to the 8 surface loops of OprD.  Thus, it was postulated that as with OprD (13, 20), these putative loop regions are  involved in determining which substrates pass through these novel porins.  Comparison of the  physical parameters of the OprD family revealed that in general members of the OpdK subfamily were shorter than the OprD sub-family (Figure 2.3). Much of this discrepancy between . the two groups was attributable to the longer surface loops and N-terminal extensions of the latter group of porins (Figure 2.2). The isoelectric points of the family varied from 4.91 to 9.07. These values correlated roughly with the amino acid composition of the extracellular loops 2 and 3, which in OprD, have been implicated in substrate binding (Tables 2.4 and 2.5). 2.3.2 Involvement of OprD in arginine uptake The sequence similarity of the OprD homologues to OprD suggested that the members of this family were specific porins. Mini-Tn5-Tc insertion mutants in each of the OprD R  52  LI  OprD OprQ OpdB OpdC Opdl OpdJ OpdP OpdT OprE OpdD OpdF OpdG OpdH OpdK OpdL OpdN OpdO OpdQ OpdR  OprD OprQ OpdB OpdC Opdl OpdJ OpdP OpdT OprE OpdD OpdF OpdG OpdH OpdK OpdL OpdN OpdO OpdQ OpdR  -DAFVSDQAEAKG ;:SSLDj NDQEAAKGi f s H L B ATlEEAKAPDYLEWH JEGEAKEG GSSLQ - DD SLGHRDNIS TGLNQRQKAEMK VPPG APDNPSYAAEVQSIPSVAKPIKGQAGATGL -EENAEPISKEG  -GHVH|AGQ©[ -E  3 ..SGSGDR [|HGRQDK gR--ADAGR-'|HASG-HDSKE I^DSPNNAGRNRFK HDTPSRRDQRE^SFTFRIPKAGG-GSQRIHQRPA.:; |KGQS S PAGG GY TP' GTASP PGASQG-DASQPGAAQGPGQ SKRfe| HDAGK SLVp!2 HDAPQGSSANPQGASGARD N-Ab| EGASQ SKAE12 GAGR AKS i  SDKT T N PVMND GKP RDD-j.SRA GA K RS AAG :|D F F K Q GDS >SAADDL S K G | A A R HA T .. LPLDS S^DNASE^SDA^AA-I TG .TS ;LPITSPSKEGYES ;;KAPDE RT. T NLPVGA D HPDHR; SGA ;i,PLDS A'-RPADS RI G G NRTLANS D *EALGE jA&G- S V D i L P Y D D Q||RPQ&D NgE PVHD DRQPGTgS F P L E S DRRAAHD L3PYSTSDSRPADD «LPIHDPRHA-DJJJRAPDT PKDS-DgRAADN PLHD-SKEPVDD TJJPFGAN-PRRSVDQ •—LLPSSGHD^GSQDD -AgGSC —L^PADGS^QPD -TgRAf. —LgKRDRE-DgSA* ESAAGD 3LPLRD;  L3 ; HFTEGKEPTTVKSRGEI, Y AT YAGET E N RFTAESRKSAEGR DSGG 3SDDi|DR R Q RFTAFKEQASSSGHGDFDGYGAS TAG JTNNSFED T T ^QVSFTKYYNQSGHRRLGSYYG SDRSFDG EG RFTAASGPGESKVRGDISTVYGR •JYHFRA E D FVEATRLRNQSGHSHLTSGYGNGTKGGIAADRE|S EE DN- S Q . SFRKVSPRTGSGDEDMTTEYGTRQVK OS AKG T Q KLNSMTQPNSTSGSDDFYSFYTGRR I . _ TD^KD - FT JJK_WQLE H SKGRN S TDNRS LSIAGANGS SSWE_DG-3A_QF_QLRQVKQRDSTNAEDLGI IFRGNS PRNDASMQDMSLFGRPAAT ISANEMAG 2MRSLSQRNSSDHQDLSVDGRGGAF ITSREHAG ^LEKTKIRDSSDSEDLALNDKNGRF-BTSKEBKD IFDAVSLRNSADMQDLSAWSAPTQK fSREL 2LWKSRTRE SAGSDDMYIMGRDKAH JTSSE JTRLE RY TARDS SDAQDIRLHCKNKRY-JTSEEl IGAHLDRNKLNSSSDYQVFSANRIGGR DVQEHDG *LKKVNQRD S SDNEDMTI 4 S M E Q D G JTEVSQRNEAGTSDLALFNRNRRF— JSSEFEG  jTSKE :;iEG ;  e  JTSGEI  :  L5  GB~FNI"RTN""E  NF IFSL  F N G :R j K L D S D F A GNL  R  fNPGAHY RV  R  K V  Q  RSR  i  DSL  LDA  R  R  S  QSR  TPWLHY  K  R  Q  RSQ  RTAFNG  H  G  T  AREAG-  ILGGANY K|VK ; Q RQVMGSSDGKNGSRSGRADGYVSSGYYGSGVTK_EJ |IRW. T3ESGS  d  JGASVGL^  ^  EEAAKs B —  DRSDSAAR-  isjj LRYij JSAJJ LRW_D _3_AgAAR LRFAR ASEDgGFR :s"tRWAR)jTi3D™SS s'  L7  p  OprD OprQ OpdB OpdC Opdl OpdJ OpdP OpdT OprE OpdD OpdF OpdG OpdH OpdK OpdL OpdN OpdO OpdQ OpdR  DIDGTKMSDNN VGYKNYGYGEDG^HH; S 5 P A D SF i Win NIKTAE TSN G^jERj IFNQSQS SPA.D T L SSFL IDGSHAPAGGAYNPLGADGRYRPLQGSGG H FAS SPF D S NVSHL H LD R LDLTRVDPDS PGYGGWYSADGKtfA1H£ , G! vLD G A S ASS AAESIYAGLYGRDG. HR «J LGF R Ki 3PLA3 A- :ASQAWH r>ra ADLTRVDPDS AGYGYLYNPNGKNAQH;!| lAFPi I P A 3 S TL WA H DIDGTHYDGDRNG AYGNYAEVRAQDGE HH }LGLMAA_K | P I (DSTF';LTYM4H SwDADYSNANSVY MRTDANGN P L T N Q G R T S "  LASY ;FAKY 1 •LAPF Q | Y ;FVA fl L l.GA ; OHQASLAF'*' %T?WSYY L>'MAAF  s  :  QVRY QLRYD QLRYD QLRYD QLRYD QVRYD QLRYD QLRYD QLRYD Q_RYD QLRYD  —KIKTARGD  HIDLLDGGGR HVRLAGVTDD ARTKAMDDNVELAGQSGEeJSNATTKAGSGIDRGAGRADIRLANGDENVARG-AANDNIDLLTTSGEGAKPKGADG-  QSEijIj  G_Ep n c PSE  L8 OprD OprQ OpdB OpdC Opdl OpdJ OpdP OpdT OprE OpdD OpdF OpdG OpdH OpdK OpdL OpdN OpdO OpdQ OpdR  Svs--  ADQG—EGgQj  NDARSYND|GS AAQAGDDI gl GGYSAVDN SGb ;NASYLDGDI"GEGYTAPGNTRGNSSSjg  I I  S  Q^JQIDGS^  DSFESpLS  G—LPAAGSSNNQR|  DFGNij DWGSNTRFI  NHAA— NFTR" SFNS-  DFERSi NFANig] NYTNjJ AFSR_I|_  iD|P3s|L^^E^PFSyFP KGSLl P D ;F D | P R ;L STIPFN "1%  420 404 411 422 427 447 459 422 431 394 397 388 391 390 391 400 384 394 392  Figure 2.2 Protein seuquence alignment of the P. aeruginosa OprD family of porins. The alignment was generated in ClustalX and manually edited with GeneDoc. Signal peptides were predicted by SignalP and are not shown. The indicated putative transmembrane regions (boxes) and surface loops are based on the published topology model of OprD. Members of the OprD sub-family are highlighted in yellow.  395 377 385 395 402 415 434 399 401 372 373 366 369 369 369 375 362 372 370  2.4 Amino acid composition of the second surface loop of the OprD homologues Amino acid (one letter code) C D E F G H I K L M N P Q R S T V W Y Neg Pos 0 5 0 0 6 0 1 5 2 1 2 2 0 3 3 4 3 0 1 5 8 0 5 0 3 9 0 2 4 2 0 1 0 l 3 5 1 1 0 1 5 7 0 4 2 0 8 1 0 2 7 0 1 1 0 2 5 1 0 0 1 6 5 0 3 3 2 10 0 3 4 3 0 0 3 0 1 6 4 0 0 0 6 5 0 3 0 0 11 3 0 1 4 0 1 2 1 4 1 3 2 0 1 3 8 0 4 0 1 10 0 0 1 6 0 0 2 0 3 5 0 1 0 0 4 4 0 1 3 0 9 0 2 2 5 0 4 0 0 5 3 2 1 1 0 4 7 0 6 0 0 10 0 0 1 4 0 0 2 2 5 4 1 2 1 3 6 6 0 3 2 2 8 1 0 3 3 0 2 3 1 3 4 3 3 0 0 4 7 2 0 1 6 0 0 3 1 0 3 0 2 0 5 10 2 1 0 2 4 8 0 5 0 1 7 1 1 3 5 0 1 2 1 3 2 1 2 0 0 5 7 0 5 0 0 4 1 0 3 6 0 1 3 1 4 4 3 1 0 1 5 8 0 4 0 0 6 0 0 5 5 0 .0 2 1 3 6 3 2 0 1 4 8 0 3 1 0 5 1 0 2 5 0 3 1 1 4 5 1 3 0 1 4 6 0 4 1 1 5 1 0 3 5 0 2 2 1 3 5 2 1 0 2 5 7 0 4 0 1 4 1 0 3 3 1 1 3 2 3 8 1 2 0 1 4 7 0 4 0 0 9 0 0 3 5 0 1 1 1 1 7 2 1 0 1 4 4 0 5 2 0 5 0 1 4 4 0 0 1 1 5 5 3 1 0 1 7 9 0 5 0 0 6 0 0 1 7 0 0 1 0 6 5 2 0 0 1 5 7  Total 40 44 40 46 39 40 43 44 45 41 39 43 40 41 40 42 41 42 40  2.5 Amino acid composition of the third surface loop of the OprD homologues Amino acid (one letter code) D E F G H I K L M N P Q R S T V W Y Neg Pos 1 7 3 6 1 0 2 4 0 0 2 3 1 3 7 1 0 2 4 8 1 7 1 6 0 2 2 3 0 1 1 0 3 6 2 0 0 1 8 5 5 1 5 6 1 1 1 2 0 0 1 4 4 5 2 0 1 2 6 6 3 2 3 6 1 0 1 5 0 3 2 2 4 5 5 2 0 4 5 6 4 3 2 12 0 1 2 7 1 0 2 0 6 5 2 4 0 3 7 8 2 4 1 8 3 1 2 •5 0 3 1 2 5 4 4 2 0 3 6 10 3 4 3 5 0 0 2 3 1 1 2 2 3 7 6 3 0 1 7 5 4 0 4 5 0 1 2 5 1 3 2 2 3 8 5 1 0 2 4 5 4 2 2 5 1 1 2 4 .0 3 1 2 3 4 5 3 0 0 6 6 3 3 2 8 1 2 2 7 0 3 2 6 5 1 3 3 1 0 6 8 2 2 4 6 0 1 0 4 2 3 3 4 4 4 3 2 0* 1 4 4 3 1 2 8 2 1 0 5 2 1 1 5 4 6 3 1 0 0 4 6 5 4 3 5 0 5 3 6 0 2 1 1 3 5 3 1 0 0 9 6 3 0 2 3 0 0 1 5 1 1 3 4 4 5 2 1 1 0 3 5 4 3 1 5 1 2 2 5 2 0 1 2 3 5 5 0 1 1 7 6 4 3 2 2 2 2 2 5 0 1 1 2 6 4 6 0 0 2 7 10 4 2 2 5 1 2 1 7 2 1 1 2 5 4 1 2 0 1 6 7 5 2 1 8 0 2 4 5 3 3 1 3 4 4 6 2 0 0 7 8 1 4 3 6 0 0 0 9 0 4 1 4 5 5 3 2 0 0 5 5  Total 46 39 47 49 59 55 48 50 46 55 51 47 51 41 48 52 46 55 53  TABLE  Porin OprD OprQ OpdB OpdC Opdl OpdJ OpdP OpdT OprE OpdD OpdF OpdG OpdH OpdK OpdL OpdN OpdO OpdQ OpdR  A 2 6 5 4 2 7 5 3 4 3 4 6 2 5 2 4 5 4 6  TABLE  Porin OprD OprQ OpdB OpdC Opdl OpdJ OpdP OpdT OprE OpdD OpdF OpdG OpdH OpdK OpdL OpdN OpdO OpdQ OpdR  A 3 3 6 1 5 5 2 2 4 3 6 2 4 5 5 6 3 2 6  C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0  56  • OpdN  • Opdl • OprE lOpcUl  o.  • OprD sub-family • OpdK sub-family  7  6  I • OpdO  OpdP I  5 4  380  400  420  440  460  Number of amino acids Physical parameters of the P. aeruginosa O p r D porin family. Both the isoelectric point and protein length were calculated from the sequence of the mature, cleaved porin protein. FIGURE 2.3  homologous genes were obtained from Pathogenesis Corporation (now Chiron Inc.).  To  determine whether any of the channels were involved in antibiotic uptake, the mutants were first screened for resistance to all of the major classes of antibiotics including, pMactams, carbapenems, cephalosporins,  quinolones, macrolides, aminoglycosides, chloramphenicol,  trimethoprim, nitrofurantoin, and rifampicin. With the exception of the OprD mutant which exhibited an 8-fold increase in MIC to imipenem and meropenem, none of the other mutants demonstrated increased resistance to any antibiotic tested (data not shown). The work of Nikaido et al demonstrated that OprD possesses a binding site specific for the basic amino acids arginine and lysine, dipeptides containing these residues, and structural analogues such as carbapenem antibiotics (37, 38). When present as a mixture, the amino acids and imipenem compete for the binding site within OprD and raise the MIC to imipenem 16-fold, emphasizing the role of this porin in amino acid uptake (12). The lack of a resistance phenotype of the remaining OprD homologue mutants precluded the use of competition assays to aid in the  57  determination of substrate specificity. Therefore, the oprD deficient mutant was used as the prototype to develop a growth assay with which to test the remaining mutant strains. Specific porins are required to facilitate diffusion in nutrient limited environments (19). Otherwise, the rates of diffusion through the outer membrane would not be sufficient to saturate the high affinity cytoplasmic membrane transporters, making outer membrane permeability ratelimiting for growth (10). The contribution of an individual specific porin is not seen in nutrient sufficient environments, as other porins are present in the outer membrane to serve as nonspecific diffusion channels. Thus, to find the optimal substrate concentration at which the loss of a specific porin would compromise growth, the OprD mutant was grown in minimal media supplemented with arginine in concentrations ranging from 10 m M to 0.1 m M . At higher substrate concentrations, (i.e. 1 m M or greater, Table 2.6), no growth differences between the mutant and wild-type strain were observed. Although the growth yield was limited at the lower carbon source concentrations, we were able to reliably follow growth as the optical density measurements correlated with CFU/mL determinations (Figure 2.4). Cultures grown in arginine concentrations below 1 m M quickly formed aggregates, obscuring quantitation by either spectroscopic or plate counting methods. This aggregation was specific to arginine as it was not observed when glucose or numerous other carbon sources were used. T A B L E 2.6  Doubling times of P. aeruginosa P A O , P A K , an oprD mutant and an opdK mutant grown in B M 2 + either arginine or vanillate 8  a  b  Carbon source Doubling time in arginine (min) Doubling time in vanillate (min) concentration (mM) PAO oprD::Tn5 PAK opdK::Tn5 jo b 0.5 63 ± 1 122 ± 0 . 5 1 34 ± 5.0 40 ± 2 . 4 34 ± 1 49 ± 3 2 43 ± 0 . 6 41 ± 3 . 2 40 ± 1.5 36 ±1.7 results shown are the means ± standard errors of two independent experiments cultures formed clumps and did not grow reproducibly at this concentration  58  0.15  7.12  14.09  21.06  28.03  35.00  CFU/mL (xlO ) 7  Correlation of optical density with colony forming units in P. aeruginosa. Wild type P. aeruginosa strain P A O l and P A K were grown in B M 2 minimal media supplemented with 1 m M arginine. Aliquots were taken every hour to determine the optical density of the culture at 600 nm and to determine the number of colony forming units. The equation used to derive the best fit line was y = 0.0042x + 0.0069. The R value of the line was 0.948. Results shown are derived form three separate experiments. FIGURE 2.4  2  Alternative assays were developed wherein various concentrations or gradients of carbon sources were added to agar, however, growth in these conditions was inconsistent and often difficult to interpret. Therefore, performing growth curves in liquid media was deemed to be the quickest and most reliable method to detect obvious growth defects.  2.3.3 Involvement of OpdK in vanillate uptake The phenotype of the opdK deficient mutant was then investigated to determine whether there was a functional basis for the observed phylogenetic clustering of the OprD family. In bacteria, genes involved in the same metabolic pathway tend to be adjacent in the genome (15). Therefore, the substrate specificity of OpdK was first investigated by examining the functions of its neighbouring genes.  The opdK gene is located in the midst of genes putatively involved in  the metabolism of vanillate and related compounds (Figure 2.5A).  The genes immediately  downstream of opdK include one that is homologous to a P. putida aldehyde dehydrogenase  59  107  PA4897  22  oprfrT PA4899  82  PA4900 mrf/C PA4902 PA4903 van/IB PA4906  PA4911  PA4914 PA4915  B Gene PA4897  opdK PA4899 PA4900  mdlC PA4902 PA4903  vanAB PA4906 PA4911 PA4914 PA4915  Putative function TonB-dependent family outer membrane protein Porin Aldehyde dehydrogenase Major facilitator superfamily transporter Benzoyl formate decarboxylase Transcriptional regulator Major facilitator superfamily transporter Vanillate O-demethylase oxidoreductase Transcriptional regulator Permease of A T P binding cassette transporter Transcriptional regulator Chemotaxis transducer  Gene 25 cycles 30 cycles 1 2 3 4 PA4897  opdK-VMm  opdK  PA4899-PA4900  mdlC  -  PA4902  n  PA4903  WKk  5  6  7  __•)  PA4900-mrf/C  PA4911 PA4915  rpsL?(99 "20 cycles  Genomic context of opdK. A . Lines above the genes indicate the location of the intragenic (solid lines) and intergenic (dashed lines) regions that were amplified by SQ-PCR. Numbers above the dashed lines indicate the size, in base pairs, of the intergenic regions. Functional classes of the genes are indicated by the following colors: Porin- black; TonBdependent family member- horizontal stripes; cytoplasmic membrane transporters- stippled; transcriptional regulators- vertical stripes; enzymes involved in carbon catabolism- dark grey; chemotaxis transducer- light grey; unknown function- white. B . Probable gene functions of some of the ORFs in the opdK gene cluster. C. Transcription of regions within the opdK gene cluster in response to vanillate. mRNA was isolated from exponential phase cells grown in BM2 + either 10 m M glucose (lanes 1, 3, 5, and 7) or 10 m M vanillate (lanes 2, 4, 6, and 8), reverse transcribed into c D N A and used as the template for SQ-PCR. F I G U R E 2.5  gene, a putative major facilitator super-family (MFS) transporter 57% similar to the BenK gene of Acinetobacter sp. ADP1, and a probable benzoyl formate decarboxylase gene (mdlC). Also found in this region are two genes highly similar to the vanAB genes which encode the subunits of vanillate O-demethylase oxidoreductase from Pseudomonas species strain HR199 (27). Thus,  60  the context of the opdK gene suggested that this porin may be involved in the uptake of small aromatic molecules. As specific porins tend to be induced by their substrates, an opdK transcriptional fusion, and for comparison, an oprD transcriptional fusion were grown in B M 2 +10 m M carbon source (Table 2.7). Other than glucose and succinate, the carbon sources consisted of benzoate derivatives with one or two more polar substituents and for the most part are metabolized by Pseudomonas species through the P-ketoadipate pathway (9). OprD was expressed in all of the carbon sources tested; in contrast, the induction of opdK was quite specific and strongly occurred in both vanillate and its aldehyde derivative vanillin (Table 2.7).  TABLE  2.7 Catechol-2,3-dioxygenase (CDO) activities of an opdK::xylE-Gm transcriptional r  fusion and an oprD: :xylE-Gm transcriptional fusion grown in various aromatic r  compounds Carbon source Vanillate Vanillin /?-hydroxybenzoate Anthranilate Benzoate Glucose Succinate /7-hydroxybenzaldehyde Phenylacetate Hydroxyphenylacetate Toluate (o- and m-) Coumarate Ferulate 2,4-dihydroxybenzoate /7-aminobenzoate Salicylate Chlorobenzoate (o-, m-, and p-) Tyrosine Quinate b  CDO activity (pmol/mg/min) opdK oprD 556 ± 3 7 435 ± 10 387 ± 18 584 ± 64 20.3 ± 5.9 205 ± 17 14.6 ± 3 . 1 158 ± 3 7 5.64 ± 1.6 146 ± 2 1 1.41 ±0.55 451 ± 5 4 0.298 ± 0 . 1 3 104 ± 10 ND <0.01 ND <0.01 ND : < o.oi ND <0.01 ND <0.01 ND <0.01 ND <0.01 < 0.01 ND < 0.01 ND ND <0.01 ND <0.01 < 0.01 ND  a  b  N D = not determined  61  With the exception of ^-hydroxybenzoate and anthranilate, the catechol-2,3-dioxygenase activity of the opdK transcriptional fusion grown in the remainder of the carbon sources was less than 10 pmol/mg/min protein and thus considered insignificant. Primers flanking the intergenic regions of the putative four gene opdK operon, and primers to 100 base-pair regions within other adjacent genes were designed to permit the evaluation of mRNA levels in glucose and vanillate by SQ-PCR (Figure 2.5C).  In vanillate,  PCR products overlapping the intergenic regions between opdK, the putative aldehyde dehydrogenase (PA4899), and the probable MFS transporter (PA4900) were generated implying that these three genes form an operon. The mdlC gene was not apparently co-transcribed with this operon, but the gene itself was strongly induced by vanillate. The putative regulator PA4902 was also highly expressed in vanillate.  The probable cytoplasmic transporter, 4903, (83%  similar to the VanK M F S transporter of P. putida) was also induced by vanillate (amplified product detectable after 30 cycles) but to a much lesser extent than PA4900 (amplified product detected after 25 cycles). The expression of PA4911 (67% similar to BraE of P. aeruginosa, the permease component of a branched-chain amino acid-specific A T P binding cassette (ABC) transporter) was not induced in vanillate.  A n unexpected result was the strong positive  regulation of the gene for an outer membrane protein belonging to the large TonB-dependent family of porins (34) as these genes are often regulated by ECF sigma factors under limiting iron conditions. In comparison to its isogenic wild-type strain, the opdK mutant was compromised in growth at low concentrations of vanillate (Table 2.6). At 0.5 m M vanillate the mutant grew at approximately half the rate of the wild type.  Similar to the results obtained with the oprD  mutant, at 2 m M carbon source, the growth rate of the opdK mutant is identical to that of the  62  wild-type strain; and at 1 m M vanillate, the mutant exhibited a very slight growth defect. The growth defect appeared to be specific to vanillate as no difference in growth between the two strains was observed when the other aromatic compounds listed in Table 2.7 were used as carbon sources (including the inducing molecule vanillin, data not shown).  Thus over a restricted  concentration range, OpdK afforded a growth advantage on vanillate.  2.3.4 Growth phenotypes of the other OprD homologues To determine whether the apparent specificity of OpdK for an aromatic compound and OprD's specificity for basic amino acids could be generalized to other members of their respective sub-groups, the substrate specificities of the 17 remaining OprD homolgues was investigated.  Using a similar approach as for OpdK, the genomic contexts of the OprD  homologous genes were investigated to see i f they were associated with any putative metabolic pathways (Figure 2.6). The genes for 12 of the 17 novel OprD homologues were adjacent to genes involved in the transport or degradation of a variety of compounds such as amino acids, carboxylic acids, and small aromatic molecules. Therefore, we were able to predict the putative substrates for these porins.  The remaining 5 OprD homologues, like OprD itself, were  neighboured by hypothetical proteins.  However, substrates were ascribed to 3 of these  remaining porins by examining the genomic contexts of their orthologous porins in P. putida. O f these 3 homologues, the orthologous genes for 2, opdL and opdQ, were adjacent to genes involved in the degradation of phenylacetic acid and nitrobenzoate respectively.  The  orthologous gene of the third homologue, OpdN, lay directly downstream of a cobF homologue. In prokaryotes, cobF is involved in cobalamin biosynthesis (28). Therefore it was postulated that OpdN might take up a vitamin B12 precursor such as glutamate or 5-aminolevulinic acid. Strains deficient in one of the 17 remaining OprD homologues were assessed for growth  63  A. OprD (PA0958)  P.aeruginosa  prolyl tRNA synthetase  OprD  P.putida  prolyl tRNA synthetase Compounds tested as substrates  OprD  Growth of an oprD mutant in glucose  I  porin  enzyme  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. A. Genomic contexts of the P.  aeruginosa oprD gene and its orthologue in P. putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glucose.  B . OprQ (PA2760)  P'.aeruginosa  P.putida siderophore receptor Compounds tested as substrates  OprQ  glutamate synthase Growth of an oprQ mutant in arginine  H  porin  enzyme  Time (minutes) Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. B. Genomic contexts of the P. aeruginosa oprQ gene and its orthologue in P. putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM arginine. ON  C . OpdB (PA2700)  P.aeruginosa [  OpdB  diterpenoid transporter  P.putida  hydrolase  OpdB  transporter Growth of an opdB mutant in glucose  Compounds tested as substrates Glucose Dehydroabiatic acid and related compounds* Octane Propionic acid Biphenyl *2 15 Quinate Anthranilate H Vanillate Hydroxyphenylacetic acid Tyrosine Histidine 0.01 Proline  transporter metabolic enzyme regulator  •  —  not used as a sole C-source by P. aeruginosa  porin  other unknown  ~~wild-type " " mutant &  66  132  198  264  330  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. C. Genomic contexts of the P. aeruginosa opdB gene and its orthologue in P.putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glucose.  D. OpdC (PAOl62)  P'.aeruginosa  cation efflux transporter  OpdC  P.putida  chemotaxis protein  protein secretion system Compounds tested as substrates Glucose Gluconate Proline Histidine Isoleucine* Valine* Leucine p-aminobenzoic acid Hydroxyphenylacetic acid 2,4-dihydrobenzoic acid Glutathione* N-acetylglucosamine*  Growth of an opdC mutant in glucose  Aspartate* Glutamate Tyrosine Propionic acid Quinate  not used as a sole C-source by P.  OpdC  0.2  T3  •  porin  CD]  transporter  ill  metabolic enzyme  IB  regulator  H  other  l~~l  unknown  IS  1-1 3  H  • e  0.02 aeruginosa  J  66  132  wild-type " mutant  198  i  264  330  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. D. Genomic contexts of the  P. aeruginosa opdC gene and its orthologue in P. putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glucose.  E . Opdl (PA0189)  P.aeruginosa  Opdl  sulfate ester A B C transporter  Growth of an opdl mutant in anthranilate  Compounds tested as substrates 0.2 Glucose DMSO* Cysteine* Methionine* SDS* Tyrosine Anthranilate Vanillate Benzoate Hydroxyphenylacetic acid  Pyruvate Proline  * = not used as a sole C-source or S-source by P. aeruginosa  0.1  acid phosphatase  •  porin  DDI  transporter  H  metabolic enzyme  IH  regulator  H  other  l~l  unknown  IS  • wild-type " " mutant e  0.01  70  140  210  280  350  420  Time (minutes)  aeruginosa O p r D p o r i n f a m i l y . E. Genomic context of the P. aeruginosa opdl gene. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM anthraniliate. F i g u r e 2.6 G e n o m i c c o n t e x t s o f t h e P.  F. OpdJ (PA2420)  P.aeruginosa  isochorismatase  Compounds tested as substrates Glucose Tyrosine Tryptophan* Phenylalanine* Proline Glutamine Glutamate Aspartate* Histidine Quinate Anthranilate Hydroxyphenylacetic acid * = not used as a sole C-source by P. aeruginosa  OpdJ  Growth of an opdJ mutant in tyrosine  9---  •  porin  mD  transporter  LU  metabolic enzyme  III  regulator  i i  other  LJ  unknown  • wild-type ~~ mutant e  100  200  300  400  500  Time (minutes)  P. aeruginosa O p r D porin family. F. Genomic context of the P. aeruginosa opdJ gene. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM tyrosine. Figure 2.6 G e n o m i c contexts of the  G. OpdP (PA4501)  P .aeruginosa  dipeptide transporter  dipeptide A B C transporter  OpdP  ^  P.putida  dipeptide A B C transporter  ^  ^  ^  •  porin  CE transporter 11  metabolic enzyme  IH  regulator other  T3  I I unknown  0.02 * = not used as a sole C-source by P.  ^  dipeptide transporter  OpdP  Growth of an opdP mutant in glucose  Compounds tested as substrates Glucose Polylysine* Valine-valine* Glycine-glutamate Glycine Glutamate Arginine  ^  aeruginosa  66  132  198  264  330  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. G. Genomic contexts of the P. aeruginosa opdP gene and its orthologue in P.putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glucose.  H . OpdT (PA2505)  P.aeruginosa  f_  catechol metabolism  OpdT  P.putida  OpdT  choline, betaine carnitine transporter Compounds tested as substrates Glucose Pyruvate Serine* Threonine* Glutamate Aspartate* Proline Arginine p-hydroxybenzoate a-hydroxybutyrate 2,4-dihyroxybenzoic acid Hydroxyphenylacetic acid  Growth of an opdC mutant in glucose  porin  DDI transporter  Benzoate Anthranilate Vanillate Histidine Tyrosine  not used as a sole C-source by P.  •  T3  j3  0.02 aeruginosa  66  132  198  264  H  metabolic enzyme  IS  regulator  H  other  I I  unknown  330  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. H. Genomic contexts of the P. aeruginosa opdT gene and its orthologue in P.putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glucose.  I. OprE (PA0291)  P.aeruginosa  <  -  l  OprE  sodium agmatine pantothenate ureohydrolase symporter  < P-alanine synthetase  -aflJnmmmnmiD <dHHnniiiffliiiinni  P.putida  OprE  taurine A B C transporter  Compounds tested as substrates  Growth of an oprE mutant in proline 0.2  Glucose Sodium pantothenate* Uracil* Arginine Proline (3-alanine Taurine Pyroglutamate p-aminobenzoic acid  0.1 "2 15  porin  Dil  transporter  El  metabolic enzyme  Hi  regulator  ^  other  I I unknown  wild-type --€>-- mutant 0.02  * = not used as a sole C-source by P. aeruginosa  •  100  200  300  400  500  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. I. Genomic contexts of the P. aeruginosa oprE gene and its orthologue in P. putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM proline.  J. OpdD (PA 1025)  P.aeruginosa  c/s-czs-muconate transporter  Compounds tested as substrates Glucose Succinate Quinate Benzoate Anthranilate Vanillate Hydroxyphenylacetic acid Pyroglutamate Propionic acid a-hydroxybutyrate y-aminohydroxybutyrate a-ketoglutarate p-aminobenzoic acid 2,4-dihydroxybenzoic acid  fatty acid metabolism  OpdD  Growth of an opdD mutant in a-ketoglutarate  •  0.2  DDI transporter  porin  H  metabolic enzyme  IS  regulator  ^  other  I I unknown  * ~ wild-type ®~~ mutant 0.02  60  120  180  240  300  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. J. Genomic context of the P. aeruginosa opdD gene. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM a-ketoglutarate.  K. OpdF (PA0240)  ^aHQmimmimiiD i  P.aeruginosa  OpdF  [>  hexuronate MFS transporter  P.putida  aromatic compound efflux Compounds tested as substrates Glucose Glucorunic acid* Galactonic acid* Propionic acid Pyruvate N-acetylglucosamine* Malate p-aminobenzoic acid 2,4-dihydrobenzoic acid p-hydroxybenzoate Hydroxyphenylacetic acid Quinate  OpdF  p-hydroxybenzoate metabolism and transport  Growth of an opdF mutant in /7-hydroxybenzoate 0.2  Glycerol Gluconate Tyrosine  0.1  porin  ED] transporter HI  metabolic enzyme  IH  regulator other  "2 15  * = not used as a sole C-source by P. aeruginosa  •  I I unknown  &  0.01  70  140  wild-type " mutant  210  280  350  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. K. Genomic contexts of the  P. aeruginosa opdF gene and its orthologue in P. putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM p-hydroxybenzoate.  L. OpdG(PA2213)  P.aeruginosa  •  nnnnnnnnrjjj]^  <Hfn  P.putida  OpdG  ]  hydrolase  <  ^—  n  <  maleate isomerase  ^  3 <  hydrolase  Growth of an opdG mutant in anthranilate 0.2  Malate Pyruvate Tartrate* Gluconate Quinate Salicylate* Tyrosine  * = not used as a sole C-source by P.  <^  MFS transporter  Compounds tested as substrates Glucose Glucaric acid* Galactonic acid* Propionic acid N-acetylglucosamine* Phthalate* Anthranilate Vanillate Benzoate Hydroxyphenylacetic acid /?-aminobenzoic acid 2,4-dihydrobenzoic acid  tartrate MFS transporter  OpdG  glucarate MFS transporter  •  transporter  •  —  H  0.01 aeruginosa  0  70  porin  m •  3  u  salicylate hydroxylase  m T3  ^  metabolic enzyme regulator other unknown  140 210 280 350 420 Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. L. Genomic contexts of the  P. aeruginosa opdG gene and its orthologue in P.putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM anthranilate.  M. OpdH (PA0755)  P'.aeruginosa  tricarboxylate transport  P.putida  OpdH  <4nDiu^ tricarboxylate transport  OpdH  Growth of an opdH mutant in glucose  Compounds tested as substrates Glucose Citrate Isocitrate cw-aconitate Succinate a-ketoglutarate Propionic acid Proline Hydroxyphenylacetic acid  •e  •  porin  dill  transporter  HI  metabolic enzyme  |H  regulator  ^  other  I I  unknown  * ~ wild-type "  mutant  198  264  e  0.02  66  132  330  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. M . Genomic contexts of the P.  aeruginosa opdH gene and its orthologue in P.putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glucose.  N. OpdK (PA4898)  <m|iniiiiiiiiiiiiiiii  P.aeruginosa  siderophore receptor  OpdK  benzoate transporter  Growth of an opdK mutant in glucose  Compounds tested as substrates Glucose Vanillate Vanillin p-hydroxybenzoate Benzoate Hydroxyphenylacetic acid Quinate  aldehyde dehydrogenase  T3  'JB 3  H  0.01  132  198  264  •  porin  HU  transporter  J l  metabolic enzyme  O  regulator  H  other  l~~l  unknown  330  Time (minutes) Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. N . Genomic context of the P. aeruginosa opdK gene. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glucose.  O. OpdL (PA4137)  P'.aeruginosa MFS transporter  OpdL  tyrosyl tRNA synthetase  phenylacetic acid metabolism and transport  Compounds tested as substrates Glucose Aspartate Quinate Tyrosine Anthranilate Vanillate p-hydroxybenzoate Phenylacetic acid* Hydroxyphenylacetic acid p-aminobenzoic acid a-ketoglutarate N-acetylglucosamine*  PhaK  Growth of an opdL mutant in hydroxyphenylacetic acid 0.2  Pyruvate  -a 3  •  porin  HDI transporter 111  metabolic enzyme  HI  regulator  ^  other  [J  unknown  H  wild-type mutant 0.02  * = not used as a sole C-source by P. aeruginosa  60  145  230  315  400  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. O. Genomic contexts of the P. aeruginosa opdL gene and its orthologue in P.putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM hydroxyphenyl acetic acid.  P. OpdN (PA4179)  P.aeruginosa  [ OpdN  peptidly prolyl cis-trans isomerase  acetolactate synthase  P.putida  vitamin B synthesis Compounds tested as substrates Glucose Pyruvate Quinate Proline Aspartate Glutamate 5-aminolevulinic acid* Hydroxyphenylacetic acid p-aminobenzoic acid a-ketoglutarate N-acetylglucosamine*  OpdN  ] 2  Growth of an opdN mutant in glutamate  TJ  not used as a sole C-source by P. aeruginosa  • n  transporter  H  metabolic enzyme  •  regulator  LI  other  • 0.01  70  140  210  280  porin  unknown  350  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. P. Genomic contexts of the P. aeruginosa opdN gene and its orthologue in P.putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glutamate.  Q . OpdO (PA2113)  P, lactam utilization protein  Compounds tested as substrates  OpdO  sialic acid transporter  Growth of an opdO mutant in glucose  porin  enzyme  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. Q. Genomic context of the P. aeruginosa opdO gene. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM glucose.  R. OpdQ (PA3038)  P'.aeruginosa (3-lactamase superfamily  OpdQ  ammonium  tranporter  <<5fflnniiiffliiin]  P.putida OpdQ  purine permease  nitrobenzoate reductase  Compounds tested as substrates Glucose Glutamate Glutamine Proline Histidine Cytosine* p-aminobenzoic acid Propionic acid Pyroglutamate a-ketoglutarate 2,4-dihydroxybenzoic acid Hydroxyphenylacetic acid  Gluconate Succinate Malate Glycogen* Serine* Threonine* Thiamine* Benzoate Allantoin  <J  aromatic compound MFS transporter  Growth of an opdQ mutant in propionic acid 0.2 0.1  T3  1 3  H  not used as a sole C-source by P. aeruginosa  •  porin  EDI  transporter  H  metabolic enzyme  I  regulator  H  other  I I  unknown  P—-a'  ~*~ wild-type " " mutant &  0.01  130  260  390  520  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. R. Genomic contexts of the P. aeruginosa opdQ gene and its orthologue in P.putida. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM propionic acid.  S. OpdR (PA3588)  P.aeruginosa  OpdR  Compounds tested as substrates Glucose Pyruvate Quinate Phenylacetic acid* Hydroxyphenylacetic acid Tyrosine Vanillate p-aminobenzoic acid 2,4-dihydrobenzoic acid  phenylacetic acid metabolism  bile induced protein  Growth of an opdR mutant in hydroxyphenyacetic acid 0.2 0.1 -  Jill  transporter  H  metabolic enzyme  •  /  TJ  porin  •  0.01  regulator other unknown  • wild-type "~ "~ mutant e  0.001 * = not used as a sole C-source by P. aeruginosa  i  150  225  300  375  450  Time (minutes)  Figure 2.6 Genomic contexts of the P. aeruginosa OprD porin family. S. Genomic context of the P. aeruginosa opdR gene. The size and spacings of individual genes are not to scale. Also shown are lists of carbon sources tested as substrates and a representative growth curve of the P. aeruginosa porin mutant in BM2 + 1 mM hydroxyphenylacetic acid.  in B M 2 minimal media supplemented with a single carbon source as discussed above. The carbon sources were largely chosen based on the genomic contexts of the porin genes in P. aeruginosa and their orthologous copies in P. putida and are listed in Figure 2.6. Growth in batch cultures demonstrated inherent variability from day to day but the reported differences between mutants and wild-type strains were consistently observed in at least three separate experiments. Representative curves from these experiments are shown in Figure 2.7.  Time (hours)  Time (hours)  2.7 Growth phenotypes of P. aeruginosa OprD homologue mutants. Mini-Tn5-Tc mutants in eight of the OprD homologous genes as well as their isogenic wild-type P A O l and P A K parent strains were grown in B M 2 minimal media supplemented with the porin's putative substrate as the carbon source. The mutants and carbon sources include opdP:Tn5 in 5 m M glycine-glutamate (A), opdC::Tn5 in 1 m M histidine (B), opdB::Tn5 in 1 m M proline (C), opdT::Jrv5 in I m M tyrosine (D), opdH::Tn5 in 10 m M czs-aconitate (E), and opdO::Tn5 in 1 m M pyroglutamate (F). Data shown are representative of at least three separate experiments. FIGURE  R  83  In addition to the reported selectivity of OprD for arginine and the growth deficiency of the opdK mutant in vanillate, growth defects were also observed for 6 of the remaining 17 OprD homologue mutants in the following substrates:  opdP- glycine-glutamate, opdC- histidine,  opdB- proline, opdT- tyrosine, opdH- cw-aconitate, and opdO- pyroglutamate. The extent of the growth defect varied amongst the 6 mutants compromised in growth, ranging from a very weak phenotype for the opdC, and opdB mutants (Figure 2.7 B, and C, respectively) to a very strong phenotype for the opdP, opdH, and opdO mutants (Figure 2.7 A , E , and F, respectively). In contrast, when 1 m M glucose or numerous other control substrates were used as carbon sources, the growth of these mutants was indistinguishable from that of the wild-type strains (shown in Figure 2.6).  The 11 other oprD homologue mutants did not consistently show any obvious  growth defects compared to the wild-type strain when grown in their predicted substrates (representative data are shown in Figure 2.6). 2.3.5 Induction of oprD homologues by their substrates Since specific porins tend to be induced by their substrates (7), we sought to support the proposed substrate specificity of the 8 oprD homologues by looking at their expression levels. Messenger R N A was isolated from wild-type P. aeruginosa grown in B M 2 minimal media using 10 m M of the putative substrate as the carbon source or 10 m M glucose as the control substrate. The mRNA was reversed transcribed into c D N A which was then used as the template for P C R using specific primers to small internal fragments of the respective oprD homologues predicted to take up the compound in question.  The resulting amplicons were  resolved on 2% agarose-TAE gels. The data shown in Figure 2.8 are representative of 2 separate experiments.  84  A OprD subfamily members  OpdH 11 12  B  OpdK 13 14  OpdO 15 16  OpdK subfamily members 5.5  1  4.4  6.8  c rpsL  F I G U R E 2.8  Expression of OprD homologues in response to their putative substrates.  mRNA was isolated from exponential phase P. aeruginosa P A O l grown in B M 2 + 10 m M carbon source, reverse transcribed into cDNA and used as the template for SQ-PCR. Panel A shows the expression patterns of the OprD subfamily members: OprD- lane 1 (glucose) and lane 2 (arginine), OpdP- lane 3 (glucose) and lane 4 (glycine-glutamate), OpdC- lane 5 (glucose) and lane 6 (histidine), OpdB- lane 7 (glucose) and lane 8 (proline), and OpdT- lane 9 (glucose) and lane 10 (tyrosine). Panel B shows the expression patterns of the OpdK subfamily: OpdH- lane 11 (glucose) and lane 12 (cw-aconitate), OpdK- lane 13 (glucose) and lane 14 (vanillate), and OpdO- lane 15 (glucose) and lane 16 (pyroglutamate). Panel C shows the expression levels of the control gene rpsL in glucose, arginine, glycine-glutamate, histidine, proline, tyrosine, cisaconitate, vanillate, and pyroglutamate (from left to right). Numbers indicate the ratios of porin gene expression in glucose and the putative substrate. Data shown are representative of two separate experiments.  In glucose, the oprD homologues were either not transcribed or transcribed at low levels. oprD, opdC, opdB, and opdT, all members of the OprD sub-family, showed a higher level of background expression than the members of the OpdK sub-family. In their respective substrates, the porin genes showed a higher level of induction. opdH was more highly expressed in cisaconitate, opdO in pyroglutamate, opdK in vanillate, oprD in arginine (confirming previous results (21)), opdP in glycine-glutamate, opdC in histidine, opdB in proline, and opdT in tyrosine.  85  2.4 D I S C U S S I O N  P. aeruginosa is an extremely versatile organism, able to metabolize over a hundred different compounds (33). This versatility can be partially attributed to the large number of transporter genes encoded by this organism (34). Many of these genes, including 16 members of the OprD family of porins, have only been identified based on homology to known transporters. At the amino acid level, the OprD homologues are 46% to 57% similar to OprD. The similarities become more apparent when the protein sequences of this family are aligned, suggesting that like OprD, the homologues form channels in the outer membrane that are specific for the uptake of related classes of molecules. The OprD family was first described in P. aeruginosa (34), but since that time it has become apparent that this family is found in many metabolically versatile soil bacteria and comprises over 100 members. In P. aeruginosa, the 19 members of the OprD family cluster into two distinct subfamilies. This phylogenetic distribution is preserved when the OprD homologues of P. putida are included in the analysis.  The majority of the homologues in the two organisms are  orthologous (i.e. more similar in sequence to counterparts in the other species than to other porins within the same species), implying that some of the OprD gene duplication events took place prior to the speciation of these two organisms and may share the same function in the two species.  Some of the channels, namely OpdJ, Opdl, OpdR, OpdO, OpdD, and OpdQ in P.  aeruginosa, as well as PP3390, PP3656, PP3168, and PP3939 in P. putida, lack a clear orthologue and may have arisen via post-speciation duplication events. Hence, these porins may have evolved to take up compounds found in niches unique to either P. aeruginosa or P. putida.  86  The specificity of the OprD homologues is implied by their genomic contexts. Sixteen of the nineteen genes were proximal to genes implicated in the uptake or catabolism of unique compounds. Also, none of the mutants deficient in OprD homologues were resistant to multiple classes of antibiotics indicating that the channels do not form general pores in the outer membrane. The genomic context of the opdK gene indicated that it may be involved in the uptake of small, aromatic compounds. The list of potential substrates was shortened to vanillate and vanillin upon examination of the induction pattern of an opdK:: xylE-Gvci transcriptional fusion grown in media containing various aromatic compounds. The induction of opdK in vanillate was confirmed by SQ-PCR, and the coregulation of adjacent genes is consistent with the possibility that they may be involved in vanillate degradation. PA4902, a putative transcriptional regulator, was positively regulated by vanillate.  In addition, vanillate induced the expression of two  cytoplasmic membrane transporters, an MFS transporter, PA4900, which was highly expressed, and PA4903, which was expressed at a much lower level. The expression of PA4911, a putative A B C transporter was negligible under the growth conditions tested. The TonB-dependent family member PA4897 was induced in vanillate. Members of this family of gated porins are responsible for the uptake of large compounds, such as iron-siderophore complexes, vitamin B12, and sulfate esters (35). Therefore, PA4897 may be involved in the uptake of a large aromatic compound resembling vanillate, such as short lignin polymers. Specific  porins  are  required  in nutrient-limited environments.  In these dilute  environments, outer membrane permeability becomes rate limiting for growth (19). We first tried to confirm this result for the well-characterized porin OprD.  Despite the evidence  suggesting a role for OprD in the uptake of basic amino acids (12, 21, 38), we were unable to  87  detect a growth defect when the oprD mutant was grown in arginine. A growth defect was detected, however, for the opdK mutant grown in limiting concentrations of vanillate. The discrepancy between these results suggested that the growth assay was useful for detecting significant differences in growth but not sensitive enough to detect subtle growth changes between two strains and emphasizes the need for more rigorous biochemical experiments to confirm the phenotypes discussed throughout this thesis. Repeated attempts to complement the opdK deficient strain were unsuccessful; therefore to address the possibility of polar effects exerted by the transposon in the mutant strains, I sought to corroborate the growth data by investigating the transcription of the OprD homologous genes in their proposed substrates by SQ-PCR. This combined approach led to the identification of six additional phenotypes. Including OprD, the probable specificities for the eight OprD family members are as follows: OprD, basic amino acids such as arginine; OpdP, the dipeptide glycineglutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH, cw-aconitate; OpdK, vanillate; and OpdO, pyroglutamate. The I m M carbon source concentration used was likely in great excess of the amounts of these compounds found in the natural environment and thus, the concentrations at which specific channels might be necessary.  Therefore, it is possible that many compounds, in addition to  binding to and being taken up by specific OprD homologues at low concentrations, may have been able to diffuse through other OprD homologues in a concentration gradient-dependent manner. Such compensatory uptake would obscure the contribution of a single channel and explain the relatively poor growth defects of some of the OprD homologue mutants and our inability to define phenotypes for 11 of the 19 mutants. Again, a more sensitive experimental  88  system, perhaps involving radiolabeled substrates or model membrane systems, should be employed to fully understand the contribution of each of these porins to membrane permeability. The members of the OprD subfamily that were expressed at a background level in glucose, particularly OprD, which was also expressed highly in vanillate and vanillin, are all likely candidates for permitting low levels of non-specific uptake through the outer membrane (Table 2.8, Figure 2.8). Thus, in order to greatly decrease outer membrane permeability, mutants deficient in multiple OprD homologues would have to be generated. The Ttg efflux family of the solvent tolerant bacterium P. putida DOT-TIE exhibits a similar type of compensatory . activity. A l l three known efflux systems must be knocked-out in order to completely abrogate toluene efflux (29). The redundancy of these homologous gene families confers a selective advantage to these Pseudomonas species, ensuring that in the event of a single gene loss or mutation, the function of that gene is not completely abrogated. The phenotypes that were found for OprD, OpdP, OpdC, OpdB, OpdT, OpdH, OpdK, and OpdO were consistent with the expression profiles of these porins in their substrates by SQPCR. This data demonstrates that like the other specific porins of P. aeruginosa, these eight OprD homologues are positively regulated by their substrates (7).  Thus, when a particular  substrate is present in the environment the expression levels, and hence the uptake activity of the specific porin will be maximal.  Limiting the numbers and types of channels in the outer  membrane when they are not required, in turn, would prevent the entry of potentially toxic compounds into the cell. Taken together, both the growth data and expression data indicate that the OprD family is comprised of semi-specialized uptake channels. The substrate specificities of these porins may form the basis of the phylogenetic clustering of this family into two sub-families. Members of  89  the family in the OprD subgroup; OprD, OpdP, OpdC, OpdB, and OpdT all take up amino acids and related molecules such as dipeptides. The members more similar in amino acid sequence to OpdK; OpdH, OpdK, and OpdO, take up a variety of structurally diverse carboxylic acids. The speculated substrate specificities for eight of the remaining OprD homologues are in agreement with the proposed basis of phylogenetic clustering (Table 2.8). The exceptions to this proposal include, OprE, which was predicted to take up either arginine or proline, and OprQ and Opdl, both members of the OprD sub-family, for which no substrates were assigned. TABLE  2.8 Predicted substrates of the P. aeruginosa OprD homologues  Porin OpdK sub-family OprE OpdD OpdF OpdG OpdH OpdK OpdL OpdN OpdO OpdQ OpdR OprD sub-family OprD OprQ OpdB OpdC Opdl OpdJ OpdP OpdT  Predicted substrate Arginine, or proline Short chain fatty acids or dicarboxylates Hexuronates Phathalate Tricarboxylates Vanillate Phenylacetate 5-aminolevulinate, or glutamate Lactam or other 5-membered rings Nitrobenzoate Phenylacetate Basic amino acids ~  Diterpenoids Basic compounds ~  Aromatic amino acids Dipeptides Aromatic compounds  OpdB and OpdC were originally predicted to take up a diterpenoid and cationic compound respectively.  However, these predictions were modified over the course of this  investigation based on their sequence similarities to OprD. Thus, the opdB mutant was predicted  90  to have a growth defect in a ringed amino acid (proline) and the opdC mutant was expected to grow slower on a positively charged amino acid (histidine). These modified predictions were verified, highlighting the power of combining the investigation of genomic contexts with phylogenetic analysis to predict gene function. Similar analyses can be applied to the OprD family members found in other soil organisms (listed in Table 2.1). We speculate that like P. aeruginosa, these organisms evolved a large repertoire of OprD homologues through a series of duplication events to specifically take up a vast array of metabolites while excluding other potentially harmful compounds.  It would be interesting and most informative to determine  whether the phylogenetic distributions of the OprD homologues of these organisms share the same functional basis as in P. aeruginosa.  91  2.5  1. 2.  3. 4.  5.  6. 7. 8.  9. 10.  11. 12.  13.  14.  15.  REFERENCES  Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J M o l Biol 215:403-10. Bagge, N., M. Schuster, M. Hentzer, O. Ciofu, M. Givskov, E. P. 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Embo J 23:3187-95.  94  3 F U N C T I O N A L C O M P E N S A T I O N W I T H I N T H EO P R D F A M I L Y  96  3.1 I N T R O D U C T I O N  96  3.2 M A T E R I A L S A N D M E T H O D S  98  3.2.1 Bacterial strains and growth conditions 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7  98  Primers and reagents Construction of mutants Quantitative and semi-quantitative P C R Outer membrane isolation and protein electrophoresis Radioactive arginine uptake assays Minimum inhibitory concentration (MIC) determinations  3.3 R E S U L T S  103  3.3.1 Expression profiles of eight OprD homologues 3.3.2 Arginine-mediated induction of OpdP 3.3.3 Involvement of OpdP in the compensatory uptake of arginine 3.4 D I S C U S S I O N  99 100 101 101 102 102  ;  103 105 106 108  3.5 R E F E R E N C E S  113  •  ;  95  *This chapter is being prepared for publication as T a m b e r , S . a n d R. E . W . H a n c o c k . 2006. Involvement of two related porins, OprD and OpdP, in the uptake of arginine by Pseudomonas aeruginosa. R.E.W. Hancock edited the manuscript.  3 FUNCTIONAL COMPENSATION WITHIN THE OPRD 3.1  FAMILY  INTRODUCTION  Pseudomonas aeruginosa is a ubiquitous organism capable of living in a wide variety of terrestrial, fresh water and marine environments.  In addition, this organism is capable of  infecting a range of animal and plant hosts, and in humans is most prevalently associated with lung infections in cystic fibrosis (CF) patients. This versatility is partially attributable to the large metabolic potential of P. aeruginosa. Roughly, 10% of the P. aeruginosa genome encodes transporter genes; another 8% is devoted to genes responsible for the metabolism of various carbon compounds, amino acids, fatty acids, phospholipids, and nucleotides (25). One of the more striking features of the P. aeruginosa genome is the presence of a large number of distinct gene families (25). The members of these gene families are likely to have arisen from a series of gene duplication events in either P. aeruginosa or an ancesteral organism. Once duplicated, one gene would be proposed to retain the original function, whereas the other copy would, through a series of mutations, diverge with respect to either function or regulation to create a paralogue. Such divergence increases the fitness of organisms by providing them with new cellular functions that can allow them to adapt to and thrive in changing environments (18). However, because it is expected that the gene copies will continue to share some common features, they should also be able to complement each other in the event of a gene loss or mutation, thus further increasing the fitness levels of the organism.  This functional  compensation has been studied most extensively in the yeast Saccharomyces cerevisiae, where it has been shown that when genes that have at least one paralogue in the genome are deleted, only 12.9% of those mutations are lethal; whereas the proportion of lethal deletions for singletons (genes that do not have homologues within the genome) is 29% (6).  96  In P. aeruginosa, an average gene family contains 2.7 members. Of particular note are three large families of outer membrane proteins (10). The TonB dependent family of gated porins has 32 members, and is proposed to be responsible for the uptake of large solutes, such as iron-siderophore complexes. The OprM family of efflux channel-tunnels has 18 members, and is involved in the secretion of various compounds from the cell (2, 3, 12). Finally, the OprD family of specific porins has 19 members which are involved in the transport of small, hydrophilic compounds into the cell (26). The large number of paralogous proteins in the outer membrane underscores their important contribution to cellular physiology, particularly when considering the intrinsic impermeability.of this structure. The outer membrane of P. aeruginosa is a formidable barrier to antibiotics having a permeability coefficient of 1-8% of that of E. coli (9). The basis of this impermeability lies in the highly anionic nature of the lipopolysaccharide molecules comprising the outer surface of the outer membrane (15), and the poor channel-forming activity of the major porin OprF (7). The majority of the small, hydrophilic molecules that enter the cell do so through specific porins. These channels are generally narrow but possess substrate-specific binding sites that selectively facilitate the uptake of structurally related classes of molecules (10). However, the non-specific diffusion of structurally diverse molecules is also permitted, provided that they are small enough to traverse the pore. In Pseudomonas, this general uptake activity has been observed with the basic amino acid specific porin, OprD and with the glucose specific channel OprB, which respectively permit the passage of gluconate and a variety of carbohydrates (1, 11). The P. aeruginosa outer membrane is rich in specific porins and includes the 19 members of the OprD family. It has been proposed that the members of this family arose, through a series of duplication and divergence events, from an ancestral gene to take up a variety of unique  97  substrates (25). Indeed, this diversity of uptake activities has been demonstrated for eight of the nineteen OprD homologues (Table 3.1, (26)). The extent of this divergence, however, is unclear and is investigated here with respect to the diffusion of arginine through the outer membrane. T A B L E 3.1  Substrate specificities of 8 members of the P. aeruginosa OprD family Porin OprD OpdP OpdC OpdB OpdT OpdK OpdH OpdO  Substrate Basic amino acids Glycine-glutamate Histidine Proline Tyrosine Vanillate cw-aconitate Pyroglutamate  Arginine is believed to traverse the outer membrane through OprD. This has been shown through the use of the liposome swelling assay, planar bilayer experiments, and competition experiments wherein basic amino acids compete with the structurally related carbapenems, imipenem and meropenem for the OprD binding site, which raises the MIC of those antibiotics towards P. aeruginosa (11, 28, 29). In addition, OprD is positively regulated in response to arginine via the ArgR regulator (16). However, contrary to these data, mutants lacking OprD are not compromised whilst growing in arginine (26). It is demonstrated here, that the unhindered growth phenotype of the oprD mutants is due, in part, to the overlapping activity of the glycineglutamate specific porin, OpdP, implying that this porin may play a greater role in outer membrane permeability than previously thought. 3.2 M A T E R I A L S A N D M E T H O D S  3.2.1 Bacterial strains and growth conditions  98  The P. aeruginosa strains used in this study are listed in Table 3.2. maintained on Luria Bertani (LB) agar plates.  Strains were  Antibiotics were provided at the following  concentrations for plasmid selection and maintenance: tetracycline - 50 pg/mL, gentamicin - 50 pg/mL.  Strains were grown in either Mueller Hinton (MH) or B M 2 liquid media (62 m M  potassium phosphate buffer (pH 7), 0.5 m M MgS04, 20 u M FeSGv) containing the specified carbon sources. The carbon source concentrations used were as follows, arginine transport - 100 uM, growth curves and quantitative PCR - 1 m M , all other manipulations - 10 m M . TABLE  3.2 B a c t e r i a l s t r a i n s a n d p l a s m i d s u s e d i n t h i s s t u d y  Strain PAOl PAK opdP oprD oprDlopdP  Description wild-type strain wild-type strain P A K opdP::mm\Jn5-Tc P A K oprD::xylE-Gm P A K opdP::Tn5-Tc oprD::xylE-Gm  Reference 26 26 26 This work This work  p E X l 007/oprD: :xylE-Gm  17  r  T  T  Plasmids pC831  r  x  Growth curves were performed by sub-culturing overnight Pseudomonas cultures grown in B M 2 media to a starting  ODeoo  of 0.01 into fresh pre-warmed growth media. These cultures  were incubated at 37°C on an orbital shaker shaking at 200 R P M .  Aliquots of the growth  cultures were taken at specified time intervals and the optical density at 600 nm was determined using an Eppendorf Biophotometer (Hamburg, Germany). 3.2.2 P r i m e r s a n d r e a g e n t s  The primers used to perform real-time P C R are listed in Table 2.3, the remaining primer sequences are listed in Table 3.3. A l l chemicals were obtained from either Sigma or Fisher with the exception of glycine-glutamate and C-arginine, which were purchased from Bachem 14  (Torrance, C A ) and American Radiolabeled Chemicals Inc. (St. Louis, M O ) respectively.  99  Imipenem was obtained from Merck-Sharp-Dohme (West Point, PA) and meropenem obtained from ICI Pharmaceuticals (MacClesfield, England). T A B L E 3.3  Primers used in this investigation  Primer Sequence (5'- 3') opdP gene F CGGGATCCCAGAAGAACGGCACGACTTCG opdP gene R AGGCCCGGGGAGCAACGGATGGAGCATGG oprD gene F T A T C T A G G A T C C A T G A A A G T G A T G A A G T G G A G C oprD gene R TTCGATGGTACCTACGCCCTTCCTTTATAG xylE gene F TGCGTGTACTGGACATGAGC xylE gene R GGATCGTCACCGTAATCTGC  Molecular biology reagents were all purchased from Invitrogen (Carlsbad, CA), with the exception of the SYBR-Green Master M i x which was purchased from Applied Biosystems (Foster City, CA).  3.2.3 Construction of mutants The plasmid, pC831 (17), was used to mobilize a copy of the oprD:\xylE-GvcL cassette, into P. aeruginosa P A K (to create an isogenic oprD mutant) or opdP (to create the double mutant). The cells were made competent through repeated washes with M g C k and CaCb, and transformed by heat shock according to the protocol of Schweitzer et al (5).  The resulting  transformants were plated onto L B agar containing 50 ug/mL gentamicin (for the oprD knockout) or 50 pg/mL tetracycline and 50 pg/mL gentamicin (for the oprDlopdP double knock-out). Since pC831 harbors a copy of the counter-selectable gene sacB, replacement of the native oprD genes with the interrupted copies was selected for by plating single colonies from the transformation onto L B agar plates containing the appropriate antibiotics plus 5% sucrose. The presence of the interrupted copies of oprD was verified by adding a solution of 10 m M catechol to a portion of the plate and observing a yellow color, which is indicative of the 2hydroxymuconic semi-aldehyde produced by the X y l E enzyme.  The insertions in oprD and  100  opdP were further verified by the amplifying each gene and the xylE-GvcH cassette by the polymerase chain reaction and confirming that the resulting products were of the expected size by agarose gel electrophoresis.  3.2.4 Quantitative and semi-quantitative PCR The isolation of mRNA, reverse transcription, and semi-quantitative P C R was carried out exactly as described in Tamber et al. (26). The amplification reactions were cycled for 25 rounds with the exception of the control gene rpsL, which encodes the S12 protein of the 30S ribosome, that was cycled 20 times and the opdB and opdT genes which were amplified for 30 cycles. The resulting amplicons were resolved on 2% agarose-TAE gels and the band intensities were quantified using ImageJ (http://rsb.info.nih.gov/ij/). Quantitative real-time P C R was carried out in an A B I Prism .7000 sequence detection system (Applied Biosystems) in I X SYBR-Green Master M i x using 200 n M of each forward and reverse primer.  3.2.5 Outer membrane isolation and protein electrophoresis Stationary phase cultures of P A K and its oprD::xylE-Gm mutant grown in B M 2 with 10 r  m M glucose, arginine, or glycine-glutatmate were harvested and the pellets were resuspended in an equal volume of 20% sucrose containing 100 pg/mL pancreatic DNase I (Pharmacia, Somerset County, NJ). After 2 hours of incubation at room temperature, the cells were broken by passing them through a French pressure cell twice at a pressure of 200 p.s.i. (Aminco, Silver Spring, MD). The cell lysates were layered onto a 2-step sucrose gradient consisting of ice-cold 50% and 70% sucrose. The gradients were spun in a SW-28 rotor (Beckman, Fullerton, C A ) at 28 000 R P M for 16 hrs at 4°C in a Beckman Optima L-100XP ultracentrifuge.  The outer  membrane band (at the interface between 50% and 70% sucrose) was collected, diluted in 3X the  101  volume of de-ionized water and collected by ultracentrifiigation at 43 000 R P M for 1 hour at 4°C. SDS-PAGE was carried out as described in Hancock and Carey (8).  3.2.6 Radioactive arginine uptake assays Exponentially growing cultures (between an absorbance at 600 nm of 0.4 - 0.6) of P A K , and its isogenic opdP, oprD, and opdPloprD mutants grown in B M 2 media containing 10 m M glucose were harvested and washed twice fresh medium. Cells were resuspended in pre-warmed fresh medium to a final absorbance of 1.0 and allowed to equilibrate for 20 min in a 37°C water bath that was shaking at 250 R P M . After this incubation period. l'OO p M total arginine (1:100 14  C-arginine, 300 mCi/mmol : unlabelled arginine) was added to the cells. Aliquots of 50 pL  were removed at the specified time points and placed in 1 mL ice cold 1M LiCI. These aliquots were immediately filtered through Whatman GF-C filters and washed with 5 mL LiCI.  The  filters were then placed in scintillation vials containing 5 mL ReadySafe scintillation fluid (Beckman, Fullerton, CA) and counted using a Beckman LS 500TA liquid scintillation counter. Prior to the addition of arginine, 1 mL of cells was removed, pelleted and stored at -20°C. The pellets were resuspended in 0.5 mL 1% sodium dodecyl sulfate followed by a 30 minute incubation at 37°C. The protein content of the cell lysates was then determined by a modified Lowry procedure (24).  3.2.7 Minimum inhibitory concentration (MIC) determinations Overnight cultures of the four P. aeruginosa strains were serially diluted in fresh M H to an inoculum size of 2 x 10 CFU/mL. Five microliters of diluted bacteria (approximately 10 6  4  cells) were then added to a 96-well culture dish containing M H plus two fold serial dilutions of imipenem or meropenem. The MICs (the lowest antibiotic concentrations which inhibited cell  102  growth) were determined after an 18 hour incubation period at 37°C and the modal values from 4 independent experiments are reported. 3.3 R E S U L T S 3.3.1  Expression profiles o f eight O p r D homologues  Members of the OprD porin family facilitate the diffusion of specific classes of compounds into the cell. To date, substrates specific to 8 of the 19 porins have been assigned (Table 3.1) and like the other specific porins of P. aeruginosa, they are induced to a higher copy number upon exposure to their respective substrates (10, 26). To determine whether or not these 8 channels were expressed under a broader range of experimental conditions, and thus be available for non-specific uptake activity, semi-quantitative P C R was done using primer pairs specific for the eight OprD homologue genes and c D N A generated from wild-type P. aeruginosa P A O grown in either glucose, cw-aconitate, arginine, glycine-glutamate, pyroglutamate, tyrosine, vanillate, proline, and histidine (Figure 3.1). The oprD, opdP, opdC, opdB, and opdT porin genes were transcribed on all 9 carbon sources tested. In contrast, the opdK and opdO genes were only transcribed on their respective substrates, vanillate, and pyroglutamate. OpdH was expressed on the presence of two compounds, cw-aconitate and vanillate; with its expression level being approximately three-fold higher in c/s-aconitate. When these expression data were examined in the context of the OprD family phylogenetic tree (Figure 3.1 A), an interesting pattern emerged. Members of the OpdK subfamily had a narrow expression profile. The three porins tested were expressed most highly on their respective substrates. The OprD sub-family members, in contrast, were highly expressed  103  on all of the carbon sources tested, suggesting that these proteins were likely candidates to permit the non-selective diffusion of nutrients into the cell.  A  B G ARDOTVP H  FIGURE 3.1 Expression profile of eight OprD homologues. A . Unrooted phylogenetic tree of the 19 members of the OprD family in P. aeruginosa. The tree was constructed using the neighbour-joining distance matrix method in ClustalX (27) as described in Tamber et al (30). Bootstrap values were all over 700 (out of 1000 trials) with the exception of the branches indicated by thinner lines. B. Semi-quantitative PCR analysis of OprD homologue transcription in various carbon sources. mRNA was isolated from mid-logarithmic phase P. aeruginosa P A O l cells grown in B M 2 + 10 m M carbon source, reversed transcribed and used as the template for PCR. Lane designations are as follows: G - glucose, A - cz's-aconitate, R - arginine, D - the dipeptide glycine-glutamate, O - pyroglutamate, T - tyrosine, V - vanillate, P - proline, and H - histidine. The bar graphs above the gel images depict the gene expression values normalized to the expression of each gene in glucose, grey bars correspond to the specific substrate of each porin. Data shown are representative of two separate experiments.  104  3.3.2 Arginine-mediated induction of OpdP Of the four OprD homologues that were expressed on arginine, opdP appeared to demonstrate the highest level of transcription (Figure 3.1, lane R). Also, of the seven homologues tested, OpdP shared the highest degree of sequence similarity with OprD (51%). Based on these criteria, OpdP was predicted to share some functional overlap with OprD and act as a conduit for the diffusion of arginine. The outer membrane protein profile of an oprD' mutant grown on arginine showed the up-regulation of a 50-kDa protein, which is the predicted molecular weight of OpdP (Figure 3.2, lanes 2 and 3). This protein appeared to co-migrate with OprD, and was masked by the latter porin when the outer membrane proteins of the wild-type strain grown on glucose or arginine (Figure 3, lanes 1 and 4). 1 2  3  4  OprD OprB OprE OprD (u) OprE (u) OprB (u)  Outer membrane protein profiles of wild-type P. aeruginosa and an oprD deficient mutant. Outer membranes were isolated from stationary phase cells grown in B M 2 + 10 m M carbon source. Outer membrane proteins were solubilized in 0.2% SDS and incubated for ten minutes at either 88°C (top panel) or 37°C (bottom panel), resolved on a 12% acrylamide gel, and stained with Coommassie Blue. Each lane contains 20 pg protein. Lane 1 - P A K glucose, Lane 2 - oprD glucose, Lane 3 - oprD arginine (* = potential candidate for OpdP), Lane 4 - P A K arginine. The positions of the OprD, OprB, and OprE porins in their heated and unheated (u) forms are indicated. F I G U R E 3.2  Therefore, to assess the level of opdP transcription in both strains, quantitative real-time P C R was employed. In the wild-type strain, the expression of OpdP was doubled on arginine relative  105  to glucose (Figure 3.3). In the P A K wild-type, the expression of opdP was doubled on arginine relative to glucose (Figure 3.3). This arginine-mediated induction was also observed in the oprD deficient strain. The level of opdP transcription on glucose was similar between the two strains. However, when grown on arginine, the expression of OpdP rose by 2-fold in the oprD mutant relative to the wild-type strain.  a.  O  4 H  glucose  arginine  PAK FIGURE  glucose  arginine oprD  3.3 Transcription of opdP in wild-type P. aeruginosa and an oprD deficient mutant.  mRNA was isolated from mid-exponential phase cells grown on B M 2 + 1 m M glucose or arginine, reverse transcribed into cDNA, which was then used as the template for PCR. Data shown are from two separate experiments with at least 4 replicates.  Arginine mediated induction of the oprD gene is carried out via the ArgR regulator (16). To determine whether this protein could have been involved in the regulation of opdP, the promoter region of the porin gene was examined for the ArgR consensus binding site (14). However, no significant similarities were found.  3.3.3 Involvement of OpdP in the compensatory uptake of arginine It has been shown that OprD has a specific binding site for basic amino acids and thus facilitates their diffusion through the outer membrane (11, 29). Despite these data, an OprD deficient mutant grows as well as its isogenic wild-type strain at arginine concentrations as low as 1 m M (26). Given the presence of OpdP in the outer membrane of the oprD deficient strain, it  106  seemed likely that this channel could participate in the uptake of arginine.  To test this  possibility, an oprDlopdP double mutant was constructed and its growth on B M 2 + 1 m M arginine was compared with the wild-type P A K strain, the oprD' mutant and the opdF mutant (Figure 3.4A). In this medium, after an initial lag phase of approximately 3 hours, the wild-type strain and the two single mutants grew at approximately the same rate. The oprDlopdP double mutant however exhibited a moderate growth defect. This defect was specific to arginine as all four strains grew equally well on B M 2 + glucose (data not shown). To investigate the cause of the double mutant's growth defect, the rate of radioactive arginine uptake by the four strains was determined (Figure 3.4B). After 5 min, the oprD mutant, the wild-type strain and the opdP mutant transported approximately the same amount of arginine. The double mutant, in contrast, only took up half the amount of arginine as the wild-type strain, and two-thirds of the amount taken up by the oprD' single mutant.  When the total arginine  concentration was decreased from 100 p M to 30 u M the differences in uptake kinetics among the four strains did not change appreciably (data not shown). A  B 2500 oo 3-S  2000 1500  B  a. 3  u  1000  c '5  <  Time (minutes)  3.4 Growth and uptake of arginine by an oprDlopdP deficient mutant. The wildtype P. aeruginosa, an oprD deficient mutant, an opdP deficient mutant and an oprDlopdP double mutant were assessed for their ability to grow in B M 2 + 1 m M arginine (A) and to take up a mixture of unlabelled and radiolabeled arginine (B). Growth data shown is representative of 3 separate experiments, n = 3 for the transport assay. FIGURE  107  In addition to basic amino acids, the binding site of OprD also recognizes the structurally related carbapenem antibiotics imipenem and meropenem and is the major route of passage for these two molecules into the cell (28). To determine whether OpdP was also involved in carbapenem uptake, the antibiotic susceptibilities of the four strains towards imipenem and meropenem were tested. The OprD deficient mutant exhibited an eight-fold increase in resistance to both antibiotics (Table 3.4). The deletion of opdP from either the wild-type strain or the oprD mutant, however, did not alter the antibiotic susceptibility of those strains.  TABLE  3.4 Major inhibitory concentrations (MIC) of imipenem and meropenem for P. aeruginosa strains lacking OprD, OpdP, or both porins  Strain PAK opdP oprD oprDlopdP  MIC (pg/mL) Imipenem Meropenem 0.5 0.125 0.5 0.06 4 1 4 1  3.4 D I S C U S S I O N  The OprD family of P. aeruginosa is comprised of 19 specific porins that are thought to have arisen from a single progenitor and diverged in function to allow the passage of structurally unique molecules into the cell.  Although this divergence may have been instrumental in  maintaining all 19 OprD homologues in the genome, at least two members of this family continued to share overlapping activity. This redundancy ensures that cellular functions are not lost when a related gene is inactivated and thus contributes to the genetic robustness of this organism. When the transcriptional profiles of the eight characterized members of the OprD family were examined, an intriguing pattern emerged. The five members of the OprD sub-family were  108  expressed highly in a broad range of carbon sources, whereas the three members of the OpdK sub-family exhibited a more narrow expression profile. Although the expression profiles of the 11 remaining members of this family remain to be determined, the data presented here suggested that these proteins are expressed along phylogenetic lines, and lead to some insights regarding the evolution of this family. Following duplication, paralogous genes are thought to diverge first with respect to expression, and then go on to establish new cellular functions (18).  Thus, based on their  expression patterns, the members of the OpdK sub-family, which promote the utilization of organic acids may represent a more specialized sub-group of porins. Given the narrow range of environments in which these porins were expressed it is unlikely that they were involved in complementing the transport defect of the oprD mutant examined here or the other oprD homologue deficient mutants examined previously (26), with the exception of OpdH, which was transcribed in vanillate.  Further experiments are required to determine whether the limited  expression of these genes encoding channel proteins alone contributes to their specialization, or whether their binding pockets differ significantly from those of the OprD sub-family. In contrast, the OprD sub-family members, which assist in growth on various amino acids or peptides, were expressed in all of the carbon sources tested. The regulation of this group of porins appears to be complex and warrants further study. As described previously, these channels are induced by their respective substrates and some were also induced by a number of other structurally unrelated compounds (26), presenting the intriguing possibility that the permeability characteristics of the outer membrane may be fine-tuned to suit particular environments. This observation is also consistent with a previous report demonstrating the nonspecific induction of OprD by a variety of amino acids (16) and suggests that, like OprD, the  109  members of the OprD sub-family may exhibit some general uptake capabilities, providing these compounds are small enough to traverse the channel. This activity would permit the uptake of a variety of unrelated metabolites into the cell while maintaining high intrinsic antibiotic resistance due to low outer membrane permeability (11). Of the four porin genes that were transcribed in arginine, OpdP bore the highest amino acid similarity to OprD (51%) and based on the relative levels of gene expression, following OprD, appeared to be the dominant channel when arginine was provided at the sole carbon source. Indeed, OpdP appeared to be induced by arginine in both the wild-type strain and the oprD deficient mutant. The induction was much more dramatic in the oprD mutant where expression was more than doubled relative to the wild-type strain. Although the physiological significance of over-expressing a protein in response to the loss of a related one is apparent, the mechanism by which this could occur is not clear. However, this is not an unusual phenomenon as the compensatory expression of other outer membrane proteins has been reported in both E. coli and P. aeruginosa (4, 13, 22). Removing either OpdP or OprD alone from the P. aeruginosa outer membrane did not affect the growth of the two single mutants on arginine as the sole carbon source or transport of this amino acid. However, when a double mutant was made, both its growth and transport abilities declined demonstrating that the two channels shared overlapping activity that was only apparent in that particular genetic context. This redundancy was limited to the uptake of arginine because the susceptibility of the oprDlopdP double mutant to the basic amino acid analogues, imipenem and meropenem, was identical to that of the oprD deficient mutant. Given the mass of arginine ( M W = 174) in comparison with that of glycine-glutamate ( M W = 204), it seems unlikely the passage of this amino acid was hindered by steric interactions within the pore and it  110  most probably traversed OpdP by simple, non-specific diffusion.  Imipenem ( M W = 317) and  meropenem ( M W = 438) are considerably larger than both arginine and glycine-glutamate and thus may require the presence of a binding site within the channel to facilitate their uptake (28). The observation that growth on or transport of arginine was not completely abrogated in the oprDlopdP double mutant suggests that the other OprD homologues, particularly the remaining 6 members of the OprD sub-family, may contribute to arginine uptake. Thus, with respect to this group of porins at least, transport through the outer membrane may be a collaborative effort with each protein contributing to uptake in small increments, providing that these porins exhibit overlapping functionality. This situation is analogous to the one observed with the efflux pumps of Gram-negative bacteria, wherein multiple homologous systems, which demonstrate broad, often overlapping specificities for antimicrobial agents, organic solvents, and other noxious agents, are present.  Mutations in multiple efflux systems are often required to  completely inactivate their activity (20, 23). Given the selective pressures exerted on the outer membrane, it is not surprising that Pseudomonas sp. and other closely related bacteria have retained multiple gene copies with overlapping functions. Many outer membrane proteins are phage receptors, are recognized by bacteriocins and/or the immune system, or can be used as portals for the transport of toxic agents. Thus, there are considerable advantages to alter a surface exposed protein or remove it completely.  For P. aeruginosa, this strategy is particularly relevant in the CF lung where,  following prolonged treatment with carbapenem antibiotics, mutants in OprD are frequently isolated (19, 21).  These strains are invariably resistant to imipenem and/or meropenem.  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J Biol Chem 265:15680-4.  114  4 FUNCTIONAL CHARACTERIZATION OF OPDH  116  4.1 I N T R O D U C T I O N  116  4.2 M A T E R I A L S A N D M E T H O D S  117  4.2.1 Bacterial strains, plasmids, primers, and media  117  4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8  118 120 121 121 122 123 124  Genetic manipulations Outer membrane isolation, protein electrophoresis and sequencing Reporter Gene Assays Semi-quantitative PCR (SQ-PCR) Biofilm Formation Assay OpdH purification •. Planar Bilayer Analysis  4.3 R E S U L T S  4.3.1 4.3.2 4.3.3 4.3.4 4.4.5 4.3.6 4.3.7  Genomic context of opdH..... Construction of an opdH knock-out and transcriptional fusion Role of OpdH in tricarboxylate uptake Tricarboxylate specific induction of OpdH Regulation of opdH by PA0756-PA0757 Involvement of OpdH in biofilm formation Channel function of OpdH  124  124 126 128 128 132 134 136  4.4 D I S C U S S I O N  140  4.5 R E F E R E N C E S  146  115 This chapter is being prepared for publication as T a m b e r , S. a n d R . E . W . H a n c o c k . 2006. Characterization of OpdH: A Pseudomonas aeruginosa porin involved in the uptake of tricarboxylates. R . E . W . Hancock edited the manuscript.  4 FUNCTIONAL CHARACTERIZATION OF OPDH 4.1 I N T R O D U C T I O N  Pseudomonas aeruginosa is a metabolically versatile organism commonly found in many terrestrial and aquatic environments. Its emergence as an opportunistic pathogen has paralleled many advances in medical care, such as the use of indwelling catheters and immunosuppressive drugs (15, 34). Once an infection has been established, it is difficult to eradicate due to the intrinsic resistance of this pathogen to many classes of antibiotics. This resistance is based on the low permeability of the P. aeruginosa outer membrane which ensures that the secondary resistance mechanisms, such as efflux pumps and (3-lactamases, of this organism are not overwhelmed (20). Depending on the compound, the permeability of the P. aeruginosa outer membrane is 10 to 500 fold lower than that of E. coli (57). This difference between the two organisms has been largely attributed to the relative abundance of specific porins in P. aeruginosa, whereas E. coli primarily uses general porins for uptake (22, 57). The P. aeruginosa channels are more selective than their general counterparts in E. coli and contribute to the barrier function of the outer membrane in two ways.  First, the selectivity imposed by their binding sites precludes the  entrance of many compounds. Second, they are not highly expressed unless in the presence of their substrates (1, 36).  This limited expression of specific porins prevents the uptake of  unrelated compounds by reducing the number of potential conduits into the cell. The cw-aconitate specific porin, OpdH, of P. aeruginosa has been recently described (47). This protein is a member of the OprD family of porins bearing 53% amino acid sequence similarity to P. aeruginosa P A O l OprD and 65% similarity to the phenylacetic acid-specific porin PhaK of P. putida KT2440 (37). Previous work has shown that an opdH deficient mutant  116  does not have the ability to grow in minimal medium with cz's-aconitate as the sole carbon source (47).  In this investigation, we expand on that work, through functional and regulatory  experiments and demonstrate that OpdH may have a general role in tricarboxylate uptake and in the formation of biofilms when citrate is the sole carbon source for growth. 4.2 M A T E R I A L S A N D M E T H O D S  4.2.1 Bacterial strains, plasmids, primers, and media The bacterial strains and plasmids used in this study are listed in Table 4.1. The opdH primers used to for the semi-quantitative P C R analysis are listed in Table 2.3 the rest of the primer sequences are listed in Table 4.2. Strains were routinely maintained on Luria-Bertani (LB) agar. Antibiotics for selection and maintenance were used at the following concentrations; tetracycline 100 ug/mL for P. aeruginosa, 12.5 pg/mL for E. coli, gentamicin 50 ug/mL for P. aeruginosa, 15 pg/mL for E. coli, carbenicillin 500 jig/mL for P. aeruginosa, and ampicillin 100 ug/mL for E. coli. For cloning experiments strains were grown in L B broth. For all other experiments P. aeruginosa strains were grown in B M 2 minimal media (62 m M potassium phosphate buffer (pH 7), 0.5 m M M g S 0 , 10 u.M FeS04) supplemented with specific carbon 4  sources at the indicated concentrations.  Overnight cultures were diluted to a starting optical  density of 0.1 for growth experiments. For reporter gene assays, the cultures were inoculated with single colonies from L B plates. When required, the pH of the carbon sources was adjusted to 7. For experiments requiring low magnesium, the M g  2 +  concentration was altered to 20 u M .  High iron conditions were achieved by adding FeS04 to a final concentration of 1 OOuM to the medium. Low iron conditions were created by culturing the bacteria in acid-washed glassware and adding dipyridyl to a final concentration of 0.3 m M .  117  TABLE  4.1 Bacterial strains and plasmids used in this study Description  P. aeruginosa strains H103 opdH 4 opdH 10 PAK opdH 26 UW-37777 UW-PAO UW-PA0756 UW-PA0757 UW-PA0756' UW-cbrA UW-cbrB Crc H851 H854 PA0748 RpoS E. coli strains DH5a S17-1 Plasmids pCR2.1 pEX18Tc pX1918GT pUCP20  Reference or source  P A O wild-type strain HI03 opdH::xylE-Grrf lopdH HI03 opdH.:xylE-Gm P A K wild-type strain P A K o/?d#::miniTn5-Tc P A O oj^ris::miniTnJ-Tc P A O reference strain P A O Pv4075r5::miniTn5-Tc P A O Pv40757::miniTn5-Tc UW-PA0756 complemented with PA0756 in pUCP20 PAO cbrA::mmiTn5-Tc P A O cbrB::miniTn5-Tc P A O crc::Tc HI 03 phoP::xylE-Gm HI 03 phoQ::xylE-Gm H103 PA0748::luxCDABE-Tc PAO rpoS::Tc  This study This study (47) (47) (26) (26) (26) (26) This study  T  r  r  r  r  (26) (26) (54) (30) (30) (29) (49)  T  r  r  r  r  r  r  General cloning strain Mobilizing strain  (19) (39)  T A cloning vector, A p , K m suicide vector containing sacB gene, Tc source of xylE-Gm cassette, Ap , G m Broad host range expression vector, Cb r  T  r  r  r  r  r  Invitrogen (45) (45) (51)  4.2.2 Genetic manipulations Routine cloning procedures were carried out as described by Sambrook et al (40). A l l enzymes and cloning kits were provided by Invitrogen (Carlsbad, C A ) , with the exception of Vent polymerase which was provided by New England Biolabs (Ipswich, M A ) . A n opdH knock-out and transcriptional fusion was constructed by amplifying the gene from in P. aeruginosa HI03 with Vent polymerase in I X reaction buffer, with 2 m M MgS04 and 5% D M S O , 0.4 m M dNTPs and 1 p M of each primer. After an initial denaturation step at 94°C for 2  118  minutes, the amplification was incubated for 25 cycles at 94°C for 1 minute, 63.5°C for 1 minute, and 72°C for 1.5 minutes followed by a final extension step at 72°C for 5 minutes. TABLE  4.2 D N A primers used in this invetigation  Primer opdH gene F opdH gene R PA0756 gene F PA0756 gene R PA0754 real-time PA0754 real-time PA0756 real-time PA0756 real-time PA0757 real-time PA0757 real-time  F R F R F R  Sequence (5' - 3') TAAATGGCTTCTCTGCCACC CTGTGGAGTTCTTGTTCTGGC GGAATTCACGAACGTACAGACCTTGAG GCTCTAGAGTTCCAGTAGGCGCTCAGG CGAACCCAAGCGTCCCGAAT CCAGCTTGCAGGTGAGGTCGA CATCCTTCTGGTGGAAGATCA CCGTCCTGC AGCAGGTCTAC AGGGCGGCAACGTGATC AATTCCCGGACCATCGTCTT  The resulting amplicon was treated with Taq polymerase and cloned into pCR2.1 TOPO using the T A cloning kit and transformed into electro-competent E. coli DH5a. The opdH gene was excised from pCR2.1 TOPO using HindUl and Xbal and cloned into the corresponding sites in pEX18Tc. The opdH gene was then cut with Sbfl and a xylE-Grrx cassette from pX1918GT was cloned into this site. The orientation of the xylE-Gm cassette was confirmed by restriction r  analysis. This plasmid was transformed into electro-competent E. coli SI7-1 and then mobilized into P. aeruginosa HI03 by biparental mating followed by successive selection on gentamicin and 5% sucrose. The replacement of native opdH with opdH.:xylE-Gm in the resulting strain T  was confirmed by PCR analysis. The opdH deficient strain was complemented by creating a merodiploid. Essentially, a mating reaction between the E. coli SI7-1 strain harbouring the opdH.:xylE-Gvrv construct and P.aeruginosa HI03 was performed.  The resulting clones were only selected on gentamicin,  leaving a wild-type copy of the opdH gene in the resulting strain. The presence amd orientation  119  of both the wild-type and the insertionally inactivated copies of the opdH gene were confirmed by PCR analysis. The PA0756 gene was cloned into the UW-PA0756 mutant strain by amplifying the gene as well as the 200 base-pair region upstream of it from U W - P A O with Platinum Pjx in 2 X reaction buffer and 2 X of the provided enhancer solution, with 1 m M MgS04, 0.3 m M dNTPs, and 0.3 u M of each primer. This mixture was incubated at 94°C for 5 minutes, followed by 25 cycles consisting of 15 seconds at 94°C, 30 seconds at 56.4°C, and 1 minute at 68°C and a final extension step at 68°C for 3 minutes. The resulting amplicon was cloned into pCR2.1 TOPO, excised with EcoRl and Xbal, and subcloned into pUCP20.  The orientation of the gene was  confirmed by restriction analysis and the resulting construct was transformed into chemically competent UW-PA0756. The presence of the PA0756 gene in the UW-PA0756' complemented strain was confirmed by both restriction and PCR analysis.  4.2.3 Outer membrane isolation, protein electrophoresis and sequencing Outer membranes were isolated from stationary phase cultures as described previously (21). Cells were harvested and the cell pellet resuspended in an equal volume of cold 20% sucrose containing 100 ug/mL freshly prepared DNasel (Pharmacia, Somerset County, NJ) and incubated at room temperature for at least an hour. The cells were passed through a French pressure cell (American Instrument Co. Silver Spring, MD) twice at a psi of 1500. The resulting lysate was layered onto a two-step sucrose gradient consisting of cold 70% and 50% sucrose, placed in a swinging bucket rotor (SW28, Beckman Instruments, Fullerton, C A ) and spun in a Beckman Coulter Optima L-100XP ultracentrifuge at 28 000 R P M for 16 hours at 4°C. The outer membrane fractionated at the interface between the 50% and 70% sucrose. This band was collected, diluted with distilled water and centrifuged in a Ti70 rotor (Beckman) at 43 000 R P M  120  for 1 hour at 4°C. Outer membranes were resuspended in 10 m M Tris-Cl (pH 8), and protein concentration was determined as described in (41).  Proteins were resolved in 11% SDS-  acrylamide gels as described in (21). The N-terminal sequence of the putative OpdH protein was determined by Edman degradation.  Following electrophoresis, the proteins were transferred onto a P V D F 1.0 p  membrane (Millipore, Billerica, M A ) .  The band corresponding to OpdH was excised and  sequenced using an Applied Biosystems 494 HT protein sequencer (Applied Biosystems, Foster City.CA). 4.2.4 Reporter Gene Assays The catechol-2,3-dioxygease activities of the opdH::xylE-Gm transcriptional fusion was r  determined as described previously (45).  Cells were harvested at the indicated times,  resuspended in 50 m M potassium phosphate buffer (pH 7.5) + 10% acetone, and lysed by sonication. Unbroken cells were removed by centrifugation. The reaction buffer consisted of 50 m M potassium phosphate buffer (pH 7.5) and 0.3 m M catechol. Aliquots of the cell extracts were added to the reaction buffer and the increase in absorbance at 375 nm was monitored on a dual beam P Y E Unicam PU8600 UV-visible light spectrophotometer (Philips, Amsterdam, NE). The change in absorbance was used to calculate the amount of 2-hydroxymuconic semi-aldehyde produced using a molar extinction coefficient of 44 000. The protein content of the cellular extracts was determined by a modified Lowry method (41). 4.2.5 Semi-quantitative P C R (SQ-PCR) SQ-PCR was carried out as described previously (47). Breifly, mRNA was isolated from exponential phase cells RNeasy mini R N A isolation kit (Qiagen, Mississauga, ON). Residual genomic D N A was removed using a DNA-free kit (Ambion, Austin, T X ) .  The R N A was  121  quantified by determining its absorbance at 260 nm and its quality was assessed by comparing its absorbance at 260 nm with its absorbance at 280 nm and by examining its appearance on a 2% agarose-TAE gel. R N A aliquots were stored at -80°C. Four micrograms of total R N A was combined with 750 ng of the random decamer (NS)s and incubated at 70°C for 10 minutes followed by 10 minutes at 25°C. A mixture containing I X reverse transcription reaction buffer, 10 u M DTT, 0.5 u M dNTPS, 500 units/mL of Superase IN (Ambion), and 10 000 units/mL of Superscript RT II (Invitrogen) was added to the R N A and incubated for an hour at 37°C and two hours at 42°C. Following reverse transcription, the R N A was destroyed by the addition of 170 m M NaOH and incubation at 65°C for 15 min. The mixture was neutralized by the addition of 170 m M HCI. One microliter of the resulting c D N A was used as the template for SQ-PCR using primers designed to either the intergenic or intragenic regions of the specified genes.  The  amplification reactions were carried out using Taq polymerase (Invitrogen) in I X reaction buffer containing 2 m M M g C l , 5% D M S O , 0.4 m M dNTPs, and 40 n M of forward and reverse 2  primers. The reactions were cycled for 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C 20 or 25 times. The amplicons were resolved by electrophoresis on 2% agarose-TAE gels stained with 50 pg/mL ethidium bromide. The intensities of the resulting bands on the agarose gels were quantified using ImageJ (http://rsb.info.nih.gov/ij/). A l l reported values were normalized to the levels of rpsL. 4.2.6 Biofilm Formation Assay Biofilms were formed in 96-well microtitre plates as described in (38).  Overnight  cultures were grown in B M 2 media containing 10 m M glucose and diluted to an optical density of 0.01 in B M 2 minimal medium. One hundred microliters the diluted cells were added to the  122  wells of a polystyrene plate (BD Biosciences, San Jose, CA).  Glucose, citrate, and benzoate  were then added at the specified concentrations to the appropriate wells.  The plates were  incubated at 37°C for 48 hours. After two days, the supernatants were decanted and the adherent cells were washed 3 times with phosphate buffered saline (PBS). The cells were then stained with 0.1 % crystal violet for 30 minutes, washed 3 times with PBS, and resuspended in 100% ethanol. The absorbance of the ethanol extracts was determined at 600 nm using a PowerWave X340 plate reader (Biotek Instruments Inc., Winooski, Vermont).  Motility assays were  performed as previously described, using solid media containing 1% agar (29). 4.2.7 O p d H purification Outer membranes were isolated from stationary phase cultures of an OprE deficient mutant grown i n B M 2 minimal media containing 10 m M citrate (pH 7) as described above. The outer membrane pellet was resuspended in 0.5 % Octyl-POE (Bachem, Torrence, CA) in 10 m M Tris-HCl (pH 8), placed on a rotating platform for 1 hour at 37°C and centrifuged at 43 000 R P M for 45 min at 4°C. The supernatant was reserved and the procedure was repeated using 3% Octyl-POE, followed by 3% Octyl-POE + 0.2 M NaCl, and then 3% Octyl-POE + 5 m M E D T A (pH 8). The supernatant from this final solubilization step contained OpdH and was dialyzed for .2 days in 0.6% Octyl-POE, 10 m M E D T A , 10 m M Tris-HCl (pH 8) at 4°C. The dialysed protein mixture was fractionated using an ion exchange FPLC column (MonoQ, Pharmacia) that had been equilibrated with the dialysis buffer. The proteins were eluted by applying a linear gradient of NaCl from 0 m M to 100 m M . The protein-containing fractions were analyzed by SDS-PAGE and those containing OpdH were pooled and dialyzed for two days in 0.6% Octyl-POE and 25 m M histidine (pH 5.5). Further purification was achieved by loading the protein mixture onto an equilibrated chromatofocussing column (MonoP, Pharmacia) and eluting with 12% Polybuffer  123  7.4 pH 3.5 (Pharmacia).  The resultant fractions were analyzed by SDS-PAGE and those  containing OpdH were aliquoted and stored at -80°C.  4.2.8 Planar Bilayer Analysis Planar bilayer experiments were performed as described previously (10). Single channel conductance experiments were carried out using a Teflon cell containing two chambers connected by a small hole (0.1 mm diameter). The chambers were filled with an equal volume of buffered salt solution (pH 7) and a calomel electrode was placed in each.  One of the  electrodes was connected to a voltmeter, which was set to 20 mV and the other to a Keithley 427 current amplifier (Keithley Instruments, Cleveland, OH). Bilayers were formed by painting a solution of 1.5% oxidized cholesterol in «-decane on either side of the hole. Once the bilayers had tuned optically black, 1 pL of concentrated OpdH (~ 100 ng) in a solution of 0.1% Triton X 100 was added to one side of the membrane. Single channel insertion events were monitored on a digital storage oscilloscope (Tecktronix, Beaverton, OR) and recorded using a Houston Instruments strip chart recorder (Bausch and Lomb, Rochester, N Y ) . For the macroscopic conductance experiments, the holes in the Teflon cells were much larger (2 mm diameter) and one of the electrodes was connected to a Keithley electrometer instead of the current amplifier. Once the protein had been added, the membrane conductance increased rapidly for 15 min and then slowed down considerably. At this point, aliquots of concentrated substrate solution (pH 7) were added to both sides of the membrane while the bathing solutions in the chambers were being stirred. Changes in the current level were recorded and once stabilized, additional aliquots of substrate were added to the chambers. 4.3 R E S U L T S  4.3.1 Genomic context of opdH  124  The opdH gene (PA0755) is upstream of three genes homologous (79% to 84% amino acid similarity) to the genes encoding the osmotic-shock sensitive TctABC tricarboxylate transport system of Salmonella enterica serovar Typhimurium (Figure 4.1 A) (52).  This system  consists of a periplasmic, tricarboxylate binding protein, TctC, and two inner membrane protein components that have yet to be characterized. Unlike the arrangement in P. aeruginosa, the S. Typhimurium system lacks an outer membrane component. membrane segments (TM) and TctB has four predicted TMs.  TctA has 12 predicted trans-  This transport system has recently  been identified as the prototype of a large family of extracytoplasmic solute receptors termed the tripartite tricarboxylate transporters (TTT, http://www.tcdb.org/tcdb/index.php?tc=2.A.80) (4).  Gene: PA0748 Homology:  PA0749  MmsR  Putative function:  Regulator  PA0754  • • •  PA0754-oprf/7  HQ  PA0750  PA0751 AmoA  PA0752 PA0753 PA0754 TctA TctB TctC  Monooxygenase  Tricarboxylate transport  opdH  PA0756 PA0757  Porin  Two-component regulatory system  TctD  TctE  B 1  2  opdH PA0756  BH  PA0757  U J  opdH. A . Lines above the genes indicate the location of the intragenic (solid lines) and intergenic (dashed lines) regions that were amplified by SQ-PCR. Functional classes of the genes are indicated by the following fills: enzyme involved in carbon catabolism- stippled; inner membrane transporter- white; porin- black; two-component regulatory system- grey. B . Transcription of regions within the opdH gene cluster. mRNA was isolated form exponential phase cells grown in either BM2 + 10 m M glucose (lane 1) or 10 m M citrate (pH 7) (lane 2), reverse transcribed into c D N A which was amplified for 25 cycles by SQPCR. Results shown are representative of two separate experiments. F I G U R E 4.1 Genomic context of  125  In Salmonella, the expression of the TctABC transport system is controlled by the divergently transcribed two-component regulatory system TctDE (53).  The P. aeruginosa  TctDE homologues, PA0756-757 shared 73% and 56% amino acid similarity to their respective counterparts in S. Typhimurium and are also closely related to the PhoPQ and PmrAB twocomponent regulatory systems from both organisms (18, 30, 32). The fifth gene in the putative opdH operon bore 70% amino acid similarity to the AmoA membrane-bound ammonia monoxygenase of P. putida (16). As the substrate specifities of these enzymes are quite broad (5), it is possible that PA0751 may be involved in the metabolism of compounds brought into the cell via OpdH, and PA0752-754. The previous demonstration of OpdH's role in cz's-aconitate uptake (47), and the high degree of similarity shared by the TctABCDE system of S. Typhimurium and PA0752-757 suggested that this group of genes in P. aeruginosa may be involved in tricarboxylate uptake. SQ-PCR analysis of the levels of PA0754 and opdH transcription with either glucose or citrate as the sole carbon source indicated that both genes were induced as a single transcriptional unit in citrate (Figure 4.IB) but not on other carbon sources, including succinate, malate, pyruvate, fumarate, arginine, and benzoate (data not shown). Applying a similar analysis to the genes of the putative two-component regulatory system PA0756-PA0757 showed that these genes were expressed in all conditions tested; the data obtained from cultures grown in glucose and citrate are shown in Figure 4.IB.  4.3.2 Construction of an opdH knock-out and transcriptional fusion A n opdH deficient mutant and transcriptional fusion was constructed in a P. aeruginosa PAO background by inserting a copy of a xylE-Gm cassette a cloned copy of the gene. This r  construct was used to replace the native chromosomal copy by homologous recombination to  126  create opdH 10. The mutant isolated after the double cross-over exhibited a small colony phenotype on both rich and minimal media. Despite its smaller size, opdH 10 shared the same colony and microscopic morphologies as the wild=type HI03 strain. Outer membranes isolated from the transcriptional fusion grown in BM2 minimal media +10 m M citrate lacked a 40 kDa protein that was present in the wild-type strain (Figure 4.2, lanes 1 and 2). The N-terminal amino acid sequence of the 40 kDa band from the wild-type strain, A X F L E D , corresponded to the first six amino acids of OpdH (AGFLED). Attempts to complement the opdH transcriptional fusion by placing a cloned copy of the opdH gene into various expression vectors resulted in clones with the gene inserted backwards or with two copies of opdH inserted in tandem. We reasoned that over-expression of the gene was lethal (46) and created a merodiploid by crossing the opdH::xylE-Gm  x  cassette into the  chromosome and selecting for single cross-over events. The resulting strain, opdH 4, contained a 40 kDa outer membrane protein as assessed by SDS-PAGE electrophoresis (Figure 4.2, lane 3), which corresponded to the size of OpdH.  FIGURE  4.2 Outer membrane profiles of wild-type P. aeruginosa, an opdH mutant, and an  opdH complemented strain. Outer membranes were isolated from stationary phase cultures grown in B M 2 + 10 m M citrate (pH 7) and 20 pg were resolved on 11% SDS-PAGE acrylamide gels. Lane 1- wild-type P. aeruginosa H103, lane 2- opdH 10, lane 3- opdH 4, lane 4Pharmacia low range molecular weight marker  127  4.3.3 Role of OpdH in tricarboxylate uptake Previously, it was shown that an opdH transposon insertion mutant {opdH 26) was compromised in its ability to grow on cw-aconitate (47). This phenotype was specific to cisaconitate as both the opdH 26 mutant and its isogenic wild-type strain P A K grew equally well on citrate and not at all on isocitrate (data not shown). It was unclear whether the growth defect on cz's-aconitate was due the polar effects of the transposon as repeated attempts to complement opdH 26 were unsuccessful. Therefore, growth curves in B M 2 minimal media + 5 m M glucose, cz's-aconitate, citrate, or isocitrate were perfomed using the opdH transcriptional fusion and opdH merodiploid created in this study. In comparison to the P. aeruginosa P A O wild-type strain, and the merodiploid, the opdH deficient strain grew much slower on all of the substrates tested. However, this growth defect could not be attributed to the. lack of OpdH because the transcriptional fusion exhibited a small colony phenotype.  Therefore, in order to obtain a  meaningful comparison, the growth of the three strains on the tricarboxylic acids was normalized to their growth on glucose (Figure 4.3).  Once the cultures reached stationary phase, after  approximately 6.5 hours, the opdH transcriptional fusion was significantly compromised in growth on both cw-aconitate and isocitrate (p < 0.05) but not on citrate. The merodiploid in contrast did not demonstrate any growth defects.  The growth profiles of the three strains on  succinate, pyruvate, fumarate, and a-ketoglutarate were also assessed but no differences were found (data not shown).  4.3.4 Tricarboxylate specific induction of OpdH As opdH was expressed on citrate (Figure 4.1) and both opdH deficient mutants (opdH 10 and opdH 26) did not exhibit significant growth defects when citrate was used as the sole  128  carbon source (Figure 4.3), this compound was used to investigate the expression of opdH throughout the cell cycle (Figure 4.4A).  complement  c/s-aconitate  citrate  isocitrate  Carbon source  FIGURE 4.3 Growth phenotype of an opdH deficient mutant. Wild-type P. aeruginosa H103, the opdH 10 mutant and opdH 4 complemented strain were grown in B M 2 + 5 m M cis-  aconitate, citrate, or isocitrate. The turbidity values obtained at the onset of stationary phase (after approximately 6.5 hours) is reported. The growth of all three strains was normalized to their growth in B M 2 + 5 m M glucose due to the small colony phenotype of the opdH mutant. Values reported are the means and standard errors of two separate experiments. * = p < 0.05  The catechol-2,3-dioxygenase (CDO) activity of the transcriptional fusion increased as the cell cycle progressed, reaching its maximal level during late exponential phase (A oo = 0.8) (Figure 6  4.4). This expression level was maintained for approximately two hours as the cells entered stationary phase and then dropped from 3805 pmol/pg/min to 2357 pmol/u.g/min after the cells had been in stationary phase for approximately two and a half hours.  The level of opdH  expression decreased even further after the culture was grown over night for a total of 29 hours to 1702 pmol/p-g/min. The decreased expression of opdH during late stationary phase suggested that it may have been under the control of the RpoS stationary phase sigma factor (48). To test this hypothesis, a mutant in the rpoS gene was grown in B M 2 media +10 m M glucose or citrate as the sole sources of carbon. Messenger R N A was extracted from both cultures, reverse transcribed into cDNA,  129  which was then used as a template for SQ-PCR. The patterns and levels of opdH expression in the mutant and in the corresponding wild-type strain were identical, suggesting that RpoS is not involved in the expression of this porin (data not shown). A  B  ] C D O activity " Turbidity  Time (hours)  C i t r a t e  concentration (mM)  Expression of O p d H in citrate. A . Expression of OpdH as a function of the cell cycle. A n opdH::xylE-Gm transcriptional fusion was grown in B M 2 +10 m M citrate (pH 7). Aliquots were taken at the indicated intervals for turbidity and CDO activity determinations. B . Dose response of opdH to citrate. C D O activities and turbidity measurements were determined from stationary phase cultures of the opdH.:xylE-Gm transcriptional fusion grown in B M 2 media + 1, 2, 5, 10, or, 20 m M citrate (pH 7). Results shown are representative of two separate experiments; n = 2 for 4A and n = 3 for B . FIGURE 4.4  r  r  During the later stages of growth, the carbon source becomes limiting and may influence porin expression. To test i f the expression of opdH was sensitive to the levels of citrate in the media, the transcriptional fusion was grown in B M 2 with 1, 2, 5, 10, or 20 m M citrate. The level of opdH expression increased with the citrate concentration, and reached a maximum at 10 m M citrate (Figure 4.4B).  Therefore, based on the results presented in Figure 4.4, all subsequent  induction assays were performed using early stationary phase cells grown in 10 m M carbon source. To investigate whether the citrate-mediated and the previously reported ds-aconitatemediated induction of opdH, could be generalized to other di- and tricarboxylic acids, the opdH transcriptional fusion was grown on all of the T C A cycle intermediates and then assayed for  130  CDO activity.  The cells grown on citrate demonstrated the highest C D O activity (925  pmol/pg/min) followed by the cells grown on c/s-aconitate (355 pmol/pg/min) and isocitrate (225 pmol/pg/min) (Figure 4.5). Cells grown on oxaloacetate, malate, and pyruvate had low levels of opdH expression ranging from 13 pmol/pg/min to 37 pmol/pg/min. The C D O activities obtained from the cells grown on succinate, fumarate, and oc-ketoglutarate were all less than 10 pmol/pg/min, thus considered insignificant.  a-ketoglutarate  succinate 2.92 ± 0.2  citrate 925 ±187 4.5 Expression of O p d H in the T C A Cycle intermediates. Representation of the T C A cycle and the C D O activities of stationary phase cultures of an opdH: :xylE-Gm transcriptional fusion grown in minimal media containing one of these intermediates (10 m M , pH 7) as the sole carbon source. For all compounds, n = at least 2. FIGURE  T  The effects iron and magnesium on opdH transcription were also examined as citrate is a known chelator of divalent cations (Figure 4.6 (17)). The opdH transcriptional fusion was grown on B M 2 + 10 m M citrate- standard, low magnesium, high iron, or low iron conditions, collected at stationary phase and assayed for C D O activity. The growth of the low iron culture was  131  severely compromised and required 2 days to reach early stationary phase. No significant effects on gene expression were detected in the presence of either excess iron or limiting concentrations of magnesium. However, in the low iron condition, the CDO activity was half of that observed in standard conditions. This result was confirmed by examining the outer membrane profiles of the P. aeruginosa wild-type HI03 strain grown in both the standard citrate or the citrate and low iron conditions by SDS-PAGE electrophoresis (Figure 4.6B). The observed down-regulation of opdH, was concomitant with the induction of several protein bands between 45 and 70 kDa. A  B  X -a a.  9  1.20  X  1  X  100  2  3  94 kDa 63 kDa  0.60  •C  0.40  u OS  0.20  MM M t r UM Fe  Standard Low M g 500 20 10 10  2+  High Fe 500 100  Low Fe 500 0  OpdH  4.6 Effect of metal ions on O p d H expression. A . The opdH transcriptional fusion was grown in B M 2 media + 10 m M citrate (pH 7) + MgS04 and FeS04 at the indicated concentrations. Low iron conditions were achieved by adding dypyridyl to the media at a final concentration of 0.3 mM. Results shown are representative of 3 experiments, n = 2, * = p < 0.05. B. Outer membrane protein profiles of wild-type P. aeruginosa HI03 grown in B M 2 + 10 m M citrate, pH 7 (lane 2) and B M 2 citrate + low iron (lane 3). Lane 1 = Pharmacia low range molecular weight marker FIGURE  4.4.5 Regulation of opdH by PA0756-PA0757 Mutants in the putative two-component regulatory system PA0756-757 were obtained to determine whether this system plays a role in the citrate induced expression of opdH. Messenger R N A isolated from exponential-phase wild-type P. aeruginosa P A O strain, the PA0756 mutant, and the PA0757 mutant grown on BM2 media + 10 m M glucose, succinate, cw-aconitate, citrate,  132  or isocitrate was reverse transcribed into c D N A and used as the template for SQ-PCR with primers designed to a 100 base-pair region within the opdH gene. In the wild-type strain, opdH was expressed highly on all three tricarboxylates tested relative to glucose and succinate (Figure 4.7A). This pattern was conserved in both the regulator (PA0756) and sensor kinase (PA0757) mutants.  The expression level of opdH did not differ  significantly among the three strains when they were grown in any one of the tricarboxylates. A striking difference between the three strains was observed, however, when they were grown on glucose and succinate (Figure 4.7A, lanes 1 and 2). On these compounds, opdH was expressed 3 and 6-fold higher in the PA0756 mutant compared to the parent strain, and 8 and 9-fold higher in the PA0757 mutant strain. The repression of opdH in succinate was restored when the PA0756 gene was over-expressed in the mutant strain (Figure 4.7A, lanes 6 and 7). These data indicate that PA0756-757 combine to repress opdH and the repression is relieved in the presence of citrate. PA0754, which is homologous to the periplasmic binding protein TctC of S. Typhimurium, was regulated by PA0756 in the same manner as opdH (Figure 4.7B). Because tricarboxylate-mediated induction of opdH was elevated in the PA0756-757 mutant strains, the possible involvement of a transcriptional activator was investigated. Candidates for this role included the CbrAB two-component regulator and the Crc protein, which are involved in the metabolism of citrate and succinate respectively (14, 33); the PhoPQ twocomponent regulatory system, which is homologous to PA0756-757 (30); and the putative regulator PA748, which is proximal to the 5 gene cluster containing opdH (Figure 4.1). The levels of opdH transcription in mutants lacking these regulators, however, were identical to those of the wild-type strain suggesting that these systems do not have an impact on opdH expression (data not shown).  133  A  B  PAO  PA0756-  PA0757-  4.7 Regulation of opdH and PA0754 by the PA0756-757 two-component regulatory system. Strains were grown on BM2 + 10 m M of the indicated carbon source. Messenger R N A was extracted from exponential phase cells, reverse transcribed and used as the template for 25 rounds of SQ-PCR. Numbers underneath the gel images indicate the ratios of gene expression in the mutant strain relative to the wild-type strain. A l l values were normalized to the levels of rpsL. Data shown is representative of at least two separate experiments. A . Expression of opdH in wild-type P. aeruginosa U W - P A O (top panel), UW-PA0756 and UW-PA0756' (middle panel), and UW-PA0757 (lower panel). Lane 1- glucose, lane 2- succinate, lane 3- citrate, lane 4- cis-aconitate, lane 5- isocitrate, lane 6- UW-PA0756' succinate, and lane 7- UW-PA0756' citrate. B. Expression of PA0754 in U W - P A O (lanes 1, 2) UW-PA0756 (lanes 3, 4) and U W PA0756' (lanes 5, 6) grown in B M 2 + succinate (lanes 1, 3, and 5) or citrate (lanes 2, 4, and 6). FiGURE  4.3.6 Involvement of OpdH in biofilm formation Previous work has shown that the type of biofilm produced by P. aeruginosa varies with carbon source.  Biofilms made on glucose tend to produce large, mushrooming, structures,  whereas biofilms produced on citrate tend to be flat and dense (27). Contrary to the events that occur during biofilm formation in glucose, the initiation of biofilm formation in citrate occurs independently of pili and flagella (27). As OpdH is a present as a surface structure when cells are grown in citrate, we sought to determine whether this protein was required for the formation of biofilms in this compound (Figure 4.8).  134  B. 0.25  0.15  0.05  5  2.5  1.25  0.63  0.3 mM  Citrate concentration  5  2.5  1.25  •rn i i i i i i i 2.5  1.25  0.63  0.3 mM  Glucose concentration  0.63  0.3 mM  Benzoate concentration  4.8 Involvement of opdH i n biofilm formation. Biofilms in B M 2 + citrate (A), glucose (B), or benzoate (C) at the indicated concentrations were formed in 96-well microtitre plates using wild-type P. aeruginosa P A K and an isogenic opdH deficient mutant. Results shown are representative of 3 separate experiments; n = 5,*=p<0.05. FIGURE  Since the opdH transcriptional fusion demonstrated a small colony phenotype, the opdH 26 mutant in a P. aeruginosa P A K background was used for the biofilm experiments. After 48 hours, the growth of these two strains in the 96-well microtitre plates was indistinguishable on all three carbon sources.  On high concentrations of citrate (up to 2.5 mM), the opdH deficient  mutant was significantly compromised in biofilm formation (Figure 4.8A).  At lower  concentrations of citrate, the mutant adhered to the polystyrene plate as well as the wild-type strain. Both the mutant and wild-type strains were assessed for twitching and swimming motility on B M 2 +10 m M citrate agar and there was no significant difference between the two strains.  135  The biofilm formation defect was not observed on either BM2 glucose or B M 2 benzoate (Figure 4.8B and 4.8C), although the growth of both strains was compromised on benzoate, relative to their growth in citrate and glucose. 4.3.7 C h a n n e l f u n c t i o n o f O p d H Given the induction of OpdH by tricarboxylates and its involvement in their uptake, I sought to determine whether it, like other specific porins, contained a binding site for its substrates by purifying the protein and analyzing its channel function in a planar bilayer apparatus. Based on its similarities to OpdH and its mobility on SDS-PAGE gels, it seemed likely that OprE was likely to contaminate preparations of OpdH (Figure 4.2, lane 1). Therefore, to circumvent this possibility, a strain deficient in the production of OprE was used to purify OpdH.  Many minor contaminating proteins were removed by the selective solubilization  procedure (Figure 4.9). 1  2  3  4  5  6  7  94 kDa — OprD _ OpdH  63 kDa 43 kDa  OprF 30 kDa  20.1 kDa  F I G U R E 4.9 Selective s o l u b i l i z a t i o n o f O p d H . Outer membranes isolated from a stationary phase culture of an oprE mutant grown on B M 2 + 10 m M citrate (lane 1) were successively solubilized in 0.5 % ocfyl-POE (lane 2), 3% octyl-POE (lane 3), 3% octyl-POE + 0.2 M NaCl (lane 4), and 3% Octyl-POE + 5 m M E D T A (lane 5). The insoluble proteins in the pellet from final solubization step are resolved in lane 6 and lane 7 contains the molecular weight markers. Proteins were resolved on an 11% acrylamide gel, Lane 1 contains 20 \xg protein, lanes 2-6 contain 10 u,L.  136  Both OprD and OpdH were soluble in Octyl-POE + E D T A , however, a large amount of both porins were retained in the final membrane pellet, indicating that these proteins were likely in strong association with the LPS of the outer membrane (Figure 4.9, lanes 5 and 6). The majority of the E D T A soluble proteins were removed from the OpdH fractions after passage through the ion exchange column (Figure 4.10, lanes 3 and 8), and were completely removed with the chromatofocussing column (Figure 4.10, lanes 10 and 11). The identity of OpdH was confirmed by comparing its mobility on an SDS-PAGE gel with the outer membrane proteins isolated from an OpdH mutant (Figure 4.10, lanes 3 and 4). Also, unlike OprD, which demonstrates two different mobilities in SDS-PAGE gels based on the temperature used to prepare the protein samples (21), OpdH was not heat-modifiable. Therefore, the mobility of heated and unheated OpdH protein samples was compared throughout the purification process to distinguish it from OprD (Figure 4.9, lanes 3 and 8, and 10 and 11). 1  2  3  4  5  6  7  8  9  10 11 12  4.10 Purification of O p d H . A n 11% acrylamide gel showing the protein composition during various stages of OpdH purification. Each lane contains 20 u.g protein. Lanes land 6 outer membranes isolated from a stationary phase culture of an oprE mutant grown in B M 2 + 10 m M citrate, lanes 2 and 7 - soluble protein fraction in 3% Octyl-POE + 5 m M E D T A , lanes 3 and 8 - proteins eluted from the MonoQ column, lanes 4 and 9 - outer membranes isolated from a stationary phase culture of the opdH 10 mutant grown in B M 2 + 10 m M citrate, lanes 10 and 11 - proteins eluted from the MonoP column, lane 5 - molecular weight marker. Samples in lanes 15 and 10 were heated at 88°C for 10 min, samples in lanes 6-9 and 11 were heated at 37°C for 10 min. The locations of OpdH and OprD in their heated and unheated (*) forms are shown. FIGURE  137  When the purified OpdH was analyzed in a planar bilayer apparatus, step-wise increases in conductance were observed (Figure 4.11 A).  These steps were not observed in control  experiments where detergent alone was added and therefore likely corresponded to the channel activity of OpdH.  After two or three channel insertion events, the insertion of subsequent  channels caused an increase in the background channel noise and the bilayer had to be reformed in order to identify channel events unambiguously.  Over one hundred channel events were  observed in 1 M KC1; the modal and average conductance through the channel was 0.7 nS (Figure 4.1 IB) approximately half the value of many general porins (Table 4.3). T A B L E 4.3  Properties of E. coli and Pseudomonas porins  Porin Organism Type Substrate Size OmpF E. coli General N / A 1.9 nS General N / A OmpC E. coli 1.5 nS PhoE E. coli General N / A 1.8 nS RafY E. coli General N / A 2.9 nS LamB E. coli Specific Maltodextrins 0.16 nS ScrY E. coli Specific Sucrose 1.4 nS BglH E. coli Specific Aryl glucosides 0.56 nS Tsx E. coli Specific Nucleosides 10 pS OprF P. aeruginosa General N / A 0.36/2-5 nS OprB P. putida Specific Carbohydrates 35 pS OprB P. aeruginosa Specific Carbohydrates 25 pS OprD P. aeruginosa Specific Basic amino acids 20 pS OprP P. aeruginosa Specific Phosphate 0.28 nS Size is based on the conductivity observed in 1M KC1 (pH 7) 3  Selectivity Cation Cation Anion Cation Cation Cation Cation Cation Cation Cation Anion Cation Anion  Reference (11) (11) (8) (2) (12) (44) (3) (3D  (13) (42) (55) (24) (23)  However, in comparison with other specific porins, OpdH was quite large, second only to the sucrose specific porin ScrY of E. coli which has a channel size of 1.4 nS. When the anionic species of the bathing solution was changed to 1 M acetate (pH 7), the average conductance of OpdH did not change appreciably (compare Figure 4.1 IB and C). However, when the cation was changed to 1 M N a , the average single-channel conductance decreased to 0.3 nS (Figure +  138  4.1 ID). When the sodium ion was replaced with larger monovalent ions such as Tris or lithium, no channels were observed, implying that the OpdH channel is selective for cations.  Channel size (nS)  Channel size (nS)  4.11 Single channel conductance of O p d H . A . Chart recording of the step-wise increases in conductance formed upon the insertion of OpdH into a bilayer composed of 1.5% oxidized cholesterol and bathed in 1 M KC1 (pH 7). B - D . Histograms the single channel conductance observed in 1 M KC1 (pH 7) (B), 1 M K-acetate (pH 7) (C), and 1 M NaCl (pH 7) (D). FIGURE  A multichannel experiment was performed to assess whether OpdH possessed a binding site specific for tricarboxylates. A bilayer with a large surface was prepared and when added, the OpdH porins inserted into it, rapidly increasing the conductance by 3-log orders within 10 to 15  139  minutes (Figure 4.12, closed symbols). When the conductance had stabilized, potassium citrate (pH 7) at final concentrations ranging from 20 p M to 100 m M was added to the bathing solution. Rather than observing a decrease in conductance, which is indicative of channel binding and blocking, a slight increase in conductance was noted (Figure 4.12, open symbols). This result was also obtained when m-aconitate was used instead of citrate.  5  10  15  20  Time (min) FIGURE  4.12 Macroscopic conductance experiment of OpdH. Purified OpdH was added to a  Teflon cell containing 1 M KC1 and the increase in conductance was monitored for 10 min until it plateaued (squares). At this point, concentrated aliquots of citrate (pH 7) was added to both sides of the membrane and the change in conductance was monitored for two min (triangles). A further aliquot of citrate was added after 2 min and the change in conductance noted (circle).  4.4 D I S C U S S I O N  OpdH belongs to the OprD family of specific porins in P. aeruginosa. Previous work with this family showed that several of these channels are responsible for the uptake of specific compounds, including OpdH, which was implicated in c/s-aconitate uptake. Here, we elaborate on that work and show that in P. aeruginosa the growth of an opdH deficient mutant is compromised on isocitrate in addition to ds-aconitate.  A significant growth defect was not  observed when citrate or other structurally related dicarboxylates were used as the sole carbon source suggesting the possibility that these compounds traverse the outer membrane through alternate porin(s).  140  A purified preparation of OpdH demonstrated a single channel conductance in 1 M KC1 of 700 pS, however, despite its homology with the specific porin OprD, and the seemingly specific nature of its uptake capabilities, we were unable to demonstrate the presence of a tricarboxylate binding site in the channel.  Assuming that like other specific porins, OpdH  possesses a binding site and is a single-file channel (3, 9, 12), the addition of either citrate or cisaconitate to the planar bilayer apparatus would have blocked the pore and caused a decrease in conductance rather than a slight increase.  In addition, the observed cation selectivity of the  channel was inconsistent with the expected composition of a tricarboxylate specific binding site within in the channel. Although there is a possibility that the OpdH substrate may be a metaltricarboxylate chelate, the presented data suggests that the channel functions as a general pore which supports the non-specific diffusion of tricarboxylates.  These results, however, do not  eliminate the possibility of binding sites existing among the extracellular loops of the channel. A comparison of the amino acid sequences of OprD and OpdH revealed that loop 3 demonstrated the greatest variability and would be a likely candidate for containing a tricarboxylate binding site (Figure 4.13). As with the extracelluar binding sites of other porins, it is predicted that the primary role of these sites would be to bring substrates to the vicinity of the channel and would not impact the transport rates through the porin (28, 56). The protein sequence alignment of OprD and OpdH also revealed that the latter porin bears three deletions in each of its C-terminal loops. Thus it is probable that differences in the loop arrangements of these two porins may explain the disparity observed in their channel sizes. The mouth of OprD is constricted by the presence of loops 5, 7, and 8, resulting in an average single channel conductance of 20 pS in 1 M KC1. When an 8 amino acid deletion is introduced into loop 5 of OprD, the single channel conductance of this porin rises to 675 pS (24). It is  141  believed that this situation is analogous to that of OpdH, which because of the decreased length of its loops 6, 7, and 8, is expected to have a mouth that is relatively unobstructed and open. Li OprD OpdH  :  DAFVSDQAEAK33g^SLEP  |FJ3^GgsgsgD  68 60  DfflTSIgSSTTYl  gFSDSPjQBKJTJEEEJAS L2  OprD OpdH  ISGRGS  DS3j  L3  LPjgT;  SGAVg TAE  138 130  S TAT SIFE  L3 OprD OpdH  FQjQgsgFEGjbLEf^H HH F FT TEEGGj H E P T H V K S R  OprD OpdH  [WjlWjjA^NSNYniOTASDnsjGFgFNI  GELYAT L f l T : K[2 IKD3GFTE ! R L E K T g I R D J S D j E : Dl L A L N D K N G R L4  s A B F I HGR2AP^2N2 jJjDHF^LGgLDQK^^Qj  L5 J^NgEgKg^gDnsWrgw  •TO^HS5lfflGLLHBwSGPG"-aTs5LRFa™T"s"s^^BGHDSK§L:  OprD OpdH  :  OprD OpdH  :  OprD OpdH  : QPnSDL :  YGA HYS  NBABGAM^  : :  204 200 274 269  HJjQP NJJDA  D6TKMjDNNVGYKNYGYj ELAGQ;  : 42 0 QTQPQW : 391  4.13 Alignment of OprD and O p d H . Signal peptides were predicted using SignalP and removed from the protein sequences which were then aligned using ClustalX. The location of the surface loops are based on the published topology map of OprD (outlined) (25, 35).  FIGURE  The specificity of tricarboxylate transport may have arisen, in part, from interactions between OpdH and the putative tricarbxylate transport system encoded by PA0754-0752. The putative periplasmic binding protein PA0754, was co-transcribed and co-regulated with opdH. As the intergenic regions between PA0754, PA0753, and PA0752 were not very large and the homologous tricarboxylate transport system in S. Typhimurium forms an operon, it seems probable that all three genes are co-regulated and form an operon with opdH. Transport systems similar to the tct operon have been conserved in both sequence and arrangement in many bacterial species (4). The inclusion of a porin gene within these putative operons is unique to the pseudomonads; however, fractionation studies have demonstrated that the TctC periplasmic  142  binding protein of S. Typhimurium interacts with both the outer and inner membranes, and may funnel substrates from one membrane to the other (52).  Thus, it is possible that the P.  aeruginosa PA0754 protein acts in an analogous manner; however, further studies are needed to confirm this hypothesis. The selective uptake of tricarboxylates by OpdH may have also been a consequence of its tight regulation. O f the nine di- and tricarboxylates tested, only the latter group of compounds led to high levels of opdH production. The level of opdH transcription on oxaloacetate, malate, and pyruvate was quite low (approximately less than 10% of the amount observed on the tricarboxylates), and negligible on fumarate, succinate, a-ketoglutarate, and on other structurally diverse compounds including amino acids and pyroglutamate. A n inspection of the chemical structures of these compounds revealed that at least three carbonyl groups were required for efficient opdH transcription. Compounds, without three carbonyl groups, but possessing a high density of carbonyl or alcohol groups (i.e. less than two carbon atoms/functional group) led to low levels of opdH transcription. In the presence of compounds lacking these structural features, there was virtually no opdH transcription, limiting the range of environments where this channel is expected to be functional. The expression profile of OpdH differed markedly from that of OprD. OprD is expressed on a broad range of carbon sources and is induced to higher levels in response to its substrate, arginine, via the ArgR regulator (36). OpdH, however, was only expressed on the presence of its substrates and rather than being induced in response to these compounds, was normally repressed by the PA0756-757 two-component regulatory system. These differences in regulation may be a reflection of the differences in channel architecture between the two porins. In 1 M KC1, the average single channel conductance of OpdH was 35 times larger than that of OprD-  The  143  constitutive presence of such a large pore in the outer membrane would significantly increase its permeability to a variety of noxious agents, and hence, limiting its expression is of considerable advantage to P. aeruginosa. E. coli employs a similar strategy with its sucrose specific porin ScrY and RafY, which is a general porin involved in the uptake of large carbohydrates. Both channels are relatively large, 1.4 nS and 2.9 nS respectively, and their expression is tightly controlled by repressor proteins (6, 43). The repression of opdH by PA0756-757 was not sufficient to explain the induction of this porin in response to tricarboxylates, as the induction was observed in mutants lacking this putative two-component regulatory system. A number of candidate regulators were investigated based on their involvement in carboxylate metabolism (CbrAB, Crc), homology to PA0756-757 (PhoPQ), or proximity to opdH (PA0748), however, none of them demonstrated a role in opdH induction. The P. aeruginosa genome hosts a large number of alternative regulators, any one of which may play a role in the expression of opdH. Future candidates for investigation include genes that are homologous to PA0756-757 (PA2479-2480, PA2809-2810, PA4776-PA4777, and PA4885-4886), genes that are homologous to regulators involved in di-and tricarboxylate metabolism in other organisms (PA5511-5512), and putative regulator genes that are proximal to homologues of di- and tricarboxylate transporters from other bacteria (PA0877, PA2206, PA3782, PA4341, PA5166). However, future investigations should not be limited to genes involved in carboxylate metabolism as opdH is expressed in vivo (50), and is upregulated in biofilms that have been exposed to imipenem (7). A potential role for OpdH in the formation of biofilms was established in this investigation. The effect was specific to citrate correlating with the expected number of OpdH molecules in the outer membrane. On citrate, biofilm formation initiates independently of pili  144  and flagella (27). Therefore, because the defect in biofilm formation by the opdH mutant was modest, it seems likely that in the presence of tricarboxylates, other adhesive proteins act in conjunction with the porin to initiate biofilm formation. It remains to be determined whether this role of OpdH can be generalized to other environments.  145  4.5 R E F E R E N C E S  1.  2.  3.  4.  5.  6. 7.  8.  9.  10.  11. 12.  13.  14. 15.  Adewoye, L. O., and E. A. Worobec. 1999. 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J Bacteriol 152:636-42.  149  5 DISCUSSION  151  5.1 IDENTIFICATION OF A NOVEL FAMILY OF SPECIFIC PORINS IN PSEUDOMONAS AERUGINOSA  151  5.2 M E M B E R S OF T H E O P R D FAMILY EXHIBIT DISTINCT Y E T COMPLEMENTARY FUNCTIONS  152  5.3 COMPARISON OF T H E CHANNEL PROPERTIES OF O P R D AND O P D H  154  5.4 R E - E V A L U A T I O N OF O P D H AS A SPECIFIC PORIN  155  5.5 BASIS OF T H E PHYLOGENETIC RELATIONSHIPS BETWEEN O P R D FAMILY MEMBERS  156  5.6 DIFFERENTIAL REGULATION OF O P R D FAMILY MEMBERS  157  5.7 CONTRIBUTION OF T H E O P R D FAMILY TO PSEUDOMONAS AERUGINOSA PHYSIOLOGY  158  5.8 R E F E R E N C E S  161  150  5 DISCUSSION 5.1  IDENTIFICATION O F A N O V E L  F A M I L Y O F SPECIFIC PORINS IN  PSEUDOMONAS AERUGINOSA  The OprD family of P. aeruginosa is comprised of 19 specific porins that are proposed to have arisen through a series of gene duplication events.  During the course of this work, it  became apparent the OprD family was not unique to Pseudomonas but was found in a variety of organisms. The family is most predominant in soil organisms, however, a few representative members are found in the enterobacteria. To date 109 different paralogues have been identified, and more are anticipated as the genomes of additional bacterial species are being sequenced. The degree of amino acid similarity among the OprD family in P. aeruginosa ranges from 46% to 57% which is quite high in comparison with other porins. The E. coli porin that bears the most similarity to OprD, for example is OmpF, which is only 15% similar at the amino acid level. A protein sequence alignment of the members of this protein family did not reveal any consensus sequences, however, several regions of high similarity, corresponding to the transmembrane P-strands of OprD were identified.  The most variable regions of the protein  sequences corresponded to the 8 predicted extracellular loops of the porins. Most dramatic were the large deletions in loops 1-3 and 5-8. Based on these sequence comparisons, it was predicted that like, OprD, the 18 homologues were specific porins with a conserved P-barrel structure. Any functional diversity observed in this family, then, would be attributed to the differences in the arrangement and/or composition of the extracellular loops. Phylogenetic analysis of the OprD homogues from both P. aeruginosa and P. putida, revealed that the members of this family fell into one of two major sub-groups. One group was more similar in sequence to OprD and the other bore a higher similarity to the phenylacetic acid specific porin PhaK of P. putida (16).  Thus, it appeared as i f the members of this family  151  exhibited differing degrees of divergence from each other. A key question that arose from this analysis was whether this division among the family members had a functional basis and how this had impacted cellular physiology. 5.2 M E M B E R S OF T H E O P R D FAMILY EXHIBIT DISTINCT Y E T COMPLEMENTARY FUNCTIONS  The retention of the 19 OprD homologues in the genome suggests their importance to cellular physiology and I attempted to discern their role by first examining their substrate selectivity. Preliminary analysis of the genome contexts of the 19 members in P. aeruginosa indicated the functions of these porins were diverse as the genes for many of them were found in or associated with putative operons involved in the metabolism of unique compounds (Figure 2.6). The substrate selectivity of the OprD homologues was assessed by performing growth curves with porin deficient mutants on minimal media with limiting concentrations of carbon sources. Distinct substrates were found for 7 of the novel OprD homologues, illustrating the functional divergence among this family. Including OprD, the selectivities are as follows: OprD - basic amino acids, OpdB - proline, OpdC- histidine, OpdP - glycine-glutamate, OpdT tyrosine, OpdH - cz's-aconitate, OpdK - vanillate, and OpdO - pyroglutamate. These growth data were corroborated by observing the induction of the specific porins in their respective substrates. The growth defect of the opdH mutant in cz's-aconitate was complemented by providing a copy of the gene in cis. However, this analysis was not performed on the other 7 mutant strains, as repeated attempts to complement the opdK mutant were unsuccessful. Despite the specialized functions of the OprD family members, some redundancy was also expected because of their shared evolutionary history. This functional overlap was observed with OprD and OpdC, which were both implicated in the uptake of histidine (23). In addition, all  152  of the porin deficient mutants were capable of growth on their substrates, even the opdH mutant, which after a prolonged period was able to use cw-aconitate as a sole carbon source, implying the presence of complementary uptake routes, most likely OprD homologues, in the outer membrane. As any permutation of the 19 porins could have shared overlapping functions, the redundancy of the family was established by determining the mechanism of arginine transport in an OprD mutant.  To shorten the list of candidate porins, the expression profile of the  characterized family members in each of the eight substrates was investigated. A n interesting pattern emerged from this analysis. Members of the OprD sub-family, OprD, OpdB, OpdC, OpdP, and OpdT, were expressed on all of the substrates tested, whereas members of the OpdK sub-family, OpdH, OpdK, and OpdO, were exclusively expressed in their substrates, with the exception of OpdH which was also transcribed on vanillate. This pattern suggested that the OprD subfamily members were more likely to exhibit complementary activity as they were expressed in a broader range of conditions. Indeed, this overlapping activity was demonstrated with one OprD homologue, OpdP, which was shown to permit the passage of arginine. As with the single porin mutants, the oprDlopdP double mutant was still capable of arginine transport. Therefore, it seemed plausible that uptake through the outer membrane with respect to arginine transport at least, does not exclusively occur through one channel but rather is the combined effort of many porins.  This hypothesis remains to be tested, possibly through the use of  pleiotropic OprD homologue mutants. From the expression data, it appears that any one or all of OpdB, OpdC, and OpdT can also be involved in arginine transport; however, the potential contributions of the 11 uncharacterized OprD homologues should also be investigated.  153  5.3 COMPARISON OF T H E CHANNEL PROPERTIES OF O P R D AND O P D H  OprD and OpdH share considerable amino acid similarity (53%). conservation, the channel properties of the two porins differed markedly.  Despite this Previous work  demonstrated that OprD forms small, cation selective, 20 pS channels in a planar bilayer system containing 1 M KC1 (8). The porin also possesses a binding site within its channel interior that recognizes basic amino acids, peptides containing these residues, and structural analogues such as the carbapenem antibiotic imipenem. As in other specific porins, it is believed that the binding site facilitates the transport of molecules by orienting them in favorable conformations as they traverse the channel. A model describing the arrangement of the extracellular loops of OprD has been proposed (9). Mutants harbouring OprD proteins with deletions in loops 5, 7, and 8 are hypersusceptible to a variety of antibiotics, implying that these three loops fold over the mouth of the channel and constrict its opening. Analysis of the loop 5 deletion mutant demonstrated a single channel conductance of 675 pS in 1 M KC1 (8). Two additional loops, 2 and 3, are proposed to fold into the channel interior where they form part of the imipenem binding site (9, 15). Systematic replacement of the acidic amino acid residues in both of these loops, alone or in combination, did not have an impact on imipenem susceptibility, suggesting that amino acids from the barrel wall may also contribute to imipenem binding. Based on these results, OprD appears to be a quintessential specific porin.  Its binding site facilitates the  diffusion of specific molecules and its small channel size excludes the non-specific passage of many others. OpdH, in contrast, was much larger than OprD, having an average single channel conductance of 700 pS in 1 M KC1. This size was reminiscent of the conductance of the OprD loop 5 deletion mutant, and a protein sequence alignment of the two porins revealed that OpdH  154  was essentially an OprD variant with shorter loops. This difference suggests that the mouth of the OpdH channel may resemble that of a general porin (Figure 1.3) and not be occluded by as many loops as OprD. This difference in loop arrangement may explain the larger channel size of OpdH as the two porins are expected to have a similar core structure based on the degree of sequence conservation that they share. Moreover, OpdH was cation selective like OprD and did not possess a binding site specific for citrate or c/s-aconitate, implying that the channel interior had not evolved sufficiently to specialize in the uptake of tricarboxylates. The possibility that the channel binds to tricarboxylate-metal ion chelates, however, remains to be investigated. Because of the lack of an apparent binding site, it is unclear whether loops 2 and 3 of OpdH fold into the channel, as predicted for the 2  nd  and 3 loops of OprD. The net charge of the rd  third OpdH loop is -3 and of the second is +4, however, the charges of the corresponding loops of OprD are -4 and +3 (Tables 2.4 and 2.5). Based on this analysis alone, it is difficult to predict the localization of the loops of OpdH. A more comprehensive molecular genetics approach or structure determination would resolve this issue. 5.4 R E - E V A L U A T I O N OF O P D H AS A SPECIFIC PORIN  The lack of a tricarboxylate binding site within OpdH and its large channel size suggests that it may be a general porin, despite its similarity to OprD. However, the distinction of specific and general porins is often not clear. Historically, specific porins have been defined by their small channel sizes and substrate-specific binding sites which limit the range of compounds taken up by these channels, and their association with cytoplasmic membrane transport systems (13). Exceptions to all of these criteria have been reported. The substrate specificities of LamB, ScrY, OprB, originally limited to a single type of sugar, have all been extended to include a wide variety of carbohydrates (4, 21, 25). The non-specific diffusion of amino acids through LamB  155  and gluconate through OprD has also been demonstrated (7, 14).  The single channel  conductance of ScrY is on par with that of general porins and oprD is not associated with any cytoplasmic membrane transporters. These data demonstrate that porins may exhibit general or specific features, depending on the nature of the solute in question. It has also been suggested that all porins possess binding sites, but specific substrates have only been identified for a few. This hypothesis is based on the crystal structure of the general porin of Rhodobacter capsulatus (24). The structure of this porin revealed the presence of a binding site formed between its third loop and an extracellular domain that had cocrystallized with an unknown solute. Later studies reported that the porin binds tetrapyrols (2). Similarly, it may be possible that OpdH possesses a binding site for an as yet unidentified compound. Based on its relationship with OprD and its observed cation selectivity, it may be possible that OpdH has. retained some remnants of an arginine-specific binding site.  This  hypothesis, however, remains to be tested. 5.5 BASIS OF T H E PHYLOGENETIC RELATIONSHIPS BETWEEN O P R D FAMILY MEMBERS  The phenotypes  of the OprD homologue deficient mutants  suggested that the  phylogenetic clustering of the OprD family was based on substrate specificity. Members of the OprD sub-family took up amino acids and related compounds and members of the OpdK subfamily represented more divergent channels that had evolved to take up a variety of carboxylic acids. From this phylogenetic framework, specific hypotheses regarding the substrates of the other OprD homologues in P. aeruginosa and other organisms can be generated.  These  hypotheses can be refined further by considering the unique niches and metabolic capabilities of the different species.  For example, it is anticipated that P. putida (12) may have a higher  proportion of aromatic compound-specific porins, while P. syringae (5) and P. fluorescens (17)  156  may have more porins specific for the uptake of plant derived compounds. Consotent with this prediction, analysis of the OprD family of P. putida revealed that this organism harbours a higher proportion of OpdK-like porins than OprD-like porins (13 vs. 8, Figure 2.1) 5.6 DIFFERENTIAL REGULATION OF O P R D FAMILY MEMBERS  The expression profiles of the 8 characterized OprD homologues also correlated with their phylogenetic distributions.  Members of the OprD sub-family were broadly expressed  whereas OpdH, OpdK, and OpdO were specifically induced by their substrates. This observation implied that compared to the OprD-like porins, the OpdK sub-family was a more specialized class of channels. However, subsequent analysis of OpdH suggested that the channel forms a rather unspecialized channel and the observed specificity of uptake was most likely due its restricted expression. The amino acid sequence alignment of the family indicated that most of the OpdK sub-family members bore large deletions in their loop regions, and thus like OpdH, may form relatively large pores in the membrane that not have developed substrate specific binding sites. The one exception to this observation is OprE which is predicted to have relatively long surface loops (Figure 2.2), particularly loops 2, 5, 6, and 8, and thus should form relatively small channels. Like the OprD sub-family members, OprE is constitutively expressed (26). Therefore, it may be possible that the regulatory patterns observed among the OprD family thus far are not an indication of their relative evolutionary divergence, but rather may reflect their channel sizes. Planar bilayer and gene expression analysis of the remaining 11 OprD homologues are required to substantiate this hypothesis.  157  5.7 CONTRIBUTION OF T H E O P R D FAMILY TO PSEUDOMONAS AERUGINOSA PHYSIOLOGY  Outer membrane permeability is often a compromise between its barrier and transport functions. P. aeruginosa balances the two by using specific porins to take up the majority o f hydrophilic nutrients it requires. The selective nature o f these channels permits the passage o f limited classes o f molecules to the exclusion o f all others.  Prior to the release o f the P.  aeruginosa genome sequence, 4 o f its specific porins had been characterized: OprB, OprP, OprO,  and OprD  (6).  Collectively,  these  porins  facilitate  the  specific  uptake  of  monosaccharides, phosphate, polyphosphate and basic amino acids (Figure 5.1).  histidine OpdT tyrosine  OpdK OpdO  vanillate  pyroglutamate  Vanillate transport and metabolism  OprB monosaccharides  OpdP glycine-glutamate arginine  OprD arginine  OprP phosphate  OprO polyphosphate  lysine histidine gluconate  FIGURE 5.1 Specific porins of P. aeruginosa. The physiological contributions o f the specific porins o f P. aeruginosa. Light grey - porins characterized prior to the release o f the P.aeruginosa genome, Dark grey - members o f the OprD sub-family, Black - members o f the O p d K sub-family. The induction o f the tricarboxylate transport operon and the vanillate transport and metabolism i n response to those compounds are also indicated. For simplicity, only the outer membrane of the bacterium is shown.  158  The OprD family contributes 19 members to the porin repertoire of P. aeruginosa. Each member of this family is proposed to have unique permeability characteristics that enable the outer membrane to take up a greater variety of compounds. The substrate selectivities of 7 novel OprD homologues have now been identified (Section 5.2). There are two proposed mechanisms by which the observed specificity of the OprD family might have been achieved. The first, exhibited by OprD, involves the formation of small, specialized channels that physically bar the entry of unwanted compounds. The second mechanism, as observed with OpdH, limits the production of channels to specific environments and thereby prevents chance encounters with potentially toxic compounds. There may also be other factors that limit transport through OpdH, as antibiotics do not apparently traverse the channel even when its expression is induced in citrate (data not shown). Given the intrinsic impermeability of the P. aeruginosa outer membrane and the diversity of nutrients it takes up, the involvement of OprD family members in the non-specific influx of nutrients is also anticipated (22, 27). This activity is particularly relevant to those members that were expressed in a wide array of carbon sources (i.e. OprD, OpdB, OpdC, OpdP, OpdT). Based on their similarity to OprD, it is believed that these channels will form small pores, and thus should not compromise the integrity of the outer membrane by acting as conduits for large, potentially toxic compounds. Thus far, generalized transport activity has been observed in OprD with respect to gluconate uptake (7) and in OpdP, which permits the non-specific diffusion of arginine, in the absence of OprD. In addition, the unique structural and regulatory constraints of each channel will impose size and/or charge restrictions on solutes, limiting uptake to a narrow range of small, structurally diverse nutrients in a sub-set of environments. Indeed, the complex regulation of the OprD sub-family described in Chapter 3 may reflect a novel strategy whereby  159  the outer membrane tailors its permeability characteristics to particular environments by modulating the levels of porin expression. Because of their shared evolutionary history, it is expected that the range of compounds taken up by OprD homologues will overlap with one another, analogous to the outer membrane proteins of other organisms (1, 10, 19, 20). This overlap is proposed to be the basis of the redundant activities described above in section 5.2. These activities can contribute to the overall fitness of P. aeruginosa by ensuring transport activities are maintained in diverse environmental conditions and/or in the event of a gene loss or mutation. Some OprD homologues may not even be expressed in the outer membrane unless several of the major porin genes have been deleted. This quiescence has been observed with several porins of E. coli and is it believed to act as an insurance mechanism should the major porins of the outer membrane be inactivated (3, 11, 18). It is anticipated that the continued study of this family's transport functions will shed more light on its specific and general contributions to permeability.  160  5.8 R E F E R E N C E S  1. 2.  3.  4.  5.  6. 7.  8.  9.  10.  11. 12.  13.  Bavoil, P., H. Nikaido, and K. von Meyenburg. 1977. Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein. Mol Gen Genet 158:23-33. Bollivar, D. W., and C. E. Bauer. 1992: Association of tetrapyrrole intermediates in the bacteriochlorophyll a biosynthetic pathway with the major outer-membrane porin protein of Rhodobacter capsulatus. Biochem J 282 ( Pt 2):471-6. Condemine, G., C. Berrier, J. Plumbridge, and A . Ghazi. 2005. Function and expression of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli. J Bacteriol 187:1959-65. Death, A., L. Notley, and T. Ferenci. 1993. Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress. J Bacteriol 175:1475-83. Feil, H., W. S. Feil, P. Chain, F. Larimer, G. DiBartolo, A . Copeland, A . Lykidis, S. Trong, M. Nolan, E. Goltsman, J. Thiel, S. Malfatti, J. E. Loper, A . Lapidus, J. C. Detter, M. Land, P. M. Richardson, N. C. Kyrpides, N. Ivanova, and S. E. Lindow. 2005. Comparison of the complete genome sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato DC3000. Proc Natl Acad Sci U S A 102:11064-9. Hancock, R. E. W., and F. S. L. Brinkman. 2002. Function of Pseudomonas porins in uptake and efflux. Annu Rev Microbiol 56:17-38. Huang, H., and R. E. W. Hancock. 1993. Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa. J Bacteriol 175:7793-800. Huang, H., and R. E. W. Hancock. 1996. The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa. J Bacteriol 178:3085-90. Huang, H., D. Jeanteur, F. Pattus, and R. E. W. Hancock. 1995. Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD. Mol Microbiol 16:931-41. Li, X. Z., N. Barre, and K. Poole. 2000. Influence of the MexA-MexB-OprM multidrug efflux system on expression of the MexC-MexD-OprJ and MexE-MexF-OprN multidrug efflux systems in Pseudomonas aeruginosa. J Antimicrob Chemother 46:885-93. Misra, R., and S. A . Benson. 1989. 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