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A site-directed mutagenesis approach to study the functions of the histidine kinase CckA in Rhodobacter… Wiesmann, Christina 2016

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A SITE-DIRECTED MUTAGENESIS APPROACH TO STUDY THE FUNCTIONS OF THE HISTIDINE KINASE CCKA IN RHODOBACTER CAPSULATUS GENE TRANSFER AGENT PRODUCTION AND RECIPIENT CAPABILITY by  Christina Wiesmann  BSc, MacEwan University, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology & Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2016  © Christina Wiesmann, 2016   ii  Abstract The Rhodobacer capsulatus gene transfer agent (RcGTA) is a phage-like particle capable of packaging, and transferring, ~4 kbp fragments of DNA between different strains of R. capsulatus. This genetic transfer is dependent upon a variety of factors, including the regulation of transcription of the main structural gene cluster, particle maturation, release, and uptake (RcGTA recipient capability) by other, ‘RcGTA competent’ cells. These processes have been previously demonstrated to be regulated by the CckA-ChpT-CtrA phosphorelay pathway. I have created three CckA site-directed mutants thought to be involved in mediating kinase, phosphatase, or cyclic-di-GMP binding activities of the CckA protein. My results provide strong evidence that these three activities play roles in regulating the transcription, maturation, and RcGTA ‘competency’ of these cells. My thesis also provides evidence that the ChpT, and DivL regulatory proteins play a role in the regulation of RcGTA recipient capability. I further demonstrate that cell growth and morphology are not noticeably affected by mutations in CckA activity, and that differences in RcGTA recipient capability are not due to differences in the ability of RcGTA particles to bind, or adsorb, to cells. Overall, my thesis provides novel insights in to how RcGTA production and recipient capability are regulated, furthering our understanding of how this novel horizontal gene transfer mechanism is regulated.       iii  Preface The work in this thesis includes contributions made by fellow scientists in my lab. Figure 3.1A comes from a manuscript submitted by Westbye et al. The strain SBΔCckA was createdby Stephan Noll,, strain B10ΔChpT was created by Dr. Cedric Brimacombe. Plasmids pZWp1079 and pRCckA-YD were created by Dr. Alexander Westbye. Primers CckA(Y589D)-F, CckA(Y589D)-R, DivL-F and DivL-R were designed by Dr. Alexander Westbye. All other biological materials were created, and all experiments were carried out, by me.  I wrote the entire thesis, with edits completed by Dr. JT Beatty.     iv  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Figures ............................................................................................................................... ix List of Tables ..................................................................................................................................x List of Symbols ............................................................................................................................. xi List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiv Chapter 1: Introduction ................................................................................................................1 1.1 General properties of Rhodobacter capsulatus ........................................................... 1 1.2 Horizontal gene transfer in prokaryotes ...................................................................... 1 1.2.1 Conjugation ................................................................................................................. 1 1.2.2 Natural transformation ................................................................................................ 2 1.2.3 Transduction ............................................................................................................... 3 1.2.4 Other forms of horizontal gene transfer ...................................................................... 3 1.3 Gene transfer agents and RcGTA, the gene transfer agent of R. capsulatus .............. 4 1.3.1 Bacterial regulatory systems controlling RcGTA production .................................... 1 1.3.2 RcGTA recipient capability ........................................................................................ 1 1.3.3 The CckA-ChpT-CtrA phosphorelay pathway. .......................................................... 2 1.4 DivK, DivJ and DivL .................................................................................................. 5 1.5 Cyclic di-GMP` ........................................................................................................... 5   v  1.6 The CckA protein ........................................................................................................ 6 1.6.2 HisKA domain ............................................................................................................ 7 1.6.3 HATPase domain ........................................................................................................ 7 1.6.4 REC domain ................................................................................................................ 8 1.7 Objectives and hypotheses .......................................................................................... 8 1.7.1 CckA(H322A) ........................................................................................................... 10 1.7.2 CckA(V399P) ........................................................................................................... 10 1.7.3 CckA(Y514D) ........................................................................................................... 11 Chapter 2: Materials and Methods ............................................................................................17 2.1 Bacterial strains and growth conditions .................................................................... 17 2.2 Construction of targeted mutants and complementation plasmids ........................... 18 2.3 Construction of chromosomal reporter strains .......................................................... 19 2.4 RcGTA recipient assay ............................................................................................. 19 2.5 RcGTA adsorption assay .......................................................................................... 20 2.6 RcGTA transduction assay ....................................................................................... 20 2.7 Western blot .............................................................................................................. 21 2.8 Agarose gel electrophoresis ...................................................................................... 21 2.9 β-galactosidase assays ............................................................................................... 22 2.10 Specific activity and total protein determination by the Lowry method .................. 22 2.11 Microscopy ............................................................................................................... 23 2.12 Bioinformatic analyses.............................................................................................. 23 Chapter 3: Results........................................................................................................................27   vi  3.1 Effects of CckA(H399A), CckA(V443P), and CckA(Y589D) on the phosphorylation state of CtrA/CtrA~P ................................................................................................................ 27 3.1.1 Loss of CckA leads to a decrease in the amount of CtrA~P ..................................... 27 3.1.2 CckA(H399A) leads to low levels of CtrA~P .......................................................... 28 3.1.3 CckA(V443P) leads to high levels of CtrA~P .......................................................... 28 3.1.4 It was found that the strain containing CckA(V443P) as the only copy of CckA, had an approximately 2 to 6-fold increase in β-galactosidase activity (Figure 3.1B). This indicates that high levels of CtrA~P were present and that CckA(V433P) is impaired in its ability to dephosphorylate CtrA. Although it could be argued that the V433P mutation enhances kinase activity without affecting phosphatase activity, work on homologous CckA proteins (Chen et al., 2009; Lori et al., 2015) argues against this conclusion. A small, significant, increase in the level of β-galactosidase activity was also seen in the CckA deletion strain complemented with a WT CckA gene driven by its native promoter (Figure 3.1B, Table S1), suggesting that there are potential copy number effects of having CckA present separately on a plasmid. This data indicates that a small increase in the number of copies of the cckA gene, and a predicted increase in CckA levels, leads to an increase in cellular amounts of CtrA~P.CckA(Y589D) leads to a decrease in the amount of CtrA~P ... 28 3.2 Levels of CckA site-directed mutants affect RcGTA production and release .......... 31 3.2.1 Loss of CckA kinase activity increases RcGTA activity .......................................... 31 3.2.2 Loss of CckA phosphatase activity decreases RcGTA activity as the result of inhibition of release from cells ............................................................................................. 32 3.2.3 Loss of c-di-GMP-binding leads to a decrease in RcGTA production and release .. 34   vii  3.3 CckA site-directed mutations affect RcGTA recipient capability ............................ 35 3.3.1 Increased cckA copy number decreases RcGTA recipient capability ....................... 35 3.3.2 Loss of CckA kinase activity leads to an increase in RcGTA recipient capability .. 35 3.3.3 Loss of CckA phosphatase does not affect RcGTA recipient capability .................. 38 3.3.4 Loss of c-di-GMP-binding leads to a loss of RcGTA recipient capability ............... 38 3.3.5 Loss of ChpT leads to an intermediate phenotype .................................................... 39 3.3.6 Loss of DivL leads to an almost complete loss of RcGTA recipient capability ....... 39 3.4 Mutations in CckA kinase and phosphatase activity do not affect RcGTA adsorption.. ................................................................................................................................ 40 3.5 Cell morphology in late exponential phase is not affected by mutant CckA activities.. .................................................................................................................................. 41 Chapter 4: Discussion ..................................................................................................................44 4.1 CckA phosphatase and kinase activities affect the levels of CtrA and CtrA~P ....... 44 4.1.1 Loss of CckA leads to a decrease in the amount of CtrA~P ..................................... 45 4.1.2 The CckA mutant RcCckA(H399A) appears to be a kinase mutant that results in decreased CtrA~P levels ....................................................................................................... 45 4.1.3 The CckA mutant CckA(V443P) appears to be a phosphatase mutant that increases CtrA~P levels ........................................................................................................................ 46 4.1.4 The CckA mutant CckA(Y589D) and the problematic binding of c-di-GMP.......... 46 4.2 CckA kinase and phosphatase activities play different roles in mediating RcGTA production and release .............................................................................................................. 48 4.2.1 CckA kinase and phosphatase activities do not affect the maturation of RcGTA particles ................................................................................................................................. 48   viii  4.2.2 The effects of the CckA H399A kinase mutation on RcGTA production and release… ............................................................................................................................... 49 4.2.3 The effects of the CckA(V443P) phosphatase mutation on RcGTA production and release. .................................................................................................................................. 50 4.2.4 The effects of the Y589D c-di-GMP-binding mutation on RcGTA production and release. .................................................................................................................................. 51 4.3 The role of DivL and the CckA-ChpT-CtrA phosphorelay in mediating RcGTA recipient capability .................................................................................................................... 52 4.3.1 The effect of the CckA H399A kinase mutation on RcGTA recipient capability .... 52 4.3.2 The effects of the CckA(V443P) phosphatase mutation on RcGTA recipient capability ............................................................................................................................... 53 4.3.3 The effects of the CckA(Y589D) c-di-GMP binding mutation on RcGTA recipient capability ............................................................................................................................... 54 4.3.5 The effects of the absence of ChpT on RcGTA recipient capability ........................ 55 4.4 Differences in RcGTA recipient capability are not due to differences in the ability of RcGTA particles to bind to cells ............................................................................................... 56 4.5 Cell morphology in the late exponential phase is not affected by CckA mutations . 57 Chapter 5: Conclusion and Future Directions ..........................................................................58 5.1 Conclusion ................................................................................................................ 58 5.2 Future directions ....................................................................................................... 61 References .....................................................................................................................................63 Appendex A: Supplementary figures .........................................................................................74    ix  List of Figures  Figure 1.2. DivL and the CckA-ChpT-CtrA phosphorelay pathway. ............................................. 3 Figure 1.3. R. capsulatus CckA alignment with C. crescentus CckA, and domains  ................... 15 Figure 3.1. CckA activities differentially affect levels of CtrA and CtrA~P ............................... 30 Figure 3.2 RcGTA activity and gene expression .......................................................................... 34 Figure 3.3. RcGTA recipient capability ........................................................................................ 38 Figure 3.4 RcGTA adsorption assay. ............................................................................................ 41 Figure 3.5 Light microscopy of mutant cells in late exponential phase. ...................................... 42 Figure 5.2 Phylogenetic tree ......................................................................................................... 61 Figure S1. Multiple sequence alignment of CckA proteins and related histidine kinase proteins used in the creation of the tree in Figure 5.1. ................................................................................ 77      x  List of Tables Table 1.1 Summary of site-directed CckA mutations created. ......................................................16 Table 2.1: Strains used in this study ..............................................................................................23 Table 2.2: Plasmids used................................................................................................................24 Table 2.3: Primers used..................................................................................................................25 Table 3.1 Summary of phenotypes ................................................................................................43 Table S1. One-way ANOVA results for comparison of measured values. ...................................78         xi  List of Symbols α alpha β beta Δ delta ᵒ degrees    xii  List of Abbreviations ATP  adenosine triphosphate bp  base pair BSA  bovine serum albumin CA  catalytic and ATPase-binding c-di-GMP cyclic diguanylate monophosphate DHp  dimerization and histidine phosphotransfer DNA  deoxyribose nucleic acid EDTA  ethylenediaminetetraacetic acid ghs  GTA head spike GTA  gene transfer agent HATPase_C histine kinase-type ATPase catalytic HK  histidine kinase ICE  integrative and conjugative element IPTG  isopropyl β-D—thiogalactopyranoside kbp  kilo-basepairs ONPG  ortho-nitrophenyl-β-galactoside PAGE  polyacrylamide gel electrophoresis PAS  Per/ARNT/Sim PCR  polymerase chain rection phage  bacteriophage RcGTA R. capsulatus gene transfer agent REC  receiver domain   xiii  Rif  rifampin SDS  sodium dodecyl-sulfate STHK  signal transduction histidine kinase WT  wild type          xiv  Acknowledgements I first offer my thanks to my supervisor, Dr. JT Beatty, who has provided endless encouragement and advice and was always available and willing to answer my questions, and provide invaluable advice, feedback, and support. I would also like to thank my committee members, Drs. Charles Thompson and Michael Murphy, for their invaluable input to my project, and Dr. Michael Murphy for all of his additional support. I would also like to thank past lab members, Drs. Cedric Brimacombe and Alexander Westbye, for their invaluable scientific inputs to my project. I would specifically like to thank Dr. Westbye for introducing me to the many different varieties of coffee, and aiding and abetting my coffee addiction.  I would also like to thank Lukas Kater for his friendship and support during the brief time he was in the Beatty Lab. I would lastly like to thank the many undergraduate students who helped with various aspects of my project. I thank the national sciences and engineering research council of Canada (NSERC) for awarding me an Alexander Graham Bell Canadian Graduate Scholarship – Master’s (CGSM). I would also like to thank the Canadian institutes of health research (CIHR) for research project funding. Lastly, I would like to thank my friends and family. I would like to thank my family for their encouragement and understanding over the last two years and a half. I couldn’t have made it through these last couple of months without your understanding and support. I also need to thank the many friends who helped me through these last two years. I would first like to thank my friend, Anastasia Hyrina. She was my first friend when I moved to Vancouver and started in the program, and she provided endless advice, encouragement, and fun times. Second, I need to thank Carlos Diaz. Without his endless encouragement, I would not have been able to celebrate   xv  the highs, or make it through the lows, of completing my degree and finding a PhD lab. I also need to thank Emily Li for her unwavering advice and support, Kristy Dockstader for being my thesis writing buddy, and the Eltis and Murphy labs for accepting me as an honorary lab member when I was in need of a lab family. I lastly need to thank my many hiking friends; I hope we have many more hikes together over the course of my PhD.             1         Chapter 1: Introduction 1.1 General properties of Rhodobacter capsulatus Rhodobacter capsulatus is a metabolically versatile phototrophic alphaproteobacterium, capable of aerobic growth in the absence of illumination, and anaerobic growth in the presence or absence of illumination (Madigan and Jung, 2009). Growth is obtained with either organic substances, or CO2 as a carbon source, and H2 as an electron donor.  Phototrophy, 'light-nourishment', depends on the biosynthesis of carotenoid and bacteriochlorophyll pigments needed for anaerobic light-driven production of a proton gradient across the cell membrane, whereas chemotrophy depends on fermentative or respiratory pathways that operate in darkness (Weaver et al., 1975; Madigan and Jung, 2009). R. capsulatus has also figured prominently in the study of horizontal gene transfer. 1.2 Horizontal gene transfer in prokaryotes Horizontal gene transfer, or ‘the non-genealogical transmission of genetic material from one organism to another’ (Goldenfeld and Woese, 2007), plays a key role in bacterial evolution, with gene transfer events that provide a selective advantage to their host, such as virulence genes, being acquired and retained by natural selection over time (Thomas and Nielsen, 2005; Juhas, 2015). There is also increasing evidence that horizontal gene transfer plays a significant role in metazoan evolution, and may also occur between vertebrate species (Boto, 2014; Soucy et al., 2015). In bacteria the three classical, well-studied methods of horizontal gene transfer are conjugation, transformation, and transduction.  1.2.1 Conjugation Horizontal gene transfer by conjugation involves the transfer of bacterial DNA mediated by cell-to-cell junctions and a pore through which DNA can pass (Thomas and Nielsen, 2005).   2         Transferred DNA is typically in the form of a conjugative plasmid, however this process can also transfer chromosomal, or excised chromosomal DNA in some cases. Some gram-negative bacteria use a type IV secretion system through which to produce a mating pilus, mediating cell-to-cell contact and the formation of a pore through which DNA can pass. Conjugative plasmids often encode the entire machinery necessary for their transfer and are the most common elements transferred via conjugation (Norman et al., 2009). The diversity of genes carried on conjugative plasmids within a bacterial population provides a pool from which many potentially beneficial genes (such as genes required for antimicrobial resistance) can readily be transferred between bacteria to help ensure survival of a species facing a hostile environment. 1.2.2 Natural transformation A second type of horizontal gene transfer, natural transformation, involves the uptake and genomic integration of naked DNA from the extracellular environment (Thomas and Nielsen, 2005; Johnston et al., 2014). Natural transformation is widespread, occurring in both gram-positive and gram-negative bacteria (Johnston et al., 2014). DNA uptake and processing involves a set of genes known as the competence (com) genes, as well as the dprA (DNA protecting protein A) gene. As exogenous double-stranded DNA is passed through the ComEC transmembrane complex, one strand is degraded. This single-stranded DNA that passes through the complex is then bound by DprA before interacting with RecA, which is needed for homologous recombination into the genome. Because natural transformation involves homologous recombination, sequence homology between the incoming and genomic DNA is required. A number of other, less well characterized proteins are also involved in the process.  Regulation of competence appears to vary widely between different bacteria. For example, in Bacillus subtilis and Streptococcus pneumoniae the two competence regulatory   3         cascades respond to different, non-conserved, effectors (Claverys et al., 2006). These species also show differences in the growth stage in which competence is induced, the percentage of cells that become competent, and the regulatory pathways involved in inducing and regulating competence (Claverys et al., 2006). 1.2.3 Transduction Transduction involves the horizontal transfer of DNA mediated by bacteriophages (phages) or phage-like particles. Transduction of host genomic DNA occurs when phage particles either randomly package host DNA segments in place of a phage chromosome (generalized transduction), or package host DNA covalently linked to part of the phage chromosome present as a prophage (specialized transduction), as in phage lambda (Frost et al., 2005). The transferred DNA may then integrate into the recipient chromosome via homologous recombination, and is thus restricted to transferring genes between bacteria that share sufficient gene sequence similarity.  1.2.4 Other forms of horizontal gene transfer Transposons are mobile genetic elements that are able to ‘hop’ around in the genome of a bacterium. While transposons generally lack the ability to move themselves between cells, their ability to insert into conjugative or transducible elements enables their transfer horizontally (Frost et al., 2005). Furthermore, conjugative transposons, or integrative and conjugative elements (ICEs), also have the ability to transfer via conjugation and exist in a range of prokaryotic organisms (Burrus et al., 2002). Transposons do not integrate via the conventional homologous recombination pathway and therefore do not require significant homology to the host genome to integrate. Some transposable elements are site-specific, while others are more promiscuous and are able to integrate at multiple different sites (Burrus et al., 2002).   4         Lastly, outer membrane vesicles (OMVs), produced by gram-negative bacteria, are spherical buds of the outer bacterial membrane containing periplasmic materials (Schwechheimer and Kuehn, 2015). While they have been speculated to allow for gene transfer between bacteria, there is, as of yet, little evidence to this effect (Fulsundar et al., 2014). Staphylococcus aureus pathogenicity islands (SaPIs) (Dearborn and Dokland, 2012), casposons (Krupovic et al., 2014), integrons (Gillings, 2014), transposable phages (Harshey et al., 2012), and gene transfer agents (GTAs) (Lang et al., 2012) are additional methods of horizontal gene transfer. 1.3 Gene transfer agents and RcGTA, the gene transfer agent of R. capsulatus  Gene transfer agents (GTAs) are phage-like mediators of genetic exchange between bacteria (Lang et al., 2012). GTAs differ from transducing phage by the fact that the genes are 1) entirely encoded and regulated by genes on the host chromosome, 2) package less DNA than would be necessary to encode the entire GTA, and 3) are produced by a small sub-population of bacteria, resulting in an undetectable decrease in culture turbidity in WT cultures (Lang et al., 2012).  GTAs are currently known to exist in a range of alphaproteobacterial species, including the marine alphaproteobacteria Silicibacter pomeroyi (Biers et al., 2008), Roseovarius nubinhibens and Reugeria mobilis (McDaniel et al., 2010).  It was also demonstrated that Flavobacterium and Flexibacter possess the ability to take up genes transferred via GTA-like particles, although their ability to produce GTA particles was not investigated (McDaniel et al., 2010). GTAs also exist in more distantly related organisms, including archaea and spirochetes (Lang et al., 2012). The best understood, model GTA is that of R. capsulatus, termed RcGTA.     5         RcGTA was discovered on the basis of the transfer of antibiotic resistance between strains of R. capsulatus (Marrs, 1974). Unlike DNA transferred via natural transformation, the process was resistant to DNase, and in contrast to conjugation did not require direct cell-to-cell contact. Although it was not fully understood, this genetic exchange system was first used to map linkages between photosynthesis genes (Yen and Marrs, 1976). It was later discovered that this gene exchange occurred via the production of RcGTA. (Yen et al., 1979; Lang et al., 2012).   RcGTA morphologically resembles a small, tailed bacteriophage (Yen et al., 1979) and randomly packages ~4 kbp of the bacterial host genome, with a slight preference for genes not encoding the RcGTA structural genes themselves (Solioz and Marrs, 1977; Hynes et al., 2012). Genes required for production and release of the RcGTA particle have been found to exist in five genomic loci: the structural gene cluster, a two-gene operon of endolysin and holin genes, genes involved in host cell recognition, and regulatory and maturation genes (Figure 1) (Hynes et al., 2016).         6          Figure 1.1 RcGTA gene clusters RcGTA gene clusters and putative gene roles in particle structure and activity. Genes are colour coded by predicted function. Adapted from Hynes et al. (2016).  1          1.3.1 Bacterial regulatory systems controlling RcGTA production The RcGTA structural gene cluster is regulated by a LuxI/R-type of quorum sensing system, encoded by the gtaRI operon (Leung et al., 2012). The operon is auto-regulatory, wherein GtaR binds to and controls transcription of the operon promoter. Transcription of the main RcGTA structural gene cluster (genes rcc01682 to rcc01699) was demonstrated to be indirectly regulated by GtaR, and it was found that this gene cluster responds to other quorum sensing signals from other bacteria (Leung et al., 2012). The quorum-sensing signals are long-chain acyl-homoserine lactones (Schaefer et al., 2002), and the gtaI gene is responsible for long-chain acyl-homoserine lactone synthesis in R. capsulatus. Production of RcGTA is also regulated by the CckA-ChpT-CtrA phosphorelay (see section 1.3.3) and by the R. capsulatus homologue of the SOS response protein LexA (Kuchinski et al., 2016). Specifically, the LexA protein, a master regulator of  the SOS stress response induced by oxidative stress and subsequent DNA damage in other species (Butala et al., 2009), negatively regulates RcGTA production by directly binding to a sequence in the CckA promoter region called an SOS box (Kuchinski et al., 2016). Transcription of cckA increases in the absence of LexA, and this leads to a reduction in both RcGTA transcription, and a decrease in recipient capability (the ability of R. capsulatus cells to receive a genetic marker transferred via an RcGTA particle), suggesting that high levels of CckA inhibit both of these processes.  1.3.2 RcGTA recipient capability   The components and regulatory factors needed for RcGTA recipient capability are also partially known. The presence of a polysaccharide capsule on R. capsulatus cells is necessary for maximal RcGTA recipient capability and is regulated, in part, by the GtaR/I quorum sensing   2         system (Brimacombe et al., 2013). The number of cells capable of being recipients is growth-phase dependent and maximal during stationary phase (Brimacombe et al., 2013). Recipient capability also requires the presence of the response regulator CtrA, as well as the RecA and DprA proteins, and homologs of proteins involved in natural transformation in other species (Johnston et al., 2014; Brimacombe et al., 2014). For example, homologs of the natural competence genes comEC, comF, and comM are essential for RcGTA recipient capability (Brimacombe et al., 2015). 1.3.3 The CckA-ChpT-CtrA phosphorelay pathway. In the model system of the dimorphic (stalked and swarmer cells) C. crescentus, the sensor hybrid histidine kinase CckA is at the top of a three-component phosphorelay pathway that flows through the ChpT phosphotransfer protein and ends with the response regulator CtrA (Figure 1.2). CtrA ultimately controls chromosomal replication through binding to the chromosome replication origin, thus restricting replication to stalked cells (Quon et al., 1998). ChpT is necessary for maximal levels of CtrA expression, and for detectable levels of CtrA phosphorylation (Biondi et al., 2006; Chen et al., 2009). However, ChpT is present throughout the cell cycle, indicating that it alone is not responsible for cell-cycle differences in CtrA phosphorylation. (Chen et al., 2009). As shown in Figure 1.2, the phosphorelay pathway appears to be reversible, such that under certain conditions CtrA~P is dephosphorylated and CckA releases inorganic phosphate (Chen et al., 2009). A second response regulator protein, CpdR, is also phosphorylated through the CckA-ChpT pathway, and CpdR controls proteolysis of CtrA (Biondi et al., 2006; Chen et al., 2009).      3          Figure 1.2. DivL and the CckA-ChpT-CtrA phosphorelay pathway.  Blue arrows indicate direction of the ‘forward’ pathway. Red arrows indicate direction of the ‘reverse’ pathway. Inorganic phosphate is indicated by Pi. The CckA domains are indicated: two transmembrane segments are shown in black; yellow indicates the PAS domain(s); the HisKA domain is shown in green; red designates the HATPase domain; blue represents the receiver domain.  In Sinorizhobium meliloti, CtrA is a regulator of motility, exopolysaccharide synthesis and cyclic-di-GMP signaling, and is thought to function in the same pathway as the CckA and ChpT proteins (Francez-Charlot, Kaczmarczyk, and Vorholt, 2015). CtrA also plays a role in regulating cell cycle progression, production of cell envelope components, and many other processes (Pini   4         et al., 2015). There is also evidence that CcsA, an orphan kinase, is involved in a phosphorelay with ChpT and CtrA to control levels of phosphorylated CtrA, and may phosphorylate CtrA in the absence of CckA (Francez-Charlot, Kaczmarczyk, and Vorholt, 2015). Studies of this pathway in Sinorizhobium melonis indicate that a homologous CckA-ChpT-CtrA phosphorelay pathway functions, and that CtrA regulates motility, exopolysaccharide synthesis and cyclic-di-GMP signaling.  In Rhodobacter sphaeroides, a close relative of R. capsulatus, flagellar gene expression is regulated, at least in part, by the CckA-ChpT-CtrA phosphorelay pathway (Vega-Baray et al., 2015). The flagellum-dependent motility of R. capsulatus also requires this pathway, whereas the flagellum-independent motility does not (Lang and Beatty, 2002; Shelswell and Beatty, 2011; Mercer et al., 2012).  In R. capsulatus, deletion of either the cckA, chpT, or ctrA gene leads to a decrease in RcGTA transduction (Mercer et al., 2012). The absence of CtrA leads to a complete loss of RcGTA production, while the loss of CckA or ChpT leads to a lack of transduction, seemingly due to a lack of cell lysis. Complementation of a ctrA deletion with CtrAD51A (a point mutant version of CtrA thought to mimic non-phosphorylated CtrA) restores RcGTA production, but much of the RcGTA accumulates within cells; therefore this complementation leads to only a partial restoration of phenotype in terms of gene transduction. Complementation of a ctrA deletion with CtrAD51E (a phosphomimetic CtrA protein) leads to a > two-fold increase in transduction frequency relative to the WT strain. However, CtrAD51E does not complement CckA or ChpT deletion mutants. This suggests that CckA and ChpT play a role in RcGTA release that is independent of their role in ensuring phosphorylation of CtrA (Mercer et al., 2012).    5         1.4 DivK, DivJ and DivL  In C. crescentus, the DivK protein inhibits CckA kinase activity by binding to and inhibiting the DivL unorthodox kinase, which in the absence of DivK functions as a stimulator of CckA kinase activity (Tsokos et al., 2011). The function of CckA varies over the cell cycle as a result of temporally specific localization to the swarmer pole where PleC, the primary DivK phosphatase, is located (Tsokos et al., 2011). DivK~P has been demonstrated to negatively regulate the levels of CtrA protein, in such a way that is dependent on the presence of DivJ (Biondi et al., 2006). R. capsulatus appears to lack homologues of DivK, DivJ and PleC, although a C-terminally truncated DivL homologue encoded in the R. capsulatus chromosome has been found to regulate RcGTA production, possibly by stimulating CtrA phosphorylation (Westbye et al. submitted). 1.5 Cyclic di-GMP`   Cyclic di-GMP (c-di-GMP) is a bacterial second messenger that plays a role in regulating a number of processes including motility, the cell cycle, and cellular differentiation (Römling et al., 2013). In C. crescentus, c-di-GMP binds to CckA, inhibiting kinase activity and stimulating phosphatase activity (Lori et al., 2015). Diguanylate cyclases are responsible for synthesizing c-di-GMP while phosphodiesterases degrade it (Römling et al., 2013). A number of flagellar proteins, proteins involved in regulating the phosphorylation state of CtrA, and proteins involved in the degradation of CtrA show c-di-GMP concentration-dependent activity (Paul et al., 2004; Abel et al., 2013). A cell-cycle regulated shift in levels of c-di-GMP and the subsequent change in CtrA concentration and phosphorylation controls chromosome replication initiation and cell cycle progression (Mann et al., 2016). However, the possible function of c-di-GMP in R.   6         capsulatus was unknown at the time I started my research, and as of this writing has not been reported in the literature. 1.6 The CckA protein   The CckA protein’s catalytically active form is as a homodimer. The R. capsulatus CckA protein is composed of two transmembrane domains, along with four distinct functional domains described in the following sections: two PAS domains; a HisKA domain; an HATPase domain; and a REC domain (Figure 1.3). The N-terminus is in the cytoplasm, as are the four functional domains. This general domain organization is conserved in CckA proteins and homologues in many species (Dubey et al., 2016). The functional domains are described in more detail in the following sections. 1.6.1 Per-Arnt-Sim (PAS) domain The CckA N-terminal functional domains are two PAS domains (Figure 1.3). PAS domains generally play a role in sensing and transducing signals in signal transduction proteins (Möglich et al., 2009). PAS domains can detect a wide variety of signals, including both chemical and physical stimuli; however, their structure is broadly conserved. PAS domains contain a five-stranded antiparallel -sheet, as well as several α-helices. Signal detection creates structural changes in the -sheet which are then propagated to the α-helical and linker domains and finally to the attached effector domain (Möglich et al., 2009). In terms of CckA, it was recently reported that in C. crescentus the N-terminal PAS domain stimulates kinase activity in a 'density-specific' fashion, meaning accumulation of CckA proteins in a small space. The second PAS domain appears to be involved in localization of CckA to the cell pole, and in binding of c-di-GMP (Mann et al., 2016).   7         1.6.2 HisKA domain  HisKA domains (sometimes referred to as DHp domains) are known to be phosphorylated by ATP on a specific histidine residue, necessary for the signal transduction kinase activity of the protein (Willett and Kirby, 2012), which occurs by a bimolecular transphosphorylation reaction within the dimer (Park et al., 1998). HisKA domains contain two sub-domains. The highly helical subdomain A contains a histidine autophosphorylation site and functions as phosphoreceptor dimerization and interaction domain. Subdomain B consists of both α-helices and -sheets and functions as a catalytic kinase (Park et al., 1998). In proteins such as CckA that also contain a receiver domain (see section 1.6.4), the phosphate is transferred from the histidine to an aspartate in the receiver domain. 1.6.3 HATPase domain  In sensor kinases the Hsp90-like ATPase (HATPase) domain, sometimes also known as the catalytic and ATPase-binding (CA) domain, contains an ATP-binding Bergerat fold (Dutta and Inouye, 2000). This conserved fold utilizes the energy derived from a conformational change when binding ATP, energy derived from ATP hydrolysis, or both, to drive processes within the cell. In C. crescentus, the phosphorylation of the histidine in the HisKA domain comes from ATP hydrolysis, and it is likely that ATP binding drives the transfer of phosphate from the histidyl phosphate in the HisKA domain to an aspartate in the receiver (REC) domain. Furthermore, specific residues within the HATPase domain have been identified in the binding of c-di-GMP. Binding of c-di-GMP inhibits kinase and stimulates phosphatase activity (Lori et al., 2015; Mann et al., 2016).   8         1.6.4 REC domain  The C-terminal CheY-homologous receiver (REC) domain contains a conserved aspartate residue that is phosphorylated in homologous proteins, and can then catalyze an output response (in the case of CckA, phosphorylation of ChpT). Receiver domains are often found in response regulator proteins that propagate an output response, often resulting in transcriptional regulation (Bourret, 2010). However, REC domains can also exist in hybrid sensor kinases, as is the case in CckA. REC domains typically have a (α)5 topology. Specific motifs within such proteins are responsible for the active site conformation, metal ion binding, phosphorylation, phosphorylation-mediated conformational changes, and signal transduction. REC domains have been demonstrated to require metal ion binding for their activity, and to catalyze autophosphorylation, leading to a conformational change in the protein that elicits an output response. Dephosphorylation of receiver domains was thought to not occur intrinsically within the protein, but to be stimulated by an auxiliary phosphatase (Bourret, 2010). However, recent work has confirmed that a CckA protein may act as a kinase or a phosphatase, depending on whether c-di-GMP and ADP, or ATP, is bound in the HATPase domain (Dubey et al., 2016).  In C. crescentus, CckA is a bifunctional histidine kinase, capable of phosphorylating ChpT, as well as being able to stimulate dephosphorylation of its own receiver domain (Chen et al., 2009). However, this dephosphorylation requires the CckA HK and REC domains to be tethered together in their native state in the protein (Dubey et al., 2016).   1.7 Objectives and hypotheses   Although the CckA-ChpT-CtrA proteins are known to be involved in regulating both RcGTA production, as well as recipient capability, many questions about the roles of these proteins and their regulators in mediating these processes remained. In the first part of my   9         research, I set out to investigate the possibility that CckA functions both as a phosphatase and a kinase. My approach was to create mutants of residues that were shown in homologous proteins to be required for either phosphatase or kinase activity (Figure 1.3; Table 1). I hypothesized that: 1) A change of CckA histidine-399 to alanine (in the H399A mutant protein) would impair kinase activity 2) A  CckA(V466P) protein would lack phosphatase activity 3) The CckA(Y589D) protein would have constitutive kinase activity 4)  Furthermore, these changes in CckA activity would be manifested as changes in the degree of CtrA phosphorylation I tested these hypotheses by measuring the activity of a promotor previously demonstrated to be stimulated by phosphorylated CtrA (Westbye et al., 2016; Westbye et al., submitted).  In the second part of my research, I set out to investigate the roles of CckA phosphatase and kinase activity in mediating RcGTA production and release, assuming that these CckA activities changed the relative levels of CtrA and CtrA~P. My hypotheses were: 1) A loss of CckA kinase activity would lead to WT levels or an increase in the intracellular amount of RcGTA 2) A loss of CckA kinase activity would lead to less than WT levels of extracellular RcGTA 3) A loss of CckA phosphatase activity would lead to a decrease in the intracellular amount of RcGTA 4) A loss of CckA phosphatase activity would lead to a decrease in the extracellular amount of RcGTA   10         5) These changes in CckA activity would result in changes in the degree of CtrA phosphorylation, manifested as changes in RcGTA gene expression  I tested these hypotheses using a western blot to identify capsid levels in pellet and supernatant fractions of R. capsulatus cultures, as well as an RcGTA production (transduction frequency) assay to measure the levels of functional RcGTA released from cells.  In the third part of my research, I set out to determine the roles of DivL, ChpT, and CckA kinase and phosphatase activities in mediating RcGTA recipient capability. I hypothesized that the elimination of each of these proteins or discrete activities would have differing effects on CckA recipient capability.  I tested the roles of these proteins by measuring the frequency of RcGTA-delivered allele acquisition (recipient capability, see section 1.3.2 of the different mutant strains. Table 3.1 summarizes the site-directed CckA mutations created. 1.7.1 CckA(H322A)  In the C. crescentus CckA, a histidine 322 to alanine (H322A; in the HisKA domain) mutant has WT levels of phosphatase activity, while lacking detectable kinase activity (Chen et al., 2009). A S. melonis cckA gene deletion was not complemented by a CckA(H427A) mutant (Francez-Charlot, Kaczmarczyk, and Vorholt, 2015), suggesting that as in the C. crescentus CckA this histidine is necessary for CtrA kinase activity, and for proper regulation of cellular processes. I made the equivalent H399A change in the R. capsulatus CckA, and evaluated the effect of this change. 1.7.2 CckA(V399P)  In C. crescentus, a V366P CckA mutant has WT levels of autophosphorylation and phosphotransfer to ChpT (Chen et al., 2009). However, the mutant has been shown to have   11         undetectable phosphatase activity in vitro. In vivo, the results were less clear. The phenotype observed was consistent with the V366P mutant either having low levels of phosphatase activity, or that an alternative CtrA~P phosphatase exists (Chen et al., 2009). This phenotype was further investigated in testing the effects of c-di-GMP on cell cycle progression (Lori et al., 2015). It was found that the presence of c-di-GMP inhibited phosphorylation of the V366P mutant CckA, providing further evidence that c-di-GMP switches CckA from acting as a kinase to acting as a phosphatase. In addition, this mutant had a loss of replication symmetry. I made the equivalent V366P change in the R. capsulatus CckA, and evaluated the effect of this change. 1.7.3 CckA(Y514D)    The C. crescentus Y514D CckA mutant protein has been shown to retain normal kinase and phosphatase activities, except in the presence of c-di-GMP (Lori et al., 2015). Specifically, CckA(Y514D) fails to switch from a kinase to a phosphatase in the presence of c-di-GMP, suggesting that the residue is essential in mediating this switch. This mutant also has an almost complete loss of replication symmetry. Furthermore, NMR spectroscopy indicated residues involved in the binding of c-di-GMP, and the Y514D mutation was shown to decrease the affinity for c-di-GMP (Dubey et al. 2016). In the A. tumefaciens CckA the corresponding tyrosine is residue 674, and a Y674D mutant also showed a phenotype consistent with it being deficient in phosphatase activity (Kim et al., 2013). In the E. coli NarX transmitter phosphatase protein a mutation of a similar tyrosine at residue 551 (Y551F) lost phosphatase activity in vitro, while retaining WT levels of interaction with a known NarL receiver protein (Huynh et al., 2013). Studies of the analogous CckA mutation (Y589D) in R. capsulatus indicated an increase in phosphorylated CtrA consistent with a constitutive kinase activity, and resulted in cell   12         filamentation in a dominant manner (Westbye, 2016). There is also evidence indicating that this Y589D mutant CckA protein fails to respond to negative regulation by DivL (summarized in table 1.1). However, the effect of this mutation on the regulation of RcGTA recipient capability was unknown, and so I investigated this question.                     13          R.c        MQALGRSAAMIAPGALVLLVLGLAAGTAGYFVQDRVLMTLLLSMSGSFLL     50                                                                         C.c      1 --------------------------------------------------      0  R.c     51 LAVVAKLAQRHLARRRTQVAGAISAFIMHDASPSFTTDGEGEIGYQNRAA    100                                                   ..::..|::.: C.c      1 ---------------------------------------MADLQLQDKVS     11  R.c    101 V----ERFGS--RGGQTLTRALGEIFANPG--AMLTRLQSKAAALGAARE    142            .    .||..  .|......|...:.|.|.  |..|.|......||.|.  C.c     12 TGAPRRRFDPWLVGAAVFFVAAAALSAAPALKAGPTTLAGLLLLLGVAG-     60  R.c    143 DMVTRRGHVRLSVHQIEGGGFLWRLEDMAERPVGGRGAESISLPMLTASK    192              |...|.|.:....:.||.     .|.||..:     |:::.|...|:. C.c     61 --VAVLGLVAIRGSALSGGD-----ADQAEGFI-----EALAEPAALAAA     98  R.c    193 SGTILFMNEALRRLVGERVRTLDR------IFTE-VPIRSGEEQEITTIE    235            .|.:|..|...|.::||: |.|.:      :|.. |..|.|:..|..... C.c     99 DGRVLAANGPWREVMGEQ-RRLPKGVAGSSLFAALVQARQGQMAEGMLSA    147  R.c    236 GPLRCMVAQIEGAGGRDEVYLMPVAPGRAPSTD----PVSFEALP-----    276            |...........||||..:.|.|:........|    ||:..|.|      C.c    148 GGTDYTAKVSRLAGGRLMIRLAPIVVAEPVVEDASPAPVAERAAPPPSSL    197  R.c    277 ----------VALMRLTVDG------RVIEVNRAARGLLGQIPASTKLSE    310                      .||:    :|      ||:|.|.|...:.|     .|... C.c    198 DAFAGASPFGAALL----EGLEPFTSRVLETNPALTTMTG-----AKAGV    238  R.c    311 LFEGLGRPVDDWVVDAVAGRTERRPEVLRARRGDREVFLQVTLARVIE--    358            ||..|        :|| |.|.|....:...|.|..||.|....:|:..   C.c    239 LFGDL--------IDA-ASRAEAETRLNEGRAGPYEVRLARDPSRIAHLY    279                     R.c    359 ----EGRPGLIAVLSDATQLKTLEGQFVQSQKMQAIGQLAGGVAHDFNNL    404                |||  |:|.:.|.::.|.:|.|..|:|||||||||||||||||||| C.c    280 LYRAEGR--LVAYMIDVSEQKQIELQLSQAQKMQAIGQLAGGVAHDFNNL    327  R.c    405 LTAISGHCDLLMLRHDKGDPDYTDLDQISQNANRAASLVGQLLAFSRKQT    454            ||||....|.|:.||..|||.|..|::|.|...|||.||.:||||||||| C.c    328 LTAIQLRLDELLHRHPVGDPSYEGLNEIRQTGVRAADLVRKLLAFSRKQT    377  R.c    455 LKPRIIDLRDTLSDLTHLLNRLTGEKVVLTLTHDPNLAPIRADKRQLEQV    504            ::..::||.:.:|:...||.||..|.|.|...:..:|..:||||.|||.. C.c    378 VQREVLDLGELISEFEVLLRRLLREDVKLITDYGRDLPQVRADKSQLETA    42  R.c    505 IMNLVVNARDAM---PGGGEIRIETENLHLIEDLKRDRA-----AVPKGN    546   14                    :|||.||||||:   .|||.:||.|..      |.||.|     ....|: C.c    428 VMNLAVNARDAVRAAKGGGVVRIRTAR------LTRDEAIQLGFPAADGD    471  R.c    547 YVVVKVTDEGVGIPADKLGKIFEPFYTTKKPGEGTGLGLSTAYGIVKQTG    596            ...::|:|:|.|||.|.:||||:||:|||..||||||||:|.||||||:. C.c    472 TAFIEVSDDGPGIPPDVMGKIFDPFFTTKPVGEGTGLGLATVYGIVKQSD    521  R.c    597 GYIFCDSVLGSGTCFTLFLPAHDRPS-----EIEQEPALPTMELPSIEEN    641            |:|...|....|..|.:|||.::.|:     :...|||.|.   .:.:.: C.c    522 GWIHVHSRPNEGAAFRIFLPVYEAPAGAVAVQAVAEPAKPR---AARDLS    568  R.c    642 SAAMVLLVEDEAPVRAFASRALKLRGYTVFEAENAEEALRILEDDQLQFD    691            .|..:|.||||..||:.|:|.|:.|||.|.||.:.||||.|.|::....| C.c    569 GAGRILFVEDEDAVRSVAARLLRARGYEVLEAADGEEALIIAEENAGTID    618  R.c    692 VFVTDVIMPGMDGPTWVAEA---LKTRPDTAVVFVSGYAEDVFREGRPPT    738            :.::||||||:||||.:.:|   |.|.|   |:|:|||||..|.:..... C.c    619 LLISDVIMPGIDGPTLLKKARGYLGTAP---VMFISGYAEAEFSDLLEGE    665  R.c    739 PNSVFLPKPFSLSELTATVQNQIARRARA    767            ....|||||..:..|...|:.|:...    C.c    666 TGVTFLPKPIDIKTLAERVKQQLQAA---    691        15         Figure 1.3. R. capsulatus CckA alignment with C. crescentus CckA, and domains (A) Pairwise alignment was performed using needle (https://www.ebi.ac.uk/Tools/psa/emboss_ needle/) (32% identity). R. capsulatus transmembrane segments are highlighted in grey, R. capsulatus and C. crescentus PAS domains are highlighted in yellow, the HisKA domains are highlighted in green, the HATPase domains are highlighted in red, and the REC domains are highlighted in blue. Histidine 399 is indicated by a black arrow, valine 443 by a dark grey arrow, and aspartate 589 by a light grey arrow. (B) Schematic representation of the CckA protein. The N-terminal transmembrane domains are represented by the black triangles. Length, indicated by the number of amino acids, is indicated below.                16           Homologous protein(s) Homologous substitution R. capsulatus substitution  Affected domain  Description of activity/phenotype in homologous protein (Chen et al., 2009; Francez-Charlot et al., 2015)Huynh et al., 2013; Kim et al., 2013; Lori et al., 2015; Dubey et al., 2016) Caulobacter crescentus CckA  S. melonis CckA(H322A)   CckA(H427A) H399A  HisKA - Site of histidine phosphorylation. - Loss of kinase activity (K-). - Normal phosphatase activity (P+). - Lethal in C. crescentus.  Caulobacter crescentus CckA CcCckA(V366P) V399P HisKA - Normal kinase and autophosphorylation activity. - Partial or complete loss of phosphatase activity. - K+P- phenotype. - Responds to c-di-GMP. Caulobacter crescentus CckA  A. tumefaciens  E. coli CcCckA(Y514D)   CckA(Y674D)  NarX(Y551F) Y589D HATPase_c - Normal kinase and autophosphorylation activity. - Loss of ability to switch from kinase to phosphatase state in response to c-di-GMP - K+++ P+ phenotype - Loss of phosphatase activity in E. coli NarX.    Table 1.1 Summary of site-directed CckA mutations created.   17         Chapter 2: Materials and Methods 2.1 Bacterial strains and growth conditions R. capsulatus strain B10 is an environmental isolate (Marrs, 1974; Weaver et al., 1975). Strain SB1003 was constructed by treating strain B100 [a phage-cured strain of BI0] with GTA from BB101, which carries the rif-10 mutation that confers resistance to rifampin [RifR], and selecting for the transfer of the rif-10 marker, conferring resistance to the antibiotic rifampin (Solioz et al., 1975; Yen and Marrs, 1976; Solioz and Marrs, 1977; Yen et al., 1979). Strain DE442 is an RcGTA overproducer mutant of unknown origin; however DE442 is likely derived from a strain derived from strain B10 (J.T. Beatty personal communication). Based on pigmentation, strain DE442 also likely contains a crtD mutation affecting carotenoid biosynthesis. The genome of strains DE442 and SB1003 have been sequenced (Strnad et al., 2010; Ding et al., 2014), and the sequences indicate a close relationship, as the chromosomes differ by only about 700 to 800 single nucleotide polymorphisms. All R. capsulatus strains were cultured at 30ᵒ C. Chemoheterotrophically cultured strains were grown in RCV minimal medium (Beatty and Gest, 1981) on a wheel in standard test tubes. Photoheterotrophically grown cultures were cultured in YPS complex medium (Wall et al., 1975) in 16.5 mL screw cap culture tubes. Liquid cultures of R. capsulatus were supplemented with tetracycline HCL (0.5 ug/ml), kanamycin sulfate (10 ug/ml), or gentamicin sulfate (1.5 ug/ml), as appropriate.  All recipient and adsorption assays were done using the WT strain B10 and derivatives, as in previous recipient and adsorption assays (Brimacombe et al., 2013; Brimacombe et al., 2014). As previous studies involving RcGTA production assays have involved the use of WT   18         strain SB1003 (Mercer et al., 2010; Westbye, 2016), I chose to complete all production assays using this strain. Escherichia coli strain DH5α (Sambrook et al., 1989) lambda pir was used for cloning work. For plasmid conjugation into R. capsulatus, E. coli S17-1 (Simon et al., 1983) was used. E. coli were cultured in LB medium (Sambrook et al., 1989) supplemented with either ampicillin (100 ug/ml), tetracycline HCL (10 ug/ml), kanamycin sulfate (50 ug/ml), or gentamicin sulfate (10 ug/ml), as appropriate.  2.2 Construction of targeted mutants and complementation plasmids Genetic constructs are listed in Table 2.2. Strain SB1003 ΔCckA (Mercer et al., 2012) was used for construction of strain B10 ΔCckA. The SB1003 ΔCckA genomic segment containing a truncated CckA interrupted by a kanamycin resistance cassette was PCR-amplified using primers CckA-F and CckA-R, and the resultant ~1.8 kbp amplicon was cloned into the pUC19 plasmid SmaI site. As described by Mercer et al. (2012), ~91% of the coding region of the CckA gene was replaced by a kanamycin resistance cartridge. RcGTA-transduction was used to replace the WT B10 CckA gene with the mutant version as described previously (Aklujkar et al., 2000). Strain B10 ΔChpT was created in the same way as the SB1003 ΔChpT strain created by (Mercer et al., 2012). Briefly, the ChpT-F and ChpT-R primers (Table 2.3) were used to amplify the ChpT gene and the amplified products were cloned into the plasmid pGEM-T-Easy. A kanamycin-resistance KIXX cartridge obtained by SmaI digestion (Barany, 1985) was then inserted into the  NruI site of the amplified ChpT fragment 79bp from the predicted start of the ChpT ORF, and the resultant ChpT insertion mutant was used to replace the endogenous ChpT   19         gene by RcGTA-transduction. PCR screening and sequencing were used to verify that the correct chromosomal mutation was created. Strain B10ΔDivL was created using RcGTA-transduction from the SBΔDivL strain (Aklujkar et al., 2000; Westbye, 2016). Briefly, the DivL gene was replaced with a version of the gene carrying the gentamicin resistance aaaC1 at a NaeI site 796 bp in to the 1608 bp divL gene(Westbye et al., 2013; Westbye, 2016; Westbye et al., 2017). 2.3 Construction of chromosomal reporter strains The plasmid pZWghsA was used for construction of strains SB1079 and SB ΔCckA1079. The plasmid was created as described previously (Westbye et al., 2017). Briefly, the native ghsA/B (rcc01079/80) operon promoter was fused to a lacZ coding region reporter in the suicide plasmid pZWp1079 (Westbye et al., 2017). This suicide plasmid was conjugated into each strain (as described above) and gentamycin resistance was used for selection of strains with chromosomal integration of the plasmid. Verification of the proper insertion was verified by PCR screening, followed by sequencing of the region of interest to ensure integration at the correct site. 2.4 RcGTA recipient assay RcGTA recipient capability was measured as previously described (Brimacombe et al., 2013). The rifampin-resistant SB1003 strain was used as a donor in all experiments. SB1003 was inoculated to an OD660 of 0.15, and RcGTA was harvested from this strain following photoheterotrophic growth in YPS medium for 40 h, by passing culture supernatant through a 0.45 µm pore-size filter after 2 min of centrifugation at 16,160 x g. One hundred uL of donor RcGTA filtrate were used for the assay.    20         Recipient cultures were grown aerobically in RCV medium. Cultures were inoculated to an OD660 of 0.15 and grown for 24 h before being harvested. A sample of 1.5 mL of culture was centrifuged for 5 min at 3,500 x g. The pellet was then re-suspended in 600 uL of G-buffer [10 mM Tris-HCl (pH 7.8), 1 mM MgCl2, 1 mM CaCl2, 1 mM NaCl, 500 mg/ml BSA] (Solioz et al., 1975). A mixture of 100 uL donor RcGTA, 100 uL recipient cells, and 400 uL G-buffer was incubated with shaking at 30ᵒ C for 1 h. Nine hundred uL of RCV medium were added and incubation and shaking occurred for another 3 h. Cells were spread on RCV plates containing 80 mg/mL rifampin. Plates were incubated at 30ᵒ C for three days before the number of Rifr colonies were counted. 2.5 RcGTA adsorption assay  RcGTA adsorption assays were carried out as described previously (Brimacombe et al., 2014). Samples from cultures of mutant strains of R. capsulatus were mixed with SB1003 filtered culture supernatant as a source of RcGTA, as described in section 2.4. This mixture was incubated with shaking for 1 h at 30 ᵒC, passed through a 0.45 µm pore-size filter, and 100 uL were subsequently mixed with 100 uL of re-suspended (as described in section 2.4) WT B10 R. capsulatus cells and 400 uL of G-buffer, prior to a 4 h incubation, as described in section 2.4. Cultures were then plated and colonies were counted after incubation, again as described in section 2.4. 2.6 RcGTA transduction assay RcGTA activity was determined by harvesting RcGTA from various SB1003-derived mutants. Stains were inoculated to OD660 0.3 and grown photoheterotrophically at 30ᵒ C for 40 h. Filtrate was collected by passing the cultures through 0.45 µm pore-size filters following centrifugation at 16,160 x g for 5 min. Filtrates were mixed with recipient B10 cells, and   21         incubated as described in section 2.4, and plated on RCV medium containing Rif. RifR colonies were counted to determine the level of transduction activity (as described in section 2.4). In this case, the number of RifR colonies (minus the number of spontaneous RifR colonies obtained using sterile medium in place of culture filtrate) was used as a measure of the amount of functional RcGTA particles produced. 2.7 Western blot Cultures were inoculated to an OD660 of 0.15 in YPS medium and grown photoheterotrophically for 40 h before samples were harvested. One and a half mL of liquid culture samples, normalized to a density of ~1.12 x 108 CFU/ml, were pelleted at 16,160 x g for 5 min. The culture supernatant was concentrated 10-fold using a vacuum centrifuge to evaporate water. SDS-PAGE separation was performed using a 12% separating gel and 4% stacking gel. Transfer to a nitrocellulose blotting membrane (BioTract NT Pall Life Sciences) was performed at 120 V for 2 h in a Mini Trans-Blot apparatus (Bio-Rad) in electroblot buffer (27.5 mM Tris-base,192 mM glycine and 20% methanol). Membranes were blocked with 5% Carnation skim milk powder in TBS-T (1.2% w/vol Tris-base, 1% NaCl, 0.1% Tween-20, adjusted to pH 7.5 with HCl), probed with rabbit anti-RcGTA capsid serum (Taylor, 2004), washed three times with TBS-T, then probed with horseradish peroxidase-conjugated donkey anti-rabbit antibody (GE Healthcare). Blots were developed using luminol and p-coumaric acid (Haan and Behrmann, 2007), and exposed to X-ray film (Kodak). 2.8 Agarose gel electrophoresis DNA fragments used for screening purposes during cloning were analysed using gel electrophoresis in 0.8 or 1% agarose gels in 0.5x TBE (40 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA, pH 8.3). Gels were stained with ethidium bromide followed by UV visualization.    22         2.9 β-galactosidase assays Activation of promoter activity was determined by measuring the -galactosidase activity of cells containing a chromosomally integrated promoter-lacZ reporter construct (as described in detail in section 2.3). All cultures were grown photoheterotrophically in RCVm medium (containing 10 mM instead of the usual 9.6 mM phosphate of RCV) (Westbye, 2016) for 24 h at 30ᵒ C in 16.5 mL screw-capped glass tubes, inoculated at an OD660 of 0.30. Fifteen mL of culture were centrifuged for 30 min at 6,859 x g in 50 mL tubes. Cell pellets were then re-suspended in 2 mL of Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM -mercaptoethanol, pH 7). The 2 mL cell suspension was lysed using a FastPrep-24 bead homogenizer (MP Biomedicals). The lysed cells were centrifuged at 16,160 x g 2 min, and 500 uL of cell lysate supernatant were added to 200 uL of o-nitrophenyl-β-D-galactoside (ONPG; 4 mg/mL in 60 mM Na2HPO4, 40 mM NaH2PO4, pH 7). Absorbance was measured at 420 nm over time, and the initial, linear portion of the curve used to calculate specific activities, normalized to total protein content of the sample.  2.10 Specific activity and total protein determination by the Lowry method  Specific activity of β-galactosidase activity was determined using the formula:  Where Cprotein is the concentration of total protein (mg/mL) determined by a Lowry assay (Peterson, 1983) and V indicates volume.   23         2.11 Microscopy A phase contrast light microscope was used for the imaging. Cells were strained with crystal violet before pictures were taken using an oil-immersion 100 X objective lens. 2.12 Bioinformatic analyses The multiple sequence alignment was performed using MUSCLE (v. 3.8.31) with default accuracy settings, curation was performed using Gblocks (v. 0.91b), the phylogenetic tree was constructed using the PhyML program (v. 3.1/3.0 aLRT) using a maximum likelihood method, reliability for internal branches was tested using the aLRT test, and graphical representation of the tree was obtained using TreeDyn (v. 198.3) (Castresana, 2000; Guindon and Gascuel, 2003; Edgar, 2004; Chevenet et al., 2006; Anisimova and Gascuel, 2006; Dereeper et al., 2008; Dereeper et al., 2010). Table 2.1: Strains used in this study Strain Reference Description B10 (Marrs, 1974; Weaver et al., 1975) R. capsulatus WT B10ΔDivL This work R. capsulatus, DivL insertion mutant, GmR; DivL::aaaC1 B10ΔChpT This work R. capsulatus, ChpT insertion mutant, KmR; ChpT::KIXX B10ΔCckA (Leung et al., 2013) R. capsulatus, CckA deletion mutant, KmR; CckA::KIXX B10ΔCckA pRCckA This work B10ΔCckA containing plasmid pRCckA, Kmr, Tcr B10ΔCckA HA This work B10ΔCckA containing plasmid pRCckA-HA, Kmr, Tcr B10ΔCckA VP This work B10ΔCckA containing plasmid pRCckA-VP, Kmr, Tcr B10ΔCckA YD This work B10ΔCckA containing plasmid pRCckA-YD, Kmr, Tcr SB1003 (Solioz et al., 1975; Yen and Marrs, 1976; Solioz and Marrs, 1977; Yen et al., 1979) R. capsulatus, SB1003, RifR   24         Strain Reference Description SBΔCckA (Mercer et al., 2012) R. capsulatus, SB1003 CckA insertion mutant, RifRKmR SBΔCckAHA This work SBΔCckA containing plasmid pRCckA-HA, RifR, Kmr, Tcr SBΔCckAVP This work SBΔCckA containing plasmid pRCckA-VP, RifR, Kmr, Tcr SBΔCckAYD This work SBΔCckA containing plasmid pRCckA-YD, RifR, Kmr, Tcr SBΔCckA1079 This work SBΔCckA CckA containing integrated pZW1079 reporter construct, RifR, GmR SBΔCckA1079 pRCckA This work SBΔCckA CckA containing integrated pZW1079 reporter construct, complemented with plasmid pRCckA, RifR, GmR, TcR SBΔCckA1079 HA This work SBΔCckA CckA containing integrated pZW1079 reporter construct, complemented with plasmid pRCckA-HA, RifR, GmR, TcR SBΔCckA1079 VP This work SBΔCckA CckA containing integrated pZW1079 reporter construct, complemented with plasmid pRCckA-VP, RifR, GmR, TcR SBΔCckA1079 YD (Westbye et al., 2017) SBΔCckA CckA containing integrated pZW1079 reporter construct, complemented with plasmid pRCckA-YD, RifR, GmR, TcR  Table 2.2: Plasmids used Name Reference Description pUC19 Invitrogen    General cloning plasmid, Ampr pZWp1079 This work Suicide plasmid used for the construction of 1079-LacZreporter strains.  pRK415 (Keen et al., 1988) Broad host range plasmid, Tetr pRCckA (Westbye et al., 2013) cckA complementation plasmid, Tetr pRCckA-HA This work CckA(H399A), kinase mutant, Tetr pRCckA-HN This work CckA(H399N), kinase mutant, Tetr   25         Name Reference Description pRCckA-VP This work CckA(V433P), phosphatase mutant, Tetr pRCckA-YD (Westbye, 2016) CckA(Y589D), constitutive kinase, Tetr           Table 2.3: Primers used. Sites of site-directed mutagenesis mismatches are underlined; restriction enzyme sites are in bold. primer sequence  used in construction of restriction site Reference CCkA_H399A_F2 CGGGCGGGGTTGCGGCGGATTTCAACAACTTG pRcckA(H399A) N/A This work CckA_H399A_R2 CAAGTTGTTGAAATCCGCCGCAACCCCGCCCG pRcckA(H399A) N/A This work CckA_V433P_F CGGGCGGCCTCGCTGCCGGGGCAGCTTCTGGCG pRcckA(V443P) N/A This work CckA_V433P_R CGCCAGAAGCTGCCCCCGCAGCGAGGCCGCCCG pRcckA(V443P) N/A This work CckA(Y589D)-F GGGGCTCTCGACCGCCGACGGGATCGTCAAGCAG pRcckA(Y589D) N/A (Westbye et al., 2017) CckA(Y589D)-R CTGCTTGACGATCCCGTCGGCGGTCGAGAGCCCC pRcckA(Y589D) N/A (Westbye et al., 2017) CckA-F TAAGTAGTCGACGATCTGGTGCTGGT Amplification of cckA::KIXX fragment cckA N/A Westbye, 2016   26         CckA-R ACAATGGTCGACACGCTTTCGCACAG Amplification of cckA::KIXX fragment cckA N/A Westbye, 2016 ChpT-F CAGCACGAGATGGTCGTAAA B10ΔChpT N/A (Ryan G. Mercer et al., 2012) ChpT-R GCACGATCCGTGCGTAAG B10ΔChpT N/A (Ryan G. Mercer et al., 2012) DivL-F ATATGAGCTCCTGGCGCGAGGACGAGACCG B10ΔDivL SacI (Westbye et al., 2017) DivL-R ATATTCTAGAGCAACTCTAGCGGCCCTGCCC B10ΔDivL XbaI (Westbye et al., 2017)   27         Chapter 3: Results 3.1 Effects of CckA(H399A), CckA(V443P), and CckA(Y589D) on the phosphorylation state of CtrA/CtrA~P Site-directed mutations of CckA were created to investigate the effects of these mutations on the relative levels of phosphorylated and non-phosphorylated CtrA. The mutations designed were based on previously studied mutations in CckA homologues in other species (Chen et al., 2009; Huynh et al., 2013; Kim et al., 2013; Francez-Charlot et al., 2015; Lori et al., 2015; Westbye, 2016). The purpose of these initial experiments was to validate these mutants as having activities of either phosphatase-only (CckA(H399A)), kinase-only (CckA(V443P)), or constitutive kinase (CckA(Y589D)). I used in vivo, indirect methods to evaluate the phosphorylation state of CtrA/CtrA~P. I used the cckA null (knockout) strain SBΔCckA as a baseline, and compared the properties of this strain complemented with plasmid-borne copies of mutant cckA genes to this SBΔCckA strain complemented with the WT cckA gene. 3.1.1 Loss of CckA leads to a decrease in the amount of CtrA~P Levels of CtrA~P were indirectly measured via a β-galactosidase assay to determine the activity of the ghsA/B promoter driving a chromosomally-integrated lacZ gene, previously shown to be stimulated by CtrA~P (Figure 3.1A) (Westbye et al., submitted). I measured levels of β-galactosidase in a CckA deletion mutant, and found that there was an insignificant (~0) level of activity, comparable to that of a strain containing no β-galactosidase reporter, indicating that there is a loss of phosphorylated CtrA in this mutant (Figure 3.1B).  These results confirm previous work showing that CckA is needed to phosphorylate CtrA, and validate my use of β-galactosidase activity driven by the ghsA/B promoter as an indication of the relative amount of CtrA~P in mutants compared to a WT strain. Therefore, in   28         the following sections I use the -galactosidase activity from this chromosomally integrated reporter as an index of the phosphorylation state of CtrA: relative to WT controls, high -galactosidase activity indicates a high fraction of CtrA~P whereas low activity indicates a high fraction of non-phosphorylated CtrA.3.1.2 CckA(H399A) leads to low levels of CtrA~P  Using the activity of the ghsA/B promoter driving lacZ expression to investigate the ability of the CckA(H399A) predicted kinase-deficient mutant to phosphorylate CtrA, it was found that the strain containing a mutant CckA(H399A) as the only copy of the CckA protein had approximately 30%  of  β-galactosidase activity as compared to WT (Figure 3.1B). This indicates that low levels of CtrA~P were present, and that the CckA(H399A) mutation impairs the ability of CckA to phosphorylate CtrA. Although it could be argued that the H399A mutation enhances phosphatase activity without affecting kinase activity, work on homologous CckA proteins (Chen et al., 2009; Francez-Charlot et al., 2015) makes this argument specious. 3.1.3 CckA(V443P) leads to high levels of CtrA~P 3.1.4 It was found that the strain containing CckA(V443P) as the only copy of CckA, had an approximately 2 to 6-fold increase in β-galactosidase activity (Figure 3.1B). This indicates that high levels of CtrA~P were present and that CckA(V433P) is impaired in its ability to dephosphorylate CtrA. Although it could be argued that the V433P mutation enhances kinase activity without affecting phosphatase activity, work on homologous CckA proteins (Chen et al., 2009; Lori et al., 2015) argues against this conclusion. A small, significant, increase in the level of β-galactosidase activity was also seen in the CckA deletion strain complemented with a WT CckA gene driven by its native promoter (Figure 3.1B, Table S1), suggesting that there are potential copy number effects of having CckA present   29         separately on a plasmid. This data indicates that a small increase in the number of copies of the cckA gene, and a predicted increase in CckA levels, leads to an increase in cellular amounts of CtrA~P.CckA(Y589D) leads to a decrease in the amount of CtrA~P I found that the strain containing CckA(Y589D) as the only copy of CckA had an undetectable level of β-galactosidase activity relative to the WT control. This suggests that CckA(Y589D) results in a loss of CtrA~P. Because the analogous Y589D mutation in homologous CckA proteins was shown to affect binding of the signaling molecule c-di-GMP, it appears that this signaling molecule modulates CckA activity also in R. capsulatus. On the basis of my -galactosidase reporter activity, it appears that binding of c-di-GMP stimulates phosphorylation of CtrA, as the inability to bind c-di-GMP in this mutant leads to a loss of phosphorylated CtrA (low -galactosidase activity).   30           Figure 3.1. CckA activities differentially affect levels of CtrA and CtrA~P Rc1079 promoter activity (β-galactosidase activity, calculated as (A420 . [protein])/min) of strains containing a chromosomal promoter reporter LacZ fusion to the 1079/80 gene encoding components of the GTA head spike protein (Westbye et al., 2016; Westbye et al., Submitted). (A) Strain SBΔCtrA1079 was complemented with either non-phosphorylatable CtrA(D51A) or phosphomimetic CtrA(D51E).   31         Taken from Westbye et al., Submitted. (B) Strain SBΔCckA1079 was complemented with WT CckA, CckA(H399A), CckA(V433P) or CckA(Y589D). Error bars represent standard deviation, n = 3 for all samples.   3.2 Levels of CckA site-directed mutants affect RcGTA production and release Previous studies have indicated that the CckA-ChpT-CtrA pathway plays a role in regulating both RcGTA production, and release, with all three proteins necessary for WT levels of RcGTA transduction (the ability of cells to produce extracellular, functional RcGTA capable of transferring genes to recipients) (Mercer et al., 2012). However, the roles of CckA kinase and phosphatase activities, and the possible role of c-di-GMP in mediating this production and release were previously unknown. Therefore, to address these questions I determined the effects of the CckA mutations described in sections 3.1-3.2 on the production of RcGTA particles capable of transducing genes. 3.2.1 Loss of CckA kinase activity increases RcGTA activity The CckA(H399A) mutant, which appears to be deficient in the ability to phosphorylate CtrA (see section 3.1), increased RcGTA transduction activity to ~2.5 times that of the WT strain (Figure 3.2A). This indicates that non-phosphorylated CtrA suffices for RcGTA production and/or release. The results of a western blot probed with RcGTA capsid antiserum (Figure 3.2B) show that expression of RcGTA is highly increased in the CckA(H399A) mutant, indicating that non-phosphorylated CtrA increases expression of the GTA gene cluster. However, compared to WT cells where most of the GTA produced is released (Figure 3.2B lane 1), the decrease in kinase activity of the CckA(H399A) mutant led to a much lower percentage of RcGTA in the supernatant, as compared to the huge amount found in the pellet. This indicates that although   32         non-phosphorylated CtrA increases the amount of RcGTA produced, phosphorylated CtrA enhances cell lysis. This interpretation is consistent with data published by Mercer et al. (2012). 3.2.2 Loss of CckA phosphatase activity decreases RcGTA activity as the result of inhibition of release from cells A phosphatase mutant of CckA (CckA(V443P)) was thought to lead to an increase in the amount of phosphorylated CtrA present (section 3.1). CckA(V443P) led to a decrease in RcGTA activity (Figure 3.2A). This indicates that phosphorylated CtrA either inhibits RcGTA production and/or release, or that CckA lacking phosphatase activity is simply insufficient to stimulate RcGTA intracellular production. Western blot experiments on this mutant showed that approximately equal or greater levels of the RcGTA capsid protein are present in the cell pellet relative to a cckA gene deletion mutant (Figure 3.2B), but that a decrease in the release of RcGTA from cells leads to the approximately 80% decrease in transduction frequency observed. These results indicate that an increase in CtrA~P, and a presumed decrease in non-phosphorylated CtrA, lead to a decrease in cell lysis and therefore a decrease in RcGTA activity.   The results of the western blot (Figure 3.2B) indicate that while capsid protein is highly expressed within cells, lysis of cells and release of RcGTA to the supernatant is inhibited. This indicates that a lack of CckA phosphatase (and a predicted increase in CtrA~P) leads to an increase in stimulation of the RcGTA structural gene promoter, but to a reduction in cell lysis.   33            34         Figure 3.2 RcGTA activity and gene expression. (A) The ratio of gene transfer activity for each indicated strain relative to the strain ΔCckA pRCckA. Error bars represent the standard deviation. n = 3 in all graphs. (B) Western blot detecting the RcGTA major capsid protein in the cells (pellet) and supernatant fractions of anaerobically grown strains. Bottom two panels represent equivalent blots with varying exposure lengths. (C) Quantication of the intensities of the western blot supernatant bands, as compared to their corresponding average RcGTA transduction frequencies (as shown in part A). A correlaction coefficient of 0.958 indicates that there is a strong relationship between the amount of RcGTA capsid protein in the supernatant, and the relative transduction frequencies of the strains. 3.2.3 Loss of c-di-GMP-binding leads to a decrease in RcGTA production and release A loss of transduction seen with this CckA(Y589D) mutant (Figure 3.2A), comparable to that of a CckA deletion strain (~2% of WT levels), indicated that this mutant was either defective in RcGTA production intracellularly, RcGTA release from cells, or both. A western blot (Figure 3.2B) shows that this mutant has the ability to produce RcGTA, but is defective in cell lysis, leading to a lack of RcGTA transduction. Using the C. crescentus Y514D CckA (and analogous CckA mutants in other species) as a model, this mutant would not bind c-di-GMP, and as a consequence not be able to switch from exhibiting kinase to phosphatase activity in response to high levels of c-di-GMP. Thus it appears that an increase in c-di-GMP concentration, and a subsequent switch of CckA from kinase to phosphatase activity, is necessary for cell lysis and the release of RcGTA particles from cells. However, the result above whereby the phosphorylation state of CtrA was investigating using the ghsA/B-lacZ reporter (Figure 3.1B) indicates that this mutant stimulates phosphatase, or inhibits kinase, activity. Therefore this mutant protein appears to behave differently than its homolog in C. crescentus.   35         3.3 CckA site-directed mutations affect RcGTA recipient capability A deletion of the ctrA gene was shown to lead to a loss of the ability to receive RcGTA-transferred DNA (RcGTA recipient capability) (Brimacombe et al., 2014). It was demonstrated that a phosphomimetic CtrA (CtrAD51E) was able to partially complement recipient capability, to approximately 50% of WT levels, while expression of a non-phosphorylatable CtrA (CtrAD51A) in the ctrA deletion mutant led to an approximately 2-fold increase in recipient capability. However the possible role of CckA and other phosphorelay-related proteins was unknown. I therefore sought to test strains containing the mutant CckA proteins described above, as well as deletions in other proteins thought to be involved in regulating the phosphorylation state of CtrA, for RcGTA recipient capability.  3.3.1 Increased cckA copy number decreases RcGTA recipient capability The predicted over-expression of CckA in strains complemented with an exogenous copy of cckA, and those containing both chromosomal and extrachromosomal copies of cckA had a slight, significant, decrease in RcGTA recipient capability (Figure 3A,B, TableS1). This indicates that a slight increase in the levels of CckA~P due to this increase in CckA levels, act to decrease the level of RcGTA recipient capability; however, the effect is small.  3.3.2 Loss of CckA kinase activity leads to an increase in RcGTA recipient capability The expression of CckA(H399A) in the cckA knockout strain B10 ΔCckA that was found to be impaired in the ability to phosphorylate CtrA (see section 3.1.2) led to an approximately 3-fold increase in RcGTA recipient capability relative to the WT strain (Figure 3.3A). This indicates either that non-phosphorylated CtrA stimulates RcGTA recipient capability, or that CtrA~P is inhibitory to RcGTA recipient capability. Because a WT strain expressing the H399A CckA kinase mutant protein in addition to the WT copy exhibited an approximately 4-fold   36         increase in levels of recipient capability (Figure 3.2B), it appears that the loss of kinase activity by the H399A mutant is the primary reason for this increase, and that this is dominant over the effects of the WT protein. It therefore seems likely that the WT and H399A mutant CckA proteins can dimerize together, and the presence of one mutant H399A protein inhibits the kinase activity of the protein dimer. These results are consistent with previous data demonstrating that the presence of non-phosphorylatable CtrA(D51A) aids in increasing RcGTA recipient capability (Brimacombe et al., 2014).   37            38         Figure 3.3. RcGTA recipient capability RcGTA recipient capability relative to the WT strain B10 measured as the ability of strains to receive an RcGTA-transferred genetic marker. A WT B10 and cckA deletion strains, and the cckA deletion strain containing WT or mutant CckA proteins. B WT B10 strain containing either WT or mutant version of the CckA protein. C WT B10 and chpT deletion strains. D WT B10 and divL deletion strains. Error bars represent standard deviation.  n = 3 in all samples.  3.3.3 Loss of CckA phosphatase does not affect RcGTA recipient capability CckA(V433P), demonstrated to lead to a two to four-fold increase in CtrA~P-dependent expression of the ghsA/B-lacZ reporter, attributed to a decrease in phosphatase activity (see above), retained approximately WT levels of RcGTA recipient capability (Figure 3.3A), indicating that an increase in the level of CtrA~P does not affect recipient capability. However, cells containing both a WT and phosphatase (V433P) mutant of CckA showed an approximately 3.5-fold increase in RcGTA recipient capability relative to the WT control (Figure 3.3B). This indicates that a CckA protein deficient in phosphatase activity, although not affecting recipient capability when it is the only type of CckA in the cell, somehow in the presence of the WT CckA, stimulates RcGTA recipient capability. 3.3.4 Loss of c-di-GMP-binding leads to a loss of RcGTA recipient capability CckA proteins containing mutations analogous to my Y589D mutation have been demonstrated to lead to a decrease in c-di-GMP-binding (Huynh et al., 2013; Kim et al., 2013; Lori et al., 2015), and the Y589D mutation caused an apparent decrease in the amount of CtrA~P in R. capsulatus (see section 3.1.4). The expression of CckA(Y589D) in the cckA knockout resulted in a significant loss of RcGTA recipient capability (Figure 3.2A ). However, when both   39         the WT and Y589D mutant versions were present, WT levels of recipient capability were seen (Figure 3.2B). This indicates that the WT allele is dominant over the Y589D mutant.  3.3.5 Loss of ChpT leads to an intermediate phenotype RcGTA recipient capability was investigated in a chpT deletion mutant of the WT strain B10. It was found that there was a decrease in recipient capability to approximately 60% of the value of WT B10 cells (Figure 3.3C). This indicates that ChpT aids in, but is not sufficient for, ensuring that adequate levels of non-phosphorylated CtrA are present to result in maximal recipient capability.  3.3.6 Loss of DivL leads to an almost complete loss of RcGTA recipient capability  The C. crescentus non-canonical histidine kinase DivL plays an important role in stimulating CckA kinase activity, thereby indirectly leading to higher levels CtrA~P and the activation of the cell cycle. A truncated homolog of DivL, lacking both the C-terminal non-functional DHp and CA domains of its C. crescentus homolog has also been demonstrated to play a role in regulating RcGTA production. Specifically, the absence of DivL in SB1003 led to an increase in expression of the RcGTA gene cluster, and an increase in transduction frequency (Westbye, 2016).  The clear role of DivL in regulating the CckA-ChpT-CtrA phosphorelay pathway in both C. crescentus and R. capsulatus, suggested that DivL likely also plays a role in regulating RcGTA recipient capability. I therefore tested the effects of deleting DivL on RcGTA recipient capability. I found that a loss of DivL leads to a reduction in RcGTA recipient capability (Figure 3.3D) to approximately 3% of WT, indicating that this protein plays a role in regulating not only RcGTA production, but also recipient capability.   40         3.4 Mutations in CckA kinase and phosphatase activity do not affect RcGTA adsorption Previous studies have indicated that RcGTA binding and uptake by R. capsulatus cells is greatly enhanced by the presence of a polysaccharide capsule in cultures grown anaerobically (Brimacombe et al., 2013). To test whether or not the increase or decrease in recipient capability of the cckA mutants studied here was due to a difference in binding of RcGTA to cells in my aerobically grown cultures, I performed an adsorption assay (described in section 2.5). Although the error bars were quite large in some data-sets (Figure 3.4), these results did not yield significant differences between WT and mutant versions of R. capsulatus cultures. I suggest that the differences seen in recipient capability (section 3.3) were not due to differences in RcGTA binding to cells, but instead were due to differences in DNA-uptake processes following binding.      41         Figure 3.4 RcGTA adsorption assay. The amount of RcGTA activity present following adsorption to various mutant strains was determined as a measure of the ability of RcGTA particles to bind various mutant strains. All results are presented relative to a B10 WT control. Error bars represent standard deviation, n = 3 for all samples.  3.5 Cell morphology in late exponential phase is not affected by mutant CckA activities As the CckA-ChpT-CtrA pathway is known to be involved in regulating the cell cycle in C. crescentus and other species (as described in the introduction), and mutations in the components of this pathway often lead to cell filamentation, I sought to investigate the effects of my mutant CckA proteins on R. capsulatus cell morphology. I saw no observable differences in morphology (Figure 3.5), or growth rate (data not shown), in any of my mutants, suggesting that the kinase and phosphatase activities of CckA do not affect growth rate, or cell division at this time point and under these growth conditions.  42             Figure 3.5 Light microscopy of mutant cells in late exponential phase. A WT strain B10. B cckA strain. C cckA strain complemented with WT cckA gene. D cckA strain complemented with mutant cckA gene H399A. E cckA strain complemented with mutant cckA gene V443P. F cckA strain complemented with mutant cckA gene Y589D.   A B C D E F   43  Table 3.1 Summary of phenotypes Strain Predicted kinase activity Predicted phosphatase activity RcGTA recipient capability (%WT) RcGTA production (%WT) RcGTA release (%WT) β-galactosidase activity (%WT) RcGTA adsorption (%WT) Cell growth/ morphology WT (B10/ SB1003) Normal Normal 100% 100% 100% 100% 100% Normal ΔCckA Absent Absent ~1% >200% Undetectable ~2% 100% Normal ΔCckA pRCckA Normal Normal ~70% ~100% ~100% ~300% 100% Normal ΔCckA H399A Absent Normal ~280% >600% 200% ~50% 100% Normal ΔCckA V443P Normal Absent ~100% >300% 50% ~600% - 2200% 100% Normal ΔCckA Y589D Constitutive Absent ~1% 100% Undetectable ~2% 100% Normal B10  ΔDivL N/A1 N/A ~1% N/A N/A N/A N/A N/A B10 Δ ChpT N/A N/A ~60% N/A N/A N/A N/A N/A  1 N/A, not applicable. WT strain SB1003 was used when measuring RcGTA production, release, β-galactosidase activity, and when observing growth and morphology. WT strain B10 was used when measuring RcGTA recipient capability, and adsorption.  44  Chapter 4: Discussion 4.1 CckA phosphatase and kinase activities affect the levels of CtrA and CtrA~P In other species, notably C. crescentus, a CckA protein acts as a sensor kinase or a phosphatase to alter the phosphorylation state of the response regulator CtrA (Biondi et al., 2006), and these two opposing activities of CtrA have been dissected mainly by site-directed mutagenesis of key residues (Chen et al., 2009). In other species, hybrid histidine kinase proteins have also been shown to be involved in complex regulatory networks involving multiple phosphorelay and response regulator proteins (Stock et al., 2000). For example, studies of the Rhodospirillum centum pathway controlling cyst formation, involving the Che3s transduction cascade, present evidence that pathways involving hybrid histidine sensor kinase proteins can be regulated not only by phosphorylation of a response regulator, but also contain other checks and balances, and can even be regulated by levels of ADP/ATP within cells (He et al., 2013; He et al., 2015). These processes include the reversal of phosphate flow, the involvement of two hybrid histidine kinase proteins and two linker proteins (each containing a single REC domain), all of which work in concert to regulate phosphorylation of Che3s and regulate the formation of cysts (He et al., 2015).  Although the R. capsulatus CckA amino acid sequence is 32.3% identical (43.4% similar) to the C. crescentus CckA sequence, and key kinase and phosphatase residues are conserved (Figure 1.3A), it was unknown whether the R. capsulatus CckA functions as does the C. crescentus homologue. I set out to investigate whether the R. capsulatus CckA has kinase and phosphatase functions as in other CckA proteins, and whether these functions affect RcGTA production and recipient capability, using site-directed mutations modeled on studies of the C. crescentus CckA. These mutant cckA genes were introduced on plasmids into a cckA knockout   45  strain, to compare the effects of each mutant gene to the absence of complementation, and to the effect of complementation with the WT cckA allele. Because previous results had shown that transcription of the ghsA/B operon is stimulated by phosphorylated CtrA (Fig 3.1A), I integrated a lacZ reporter gene into the ghsA/B operon of the mutant strains as a way to measure the activity of the ghsA/B promoter, and thereby indirectly measure the relative levels of CtrA and CtrA~P. 4.1.1 Loss of CckA leads to a decrease in the amount of CtrA~P The β-galactosidase assay of the cckA knockout strain containing the chromosomal ghsA/B::lacZ reporter showed insignificant levels of promoter activity in the absence of CckA (Table 3.1). This indicates that the CckA protein is important for CtrA phosphorylation during early stationary phase under these growth conditions. These results are consistent with previous data from studies on both C. crescentus and R. capsulatus, which provided both direct and indirect evidence that CckA is necessary for CtrA phosphorylation (Lang and Beatty, 2002; Biondi et al., 2006; Chen et al., 2009; Shelswell and Beatty, 2011; Mercer et al., 2012; Vega-Baray et al., 2015). Therefore my results confirm the involvement of CckA in this pathway in R. capsulatus.  4.1.2 The CckA mutant RcCckA(H399A) appears to be a kinase mutant that results in decreased CtrA~P levels Previous studies showed that the C. crescentus CckA(H322A) protein lacks kinase activity, as expected because this mutation is at the site of histidine phosphorylation in the histidine kinase domain of the protein (Chen et al., 2009). However, this mutation was demonstrated to leave phosphatase activity intact. These previous observations led me to hypothesize that creating a homologous mutation in the R. capsulatus CckA protein (RcCckA) would lead to a loss of kinase activity, resulting in reduced CtrA phosphorylation levels. Using   46  the CtrA~P-dependent ghsA/B promoter driving a chromosomally-integrated lacZ gene, I observed a decrease in levels of β-galactosidase activity to levels approximately 30% of the WT, indicating a reduction in phosphorylated CckA (Table 3.1). This is consistent with this mutant having decreased kinase activity, consequently leading to a decrease in levels of phosphorylated CtrA. 4.1.3 The CckA mutant CckA(V443P) appears to be a phosphatase mutant that increases CtrA~P levels The C. crescentus CckA phosphatase mutant (V443P) was previously described as lacking phosphatase activity in vitro (Chen et al., 2009). I therefore hypothesized that a homologous mutation in the R. capsulatus CckA would have the same affect. Using the indirect measure of RcCtrA~P (the lacZ reporter described above), I found a two- to six-fold increase β-galactosidase activity, indicating a large increase in the levels of phosphorylated CtrA (Table 3.1). This result is consistent with the idea that CckA(V443P) fails to de-phosphorylate CtrA, resulting in an increase in phosphorylated CtrA.  4.1.4 The CckA mutant CckA(Y589D) and the problematic binding of c-di-GMP Based on homology to the C. crescentus CckA protein, I hypothesized that this mutant would have constitutive kinase activity, and lack phosphatase activity, as a previous study reported that the homologous C. crescentus CckA(Y514D) mutation  renders the protein unable to switch from a kinase to phosphatase state in the presence of c-di-GMP (Lori et al., 2015). I found that this mutation leads to β-galactosidase activity of about 2% of the WT control, indicating that it is the absence of CtrA~P that is needed to activate the ghsA/B promoter (Table 3.1). This differs from my hypothesis, in which I predicted there would be an excess of phosphorylated CtrA due to constitutive kinase activity of the protein.   47  Recently published papers by Mann et al. (2016) and Dubey et al. (2016) have greatly illuminated how c-di-GMP binds to the C. crescentus CckA, and the effects of various mutations on kinase and phosphatase activities. Mann et al. (2016) showed by deletion analysis that the C-terminal-most PAS domain (PAS-B) is important for binding of c-di-GMP. This was surprising because the Y514D mutation (analogous to my Y589D mutation) is located in the HATPase (or CA) domain. The authors suggested that perhaps c-di-GMP is bound at an interface between the PAS-B and CA domains. Dubey et al. (2016) solved the crystal structure of CckA protein segments and found that in fact c-di-GMP is bound through the concerted interaction with residues Y514, K518, W523, and main-chain atoms of I524 (all within the HATPase or CA domain); furthermore, the structure of a dimer of the HisKA/HATPase (DHp/CA) segments showed that the c-di-GMP binding site is located between these two domains. It was suggested that the absence of c-di-GMP binding resulting from the deletion of the PAS-B domain by Mann et al. (2016) was due to an indirect conformational change in the protein structure. Therefore, although the concept of c-di-GMP being bound between two domains suggested by Mann et al. (2016) was correct, their choice of domains (PAS-B and CA) was incorrect -- the correct domains are HisKA and HATPase (DHp and CA).  These recently discovered data indicate that solely mutating the Y589D residue may not have been sufficient to study the effect of c-di-GMP binding on CckA enzymatic activity. Although it is possible that the Y589D mutation decreased the binding affinity of c-di-GMP, and that in R. capsulatus this results in a decrease CtrA~P (the inverse of the effect in other species), additional experiments targeting the residues identified in the crystal structures of Dubey et al. (2016) should be done to more fully investigate the effect of c-di-GMP on the activity of the R. capsulatus CckA.   48  4.2 CckA kinase and phosphatase activities play different roles in mediating RcGTA production and release  Previous evidence indicated that the CckA-ChpT-CtrA pathway is involved in mediating RcGTA production, and release from cells. I therefore investigated the role of the CckA kinase, phosphatase, and c-di-GMP-binding mutants I created in mediating these activities. 4.2.1 CckA kinase and phosphatase activities do not affect the maturation of RcGTA particles To test the effects of mutations affecting phosphatase and kinase activities in mediating RcGTA transduction, I first used a transduction frequency assay to measure levels of functional RcGTA particles produced by and released from cells (Figure 3.2A). The data showed an increase in transduction frequency with the H399A kinase mutant, and a decrease in transduction frequency with the V443P phosphatase mutant. Previous research has demonstrated that a change in transduction frequency can occur due to: i) a change in RcGTA structural gene transcription; ii) a lack of maturation of RcGTA precursors into transduction-competent particles; iii) a change in the frequency of cell lysis and release of extracellular RcGTA particles.  I sought to determine whether or not the changes in gene transduction activity (Figure 3.2A) were due to changes in the release of functional GTA particles in the mutants. I performed western blots to detect levels of capsid protein present in the cell-free culture supernatant, compared to the cell-associated levels. The levels of capsid protein in the cell-free culture supernatant  strongly correlated with levels of RcGTA activity (frequency of gene transduction) (Figure 3.2C), indicating that the amounts of capsid protein produced in all mutants reflects the amount of mature RcGTA produced, and that the absence of CckA kinase or phosphatase   49  activity does not affect RcGTA maturation. Instead, both of these activities appear to be needed for cell lysis that is commensurate with the amount of RcGTA intracellular accumulation. 4.2.2 The effects of the CckA H399A kinase mutation on RcGTA production and release My results show that the presumed loss of CckA kinase activity in the H399A mutant results in a huge increase in the amount of capsid protein produced within cells. This increase in the amount of the capsid protein is consistent with previous data demonstrating that non-phosphorylated CtrA is primarily responsible for the induction of RcGTA structural gene cluster transcription (Mercer et al., 2012; Westbye, 2016). The huge increase may mean that in WT cells CckA kinase activity maintains a fraction of CtrA in the phosphorylated state, unable to induce RcGTA structural gene transcription, whereas in the H399A kinase mutant there is little or no CtrA~P whatsoever. This idea is consistent with the relative lack of release of RcGTA particles to the supernatant -- that is, if the same number of H399A mutant cells as the WT lysed, because of the huge amount of capsid protein within cells it would be expected to see a proportional increase in the amount released. Instead, the western blot band in the H399A extracellular lane is only slightly more intense than the band in the WT lane (Figure 3.2B). Therefore it appears that this mutant is defective in cell lysis, a process thought to be induced primarily by CtrA~P. The increase in transduction frequency (approximately 3 times that of WT; Figure 3.2A) is consistent with the relative western blot band intensities (compare WT and H399A bands in the supernatant blots in Figure 3.2B). Perhaps enough CtrA~P is produced by crosstalk with another pathway to allow for a small amount of lysis. In fact it was shown that the C. crescentus ChpT, the immediate phosphodonor to CtrA, also phosphorylates the CpdR protein that controls proteolysis of CtrA (Blair et al., 2013). Therefore, it is possible that an analogous branched pathway may result in some production of CtrA~P in the absence of CckA kinase activity. For example, the   50  widespread CheA histidine kinase phosphorylates more than one response regulator (Porter et al., 2011). 4.2.3 The effects of the CckA(V443P) phosphatase mutation on RcGTA production and release  Although the effects of the kinase mutation can be explained readily, the results regarding the predicted phosphatase mutant are more difficult to explain. The presumed increase in CckA~P in the V443P mutant resulted in high levels of capsid protein present within or bound to the outside of cells (within the pellet fraction), and low levels of RcGTA particles present in the cell-free culture supernatant. Previous studies indicated that both non-phosphorylated CtrA and CtrA~P allow for intracellular RcGTA accumulation, whereas CtrA~P was needed for release of particles from cells ( Mercer et al., 2012). My results indicate that an absence of CckA phosphatase activity results in an increase in transcription of RcGTA structural genes (thought to be stimulated by non-phosphorylated CtrA). This could be explained if there is a second phosphatase that allows for sufficient de-phosphorylation of CckA~P to allow for CtrA-induced transcription of RcGTA structural genes, and production of RcGTA particles within cells. Alternatively, CckA kinase activity may be weak in the early phase of induction, when the structural genes are expressed, and so the phosphatase may not be needed for intracellular capsid protein to accumulate. CtrA~P is thought to contribute to cell lysis, but as shown in Figure 3.2 I saw little release of RcGTA particles from V443P phosphatase mutant cells. However the Rc1079 promoter activity of the V443P mutant cells was high, consistent with high levels of CtrA~P. Perhaps an unnatural increase in the ratio of CtrA~P/CtrA inhibits cell lysis, because there is a   51  narrow range in this ratio that is needed for lysis: either too low or too high a ratio may inhibit lysis, without affecting the maturation of the RcGTA intracellular particles. 4.2.4 The effects of the Y589D c-di-GMP-binding mutation on RcGTA production and release The low levels of RcGTA production and the absence of release in the Y589D mutant (designed to be unable to bind c-di-GMP) are also puzzling. Because a ΔcckA deletion mutant produces a large amount of capsid protein, the Y589D mutant CckA protein must modulate RcGTA structural gene expression to yield an intracellular level that resembles the WT phenotype (Figure 3.2 B). However, RcGTA gene transduction activity is about 1% of the WT value. This indicates that induction of lysis genes, thought to be stimulated by CtrA~P, requires CckA to bind c-di-GMP. However this is the inverse of what would be expected if it is assumed, as in the C. crescentus Y514D mutant CckA, the Y589D mutation inhibits phosphatase activity while retaining constitutive kinase activity. One possible explanation is that there is a second pathway involved in RcGTA release that is regulated by CckA, and that it is negatively affected by the Y589D mutation. Another possibility is that there is narrow range in the ratio of CtrA~P/CtrA needed for lysis, and either too low or too high a ratio may inhibit lysis. A complicating factor is the possibility that c-di-GMP induces the proteolysis of CtrA, as in C. crescentus (Lori et al., 2015). However, we now know that relying solely on the Y589D residue that resides in what Mann et al. (2016) call the CA domain appears to have been a bad choice to investigate c-di-GMP binding. Additional experiments, targeting additional homologues of the C. crescentus residues recently demonstrated to directly bind c-di-GMP (Dubey et al. 2016) might be a better way to investigate the effect of c-di-GMP on the R. capsulatus CckA activity.   52  4.3 The role of DivL and the CckA-ChpT-CtrA phosphorelay in mediating RcGTA recipient capability After assessing the effects of the CckA mutants in mediating CtrA phosphorylation, I set out to determine the effects of these phosphatase and kinase activities on mediating RcGTA recipient capability (the ability of cells to take up DNA transferred via RcGTA particles). 4.3.1 The effect of the CckA H399A kinase mutation on RcGTA recipient capability Investigation of the role of the CckA(H399A) kinase mutant in mediating RcGTA recipient capability led to the finding that there was an approximately 2.5- to 3-fold increase in RcGTA recipient capability in this mutant (Figure 3.3A). This indicates that RcGTA recipient capability is stimulated by CtrA~P, and that the phosphorylation level is subsequently regulated by CckA. This result is consistent with previous data, which demonstrated that when non-phosphorylatable CtrA(D51A) is present as the only copy, there is an increase (~2-fold over SB103 WT) in RcGTA recipient capability (Brimacombe et al., 2014). Cells harboring both a WT (on the chromosome) and mutant (on a plasmid) copy of CckA showed a phenotype similar to that of the mutant containing CckA(H399A) as the only copy, with levels of RcGTA recipient capability enhanced ~4-5 fold over SB1003 WT (Figure 3.3B). This could indicate that having both kinase and phosphatase activities present, while having a higher ratio of phosphatase to kinase activity, further enhances recipient capability. This result could also indicate that WT/H399A heterodimers lack kinase activity, in which case there is a drastic increase in phosphatase activity in these mutants, with kinase activity retained only by WT CckA homodimers.    53  4.3.2 The effects of the CckA(V443P) phosphatase mutation on RcGTA recipient capability The CckA(V443P) phosphatase mutant had approximately the same levels of RcGTA recipient capability as SB1003 WT when it was present as the only copy (Figure 3.3A). This result is somewhat contradictory to previous data showing an ~50% decrease in RcGTA recipient capability in the presence of only phosphomimetic CtrA(D51E) (Brimacombe et al., 2014). This therefore suggests either that non-phosphorylated CtrA enhances, but is not necessary for, RcGTA recipient capability, or that this mutant retains some phosphatase activity, enough so that CtrA can be de-phosphorylated and allow for recipient capability. There are a few explanations for why this may be the case. While the results of my β-galactosidase assay indicate that the V443P mutant has increased kinase activity, the level of phosphatase activity was not measured; it is possible that this mutant primarily acts as a kinase, but is capable of switching to act as a phosphatase under the correct conditions. Furthermore, as discussed in the introduction (section 1.7.2), the homologous CckA mutation in C. crescentus retained a phenotype consistent with the V399P mutation either being an incomplete phosphatase, or with there being a second protein that can de-phosphorylate CtrA (Chen et al., 2009). When both this phosphatase mutant (on a plasmid) and a WT (on the chromosome) copy of CckA were present, there was an approximately 3-fold increase in recipient capability (Figure 3.3B). This result is unexpected, as it suggests that a WT level of kinase activity contributed by WT CckA homodimers, with proportionately less phosphatase activity, contributed by heterodimers, and/or CckA(V443P) homodimers, enhances recipient capability. This result contradicts the results observed with both the kinase and phosphatase mutants alone, which suggest that an increase in kinase activity stimulates recipient capability.    54  4.3.3 The effects of the CckA(Y589D) c-di-GMP binding mutation on RcGTA recipient capability The CckA(Y589D) c-di-GMP binding mutant had ~1% recipient capability as compared to WT, similar to what was seen in a CckA deletion mutant (Figure 3.3A). This indicates that this mutant has neither kinase nor phosphatase activity. It is possible that in R. capsulatus, the structural integrity of the Y589D mutant homodimer is compromised, and therefore does not allow for either activity to occur. When this mutant was present along with a WT copy of CckA, WT levels of recipient capability were observed (Figure 3.3B). This indicates that functional WT CckA homodimers can form, and that these homodimers are sufficient to allow for activities needed for recipient capability, leading to approximately WT levels of activity.  These results also suggest that it is the ratio of kinase and phosphatase activities, and not the total amount of CckA protein, which is important for ensuring balanced levels of CtrA/CtrA~P for recipient capability. If it is assumed that the CckA Y589D mutant and the WT CckA can form a functional heterodimer, and that there would therefore be an increase in the amount of functional CckA protein in cells, it would indicate that simply an increase in the amount of CckA protein does not affect recipient capability. Alternatively, if it is assumed that heterodimers are non-functional, and that only a small number of functional WT CckA homodimers are present, it would indicate that a lower amount of functional CckA protein does not affect recipient capability. In summary, these data indicate that it is the relative ratio of kinase to phosphatase activity, and not the total amount of functional CckA protein, which affects RcGTA recipient capability. As noted in section 4.1.4, it is possible that the Y589D mutation may function differently than the homologous C. crescentus mutation, and on its own may not have been the best, most complete, method of studying c-di-GMP binding.   55  4.3.4 The effects of the absence of the truncated (WT) DivL protein on RcGTA recipient capability Previous results of research on both C. crescentus and R. capsulatus suggest that DivL plays a role in mediating the effects of CtrA on the cell cycle, and RcGTA production, respectively (Tsokos et al., 2011; Westbye et al., 2017). In C. crescentus, the presence of DivL is necessary for proper CckA kinase activity, and phosphorylation of CtrA. Loss of DivL leads to cell cycle defects consistent with a decrease in CckA kinase activity (Tsokos et al., 2011). Further studies found that binding of DivK~P to the DHp domain of DivL leads to an inhibition of CckA kinase activity, resulting in a loss of CtrA~P, allowing for replication of cell division. In R. capsulatus, DivL lacks the nonfunctional DHp and CA domains present in its C. crescentus homolog and therefore is thought to be truncated (Westbye, 2016). In the WT strain SB1003, a loss of DivL results in increased transduction activity, decreased CckA kinase activity and levels of CtrA~P, and it was suggested that DivL also affects RcGTA production through a post-transcriptional process involving the ClpX protein as a chaperone and/or a component of the ClpXP protease (Westbye et al., submitted). This assumption led me to hypothesize that a divL knockout strain should exhibit an increase in the level of recipient capability, as seen with the H399A kinase mutant. I did not observe this, but instead observed a decrease in recipient capability to levels similar to that of a CckA deletion, or the Y589D mutant (~1% of WT levels). Because these two mutations seemingly lead to non-functional CckA protein being present, the data indicate that in the absence of DivL, CckA fails to retain kinase and phosphatase activities. 4.3.5 The effects of the absence of ChpT on RcGTA recipient capability  Biondi et al. (2006) demonstrated in vitro that ChpT can act as a phosphorelay protein to transfer a phosphate from CckA to CtrA as well as CpdR. However, they did not investigate de-  56  phosphorylation of CtrA. Chen et al. (2009) demonstrated that ChpT can facilitate, and is necessary for, de-phosphorylation of CtrA ending in the production of inorganic phosphate by CckA. Based on these previous findings I hypothesized that a ChpT deletion would lead to a loss of CtrA~P, and an increase in the levels of CtrA, because in the absence of ChpT there would be no transfer of phosphate from CckA to CtrA. Based on what I observed with the CckA(H399A) mutant kinase protein, which led to a decrease in the level of phosphorylated CtrA, I expected to see an increase in RcGTA recipient capability. However, I saw a decrease to ~60% of WT levels. This phenotype opposite of what I expected indicates that in R. capsulatus the absence of ChpT prevents de-phosphorylation of CtrA and leads to a decrease in recipient capability.  4.4 Differences in RcGTA recipient capability are not due to differences in the ability of RcGTA particles to bind to cells Previous data indicated that recipient capability in anaerobically grown RcGTA cultures is inhibited by the absence of a polysaccharide capsule on cells  (Brimacombe et al., 2013). Because of this, I set out to investigate whether or not differences in RcGTA recipient capability in CckA kinase and phosphatase mutants were due to differences in RcGTA binding to cells. I measured this by using a gene transduction assay to quantify the residual amounts of RcGTA particles present after mixing them with cells containing WT or mutant versions of CckA. I found no significant differences in residual levels of RcGTA after incubation with WT or CckA mutant cells (Figure 3.4). These results, therefore, indicate that the differences in RcGTA recipient capability seen are due to an intracellular uptake mechanism, and not due to changes in the binding of RcGTA particles to cells. This supports the conclusion that the CckA-ChpT-CtrA phosphorelay pathway   57  plays an integral role in regulating the uptake and integration of DNA transferred via RcGTA particles. 4.5 Cell morphology in the late exponential phase is not affected by CckA mutations Investigation of cell morphology of WT and mutant cells in the late exponential phase (Figure 3.5) yielded no significant differences between the cell morphology of WT or mutant cultures. I also saw no observable differences in culture growth rate with any of the strains (data not shown). This indicates that these mutant kinase, phosphatase, and c-di-GMP-binding activities do not affect the growth or cell division of R. capsulatus under these conditions.     58  Chapter 5: Conclusion and Future Directions 5.1 Conclusion In summary, my results demonstrate that the CckA-ChpT-CtrA phosphorelay pathway is regulated in complex way, involving all three phosphorelay proteins, as well as DivL (Figure 5.1).   Figure 5.1 Model for regulatory pathways involved in mediating RcGTA production, release, and recipient capability. A Phosphorylated CtrA allows for RcGTA recipient capability (as indicated by the light arrow), while an excess of non-phosphorylated CtrA allows for an increase in RcGTA recipient capability (as indicated by the heavy arrow). Deletion of either CckA, ChpT, or DivL, leads to a disruption of the phosphorelay pathway and a disruption in RcGTA recipient capability. B The presence of excess phosphorylated CtrA allows for increased RcGTA gene transcription, while a reduction in phosphorylated CtrA leads to a large increase in RcGTA  production (as indicated by arrow thickness). Deletion of CckA has a small effect on the levels of RcGTA production. C The presence of either excess phosphorylated CtrA, excess non-phosphorylated CtrA, or deletion of CckA, leads to and decrease in cell lysis (as indicated by the light arrows).   59  In the case of RcGTA production and release, while the phosphatase and kinase states of CtrA do affect RcGTA production and release, the results are not as easy to interpret as originally hypothesized. For example, while the CckA(H399A) kinase mutant protein leads to a decrease in levels of CtrA~P, and an increase in RcGTA production within cells, it has a relatively reduced level of RcGTA release as expected. In contrast, while the CckA(V443P) protein causes an increase in the level of CtrA~P, there is also a slight increase in RcGTA production, and a decrease in RcGTA release. And while the CckA(Y589D) mutation fails to produce significant levels of CtrA~P, there is a decrease in RcGTA production and release, below the levels seen when CckA is absent. Similar complexities are seen with regards to RcGTA recipient capability. While the CckA(H399A) kinase mutant leads to increased levels of recipient capability, the CckA(V443P) phosphatase mutant does not lead to a decrease in RcGTA recipient capability. And when both WT and phosphatase mutant CckA(V443P) proteins are present, there is an increase in RcGTA recipient capability. However, similarly to the case of production, the Y589D mutant lacks detectable recipient capability. As well, deletion of the DivL protein, thought to stimulate CckA kinase activity, leads to a loss of recipient capability, and the deletion of chpT leads to a ~40% decrease in recipient capability. In summary, these results suggest that the kinase and phosphatase functions of CckA, as well as the DivL regulatory protein, and ChpT phosphotransferase proteins, all play a role in mediating RcGTA recipient capability, and release. Considering the results reported by Kuchinski et al. (2016), in which a lexA mutation appeared to result in the overexpression of CckA that correlated with a decrease in both RcGTA production and recipient capability, it seems as though the relative levels of both CtrA and CtrA~P play a key role in the regulation of   60  both processes. However, the exact functions of each of these proteins in RcGTA production and recipient capability are unclear and will require further studies to fully explain. The results presented in this thesis help to further our knowledge of how RcGTA recipient capability and production are regulated. Firstly, I have demonstrated that the R. capsulatus and C. crescentus CckA proteins are sufficiently homologous such that some equivalent mutations in both proteins affect CckA in similar ways, and yet others differ in some ways. I have also demonstrated that mutating specific residues in CckA does, indeed, appear to affect the phosphorylation state of CtrA, and that changes in phosphorylation state have drastic effects on RcGTA recipient capability and production. The conservation of the CckA protein among many α-proteobacteria known or suspected to produce RcGTA-like particles (Figure 5.1 and Appendix A Figure S1) may allow for the extension of these findings to other bacteria. The generalization of these findings also furthers the evidence that RcGTA-like particles have played a role in the evolution of many α-proteobacterial species.       61   Figure 5.2 Phylogenetic tree of selected CckA and related histidine kinase proteins, created using a maximum likelihood method. This distance-based tree shows the predicted evolutionary relationships between histidine kinase or hybrid histidine kinase proteins mentioned in my thesis, and in closely related bacterial species known to either produce functional GTA particles, or to contain homologues of RcGTA genes. ‘G’ indicates bacterial species’ containing a functional gene transfer agent. ‘g’ indicates species’ containing homologues of RcGTA genes. ‘–’ indicates bacterial species lacking a functional GTA and putative GTA genes.  5.2 Future directions The mutants created here could be tested for a variety of activities to further understand their involvement in regulating CckA kinase activity, and their involvement in CtrA phosphorylation. Using a promoter known to be activated by non-phosphorylated CtrA, such as a promoter for the main RcGTA cluster, would give a better understand of exactly how these mutants affect levels of both CtrA and CtrA~P. Using such reporters, measurements of the relative levels of CtrA and CtrA~P over the progression of a culture's growth phases would give   62  information on how these levels change over time, as the levels of RcGTA produced and released have previously been demonstrated to be regulated by culture growth phase, and CtrA  phosphorylation is differentially regulated over the cell cycle in C. crescentus (Domian et al., 1997; Domian et al., 1999). Alternatively, levels of CtrA and CtrA~P could be directly measured using a phosphor-tag method (Bekesova et al. 2015): briefly, a phosphorylated protein migrates more slowly than non-phosphorylated protein in a phos-tag SDSPAGE, and the relative amounts of the two forms of the protein are detected in a western blot. It would also be interesting to express the mutant cckA genes in a WT background, to see if the results resembled the effects of such expression on recipient capability (Figure 3.3B). Another experiment that could be used to clarify the results in this thesis would be a western blot to investigate the total levels of CtrA and CtrA~P present in my various mutant strains. Although I have hypothesized that mutations in CckA simply affect levels of CtrA and CtrA~P, it is also possible that the levels of the CtrA protein differ over culture growth phases, and are affected by the phosphorylation state of CckA. Certainly ctrA gene expression changes greatly as a culture progresses through the growth phases (Leung et al., 2013). A time course of CtrA western blots over a culture growth may provide key insights into how the kinase and phosphatase states of CckA affect the levels of CtrA over the growth phases of a culture. Because there is likely to be a difference in CckA, CtrA and other activities between the small percentage of cells producing RcGTA and the vast majority of cells capable of receiving RcGTA-borne genes, it would be most insightful to sort donor and recipient cells (using FACS, as done in unpublished work [Ding et al., in preparation]) to obtain a clearer picture.    63  References Abel, S., Bucher, T., Nicollier, M., Hug, I., Kaever, V., Abel Zur Wiesch, P., and Jenal, U. (2013) Bi-modal distribution of the second messenger c-di-GMP controls cell fate andasymmetry during the Caulobacter cell cycle. PLoS Genet 9: 1-17 Aklujkar, M., Harmer, A.L., Prince, R.C., and Beatty, J.T. (2000) The orf162b sequence of Rhodobacter capsulatus encodes a protein required for optimal levels of photosynthetic pigment-protein complexes. J Bacteriol 182: 5440–7  Anisimova, M., and Gascuel, O. (2006) Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. 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J Bacteriol 126: 619–29                         74  Appendex A: Supplementary figures    75       76             77    Figure S1. Multiple sequence alignment of CckA proteins and related histidine kinase proteins used in the creation of the tree in Figure 5.1. G indicates bacterial species containing functional gene transfer agents. g indicates species containing putative GTA genes. – indicates bacterial species lacking a functional GTA or putative GTA genes. Red box highlights site of histidine to alanine mutation. Yellow box indicates the site of valine to proline mutation. Purple box indicates site of tyrosine to aspartate mutation.      78  Table S1. One-way ANOVA results for comparison of measured values. The relevant figure, strains, and comparisons are indicated. Figure 3.1 B   Strain 1 Strain 2 p-value SB1079 SBΔCCkA 1079 0.098193 SB1079  SBΔCckA 1079 pRCckA 0.014392 SB1079  SBΔCckA 1079 HA 0.398019 SB1079  SBΔCckA 1079 VP 0.035843 SB1079  SBΔCckA 1079 YD 0.108622 SBΔCckA 1079 pRCckA SBΔCckA 1079 HA 0.004073 SBΔCckA 1079 pRCckA SBΔCCkA1079 VP 0.059531 Figure 3.2 A   Strain 1 Strain 2  SBΔCckA pRCckA SB1003 0.159864 SBΔCckA pRCckA SBΔCckA  0.000865 SBΔCckA pRCckA SBΔCckA  H399A 0.001485 SBΔCckA pRCckA SBΔCckA V443P 0.001557 SBΔCckA pRCckA SBΔCckA Y589D 0.000896 Figure 3.3   Strain 1 Strain 2  B10 B10 pRCckA 1.3E-05 B10 B10 H399A 4.12E-05 B10 B10 V443P 0.003238 B10 B10 Y589D 0.397262 B10 B10 ΔCckA 3.34E-05 B10 B10 ΔCckA pRCckA 0.003675 B10 B10 ΔCckA H399A 0.000542 B10 B10 ΔCckA V443P 0.34439 B10 B10 ΔCckA Y589D 1.44E-06 B10 B10 ΔDivL 2.17E-07 B10 B10 ΔChpT 1.74E-05 Figure 3.4   Strain 1 Strain 2  B10 B10 ΔCckA 0.1032 B10 B10 ΔCckA pRCckA 0.0316 B10 B10 ΔCckA H399A 0.1399 B10 B10 ΔCckA V443P 0.2239 B10 B10 ΔCckA Y589D 0.115 B10 No cell control <0.0001  

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