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Environmental conditions regulating gene transfer in Rhodobacter capsulatus Bernelot Moens, Rachel 2012

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  ENVIRONMENTAL CONDITIONS REGULATING GENE TRANSFER IN RHODOBACTER CAPSULATUS  by  Rachel Bernelot Moens  B.Sc., McGill University, 2010   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies  (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2012   © Rachel Bernelot Moens, 2012  ii  ABSTRACT  Rhodobacter capsulatus is a metabolically versatile α-proteobacterium that produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that is capable of mediating horizontal gene transfer. RcGTA particles transfer random 4.5 kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This thesis addresses certain environmental conditions, in particular carbon limitation and the presence of subinhibitory concentrations of antibiotics, that influence gene transfer by RcGTA. A new transduction assay was developed to test the effects of various substances on gene transfer. Using this transduction assay, both carbon limitation and low levels of DNA gyrase inhibitors were found to increase the frequency of gene transfer, although by different mechanisms. Carbon limitation caused an increase in production and release of RcGTA. This effect was a general response to carbon limitation, and was independent of carbon source. Gyrase inhibitors, on the other hand, did not influence production or release of RcGTA and instead were thought to act on the recipient cells via DNA gyrase. GyrB overexpression constructs were made in order to confer resistance to novobiocin. The presence of these constructs negated the novobiocin-mediated increase in gene transfer. The results of this thesis suggest that certain antibiotics as well as carbon limitation affect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange in natural ecosystems.  iii  TABLE OF CONTENTS  Abstract ........................................................................................................................................... ii Table of Contents ........................................................................................................................... iii List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Abbreviations ................................................................................................................................. ix Acknowledgements ......................................................................................................................... x 1.0 Introduction .......................................................................................................................... 1 1.1 Rhodobacter capsulatus ........................................................................................................ 1 1.2 Horizontal gene transfer ........................................................................................................ 1 1.3 Gene transfer agents .............................................................................................................. 2 1.4 RcGTA .................................................................................................................................. 3 1.5 RcGTA-like genes in the α-proteobacteria ........................................................................... 6 1.6 Expression and regulation of RcGTA ................................................................................... 8 1.6.1 Nutrient availability and growth conditions .................................................................. 8 1.6.2 CtrA and CckA .............................................................................................................. 9 1.6.3 Quorum sensing ........................................................................................................... 10 1.6.4 Subinhibitory concentrations of antibiotics ................................................................. 11 1.7 Type II DNA topoisomerases ............................................................................................. 12 1.7.1 DNA gyrase and topoisomerase IV ............................................................................. 12 1.7.2 DNA gyrase inhibitors ................................................................................................. 13 1.7.3 Effects of DNA gyrase inhibitors on gene expression ................................................. 14 1.8 Goals of my research .......................................................................................................... 15 2.0 Materials and Methods ....................................................................................................... 17 2.1 Bacterial strains and growth conditions .............................................................................. 17 2.2 Recombinant DNA techniques, plasmids, and primers ...................................................... 18 2.3 Transduction assay .............................................................................................................. 20 2.4 RcGTA bioassay ................................................................................................................. 20 2.5 RcGTA attachment assay .................................................................................................... 22 iv  2.6 SDS-PAGE and Western blot ............................................................................................. 22 2.7 Construction of g4 gene disruption strains ......................................................................... 23 2.8 Conjugation ......................................................................................................................... 23 2.8.1 Conjugation efficiency assay ....................................................................................... 23 2.9 UV mutagenesis for isolation of Nb R  mutants .................................................................... 24 2.10 DNA sequencing ............................................................................................................... 24 2.11 Site-directed mutagenesis ................................................................................................. 25 2.12 Construction of Rho expression constructs....................................................................... 25 2.13 Sequence alignment .......................................................................................................... 25 2.14 Statistical analysis ............................................................................................................. 25 3.0 Results ................................................................................................................................ 26 3.1 Development of a new transduction assay .......................................................................... 26 3.2 Subinhibitory concentrations of antibiotics ........................................................................ 26 3.2.1 Effects of subinhibitory concentrations of antibiotics on gene transfer ...................... 26 3.2.2 Effects of subinhibitory concentrations of novobiocin on RcGTA production and release ................................................................................................................................... 29 3.2.3 Effect of a subinhibitory concentration of novobiocin on DNA uptake and recombination ....................................................................................................................... 32 3.2.4 Effect of novobiocin on RcGTA attachment to recipient cells .................................... 35 3.2.5 Direction of gene transfer between the strains DW5 and ΔRC6 ................................. 36 3.2.6 Cell-to-cell contact is not involved in the novobiocin-induced increase in gene transfer ............................................................................................................................................... 38 3.2.7 Effect of a subinhibitory concentration of novobiocin on the kinetics of RcGTA production ............................................................................................................................. 39 3.2.8 Creation of Nb R  mutants and characterization in the transduction assay .................... 39 3.3 Effect of novobiocin on mutation rate ................................................................................ 47 3.4 Effects of DNase on RcGTA production, release, uptake and integration ......................... 48 3.5 Carbon limitation ................................................................................................................ 49 3.5.1 Effects of carbon limitation on production of RcGTA ................................................ 49 3.5.2 Effect of carbon limitation on gene transfer ................................................................ 51 3.5.3 Production of RcGTA in regulatory gene mutants subjected to carbon deprivation ... 52 v  3.5.4 Effects of culture growth phase on stimulation of RcGTA production by carbon deprivation ............................................................................................................................ 53 4.0 Discussion .......................................................................................................................... 55 4.1 A new transduction assay.................................................................................................... 55 4.2 Subinhibitory concentrations of antibiotics ........................................................................ 56 4.2.1 Substances affecting gene transfer in R. capsulatus .................................................... 57 4.2.2 Effects of novobiocin on different aspects of gene transfer ........................................ 58 4.2.3 Novobiocin resistance and DNA gyrase ...................................................................... 62 4.3 Effect of carbon limitation on RcGTA expression and gene transfer ................................. 65 4.4 Future Research .................................................................................................................. 67 4.5 Concluding remarks ............................................................................................................ 68 References ..................................................................................................................................... 69 Appendix A ................................................................................................................................... 75 Survival of UV-irradiated cells ................................................................................................. 75   vi  LIST OF TABLES  Table 2.1 R. capsulatus strains used in this study......................................................................... 18 Table 2.2 Plasmids used in this study ........................................................................................... 19 Table 2.3 Primers used in this study ............................................................................................. 20 Table 3.1 A variety of substances tested in the transduction assay .............................................. 28  vii  LIST OF FIGURES  Figure 1.1 Comparison of gene transfer by GTAs and transducing phage ..................................... 4 Figure 1.2 Representation of the RcGTA gene cluster and neighbouring genes ............................ 4 Figure 1.3 Electron micrographs of GTA particles ........................................................................ 6 Figure 1.4 A 16S phylogenetic tree of α-proteobacteria ................................................................. 7 Figure 1.5 Western blot of RcGTA capsid protein in intracellular and extracellular fractions ...... 9 Figure 1.6 Proposed model for the R. capsulatus CtrA signal transduction system ..................... 10 Figure 2.1 Schematic of transduction assay .................................................................................. 21 Figure 3.1 Representative plates from a transduction assay ......................................................... 27 Figure 3.2 Representative results from selected transduction assays ........................................... 27 Figure 3.3 Transduction assay in the presence of Nb and/or DNase I .......................................... 30 Figure 3.4 Production and release of RcGTA in the presence or absence of novobiocin ............ 32 Figure 3.5 Bioassay of the effects of novobiocin on gene transfer ............................................... 34 Figure 3.6 Conjugation assay of the effect of novobiocin on recombination ............................... 34 Figure 3.7 Effect of novobiocin on the attachment of RcGTA to strains DW5 and ∆RC6 .......... 35 Figure 3.8 Transduction assay with alternative strains ................................................................. 37 Figure 3.9 Effects of cell-cell contact on gene transfer in a bioassay........................................... 38 Figure 3.10 Time course of transduction assays in the presence or absence of 2 µg/ml Nb ........ 40 Figure 3.11 Alignment of the GyrB proteins from R. capsulatus (R.c), E. coli (E.c), and S. aureus (S.a) ............................................................................................................................................... 42 Figure 3.12 Growth of strain DE442 containing pRhoK constructs in the presence of varying amounts of Nb ............................................................................................................................... 43 Figure 3.13 Induction of gyrB and growth curve of strain B10S-T7 containing pRhoT constructs compared to B10S-T7 ................................................................................................................... 45 Figure 3.14 Transduction assays with Nb R  strains........................................................................ 47 Figure 3.15 Effect of Nb on the mutation rate .............................................................................. 48 Figure 3.16 Effect of DNase I on RcGTA production and release ............................................... 49 Figure 3.17 Western blot of RcGTA capsid protein production by strain SB1003 grown in varying amounts of malate ............................................................................................................ 50 Figure 3.18 Western blot of SB1003 grown in RCV with alternative carbon sources ................. 50 Figure 3.19 Bioassay examining the effect of carbon source depletion on gene transfer ............ 51 viii  Figure 3.20 Effect of carbon depletion on gene transfer and RcGTA production ........................ 52 Figure 3.21 Time course of RcGTA production in RCV and carbon limited culture .................. 54  ix  ABBREVIATIONS  Cb  clorobiocin Cip   ciprofloxacin GTA   gene transfer agent HSL  homoserine lactone kb  kilobase pair kDa  kilodalton KU  Klett unit LB  lysogeny broth Nb  novobiocin OD  optical density ORF  open reading frame RcGTA Rhodobacter capsulatus gene transfer agent RCV  Rhodobacter capsulatus minimal growth medium SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis YPS  yeast extract/peptone/salts medium  x  ACKNOWLEDGEMENTS   Over the course of my graduate studies at UBC, I have been helped and supported by a number of people. To these people I owe my thanks and gratitude. The past and present members of the Beatty lab have been invaluable for their willingness to share knowledge and ideas with me. In particular, I wish to thank Tom Beatty for his guidance throughout my time here. His support and enthusiasm for my project and for my development as a scientist have been of great assistance to me. Erin Gaynor and Julian Davies provided much appreciated advice and direction as members of my advisory committee. I also thank the Davies lab for tolerating my frequent raids of their abundant supplies of antibiotics. My family has been a great source of comfort and support throughout my time at UBC. Their support includes, but is not limited to feeding me, taking care of my dog, and listening politely while I talked about a certain species of bacterium that very few people are as interested in as me. My friends have been largely responsible for keeping me sane over the past two years, and they have done a remarkably good job. They have stood by me for the ups and downs of my project, and were there cheering me on as I reached the finish line. I thank them all for being there for me.  1  1.0 INTRODUCTION  1.1 Rhodobacter capsulatus Rhodobacter capsulatus is a metabolically versatile α-proteobacterium capable of growth under many conditions of energy generation and nutrient assimilation. Growth is fastest under anaerobic, photoheterotrophic conditions, meaning in the absence of oxygen and in the presence of light and various organic substrates. R. capsulatus can also be grown photoautotrophically, using light as the energy source, molecular hydrogen or sulfide as the electron donor, and CO2 as the sole carbon source. Chemoheterotrophic growth can also occur under dark, aerobic or anaerobic conditions. R. capsulatus is able to rapidly switch between photo- and chemotrophic growth when environmental conditions change (Imhoff, Blankenship et al. 2004). This metabolic flexibility allows for growth in many aquatic environments under a range of illumination and oxygen availability conditions. R. capsulatus contains a cluster of phage-related genes that encode a gene transfer agent (RcGTA), a virus-like element that seems to exist solely for the purpose of mediating horizontal gene transfer, and this thesis addresses questions relating to the environmental conditions affecting gene transfer by RcGTA.  1.2 Horizontal gene transfer Bacteria need to be able to adapt to changing environments, and horizontal gene transfer (HGT) is one mechanism that they use for this purpose. HGT encompasses conjugation, transformation, and transduction, and plays an important role in the evolution of prokaryotic genomes. In a study of 116 complete prokaryotic genomes, it was found that 14% of open reading frames were the result of recent HGT (Nakamura, Itoh et al. 2004). Transformation is a 2  process by which cells enter a time-limited, regulated physiological state known as ‘competence,’ which allows them to actively take up, integrate, and express extracellular DNA. The source of extracellular DNA may be naturally decomposing cells, excretion from living cells, or cells that have been disrupted in some manner, such as by lysins (Johnsborg, Eldholm et al. 2008) or bacteriophages (Rohwer and Thurber 2009). Cell-cell contact is necessary for conjugation to occur. Conjugative plasmids and integrated conjugative elements can be donated from one cell to another via a specialized transfer pore. Many of these conjugative elements can also effect transfer of chromosomal DNA (Wilkins, Frost et al. 2002). Transduction is the transfer of non-viral DNA from a donor to a recipient via a bacteriophage. It is a key player in HGT, with integrated prophages accounting for as much as 10-20% of some bacterial genomes (Casjens 2003). These prophages have been found to increase bacterial survival in adverse conditions such as the presence of antibiotics and osmotic, oxidative, and acid stresses, as well as to increase growth and influence biofilm formation (Wang, Kim et al. 2010). Transduction has a role in the spread of both virulence (Cheetham and Katz 1995; Karaolis, Somara et al. 1999) and antibiotic resistance (Davies 1994).  1.3 Gene transfer agents GTAs are a class of horizontal gene transfer mechanism resembling generalized transduction in which genomic DNA fragments are packaged into particles resembling small bacteriophages that are released into the environment and can be introduced into other cells (Lang and Beatty 2007; Stanton 2007). R. capsulatus GTA (RcGTA) was the first GTA to be discovered (Marrs 1974). GTA-like systems have been identified in species of Brachyspira (Humphrey, Stanton et al. 1997), Methanococcus (Bertani 1999; Eiserling, Pushkin et al. 1999), 3  and Desulfovibrio (Rapp and Wall 1987), although these are not homologous to the R. capsulatus system. Additionally, GTA-like systems that package random genomic DNA but have not been shown to transfer genes have been identified in Bartonella (formerly Rochalimaea) henselae and B. bacilliformus (Anderson, Goldsmith et al. 1994; Barbian and Minnick 2000). GTA production is similar but not identical to generalized transduction. The differences are contrasted in Figure 1.1. Unlike a phage infection, the GTA genes are contained in the genome of the GTA-producing cell. The GTA particles produced contain random fragments of the donor cell’s genome that are too small to encode the complete GTA gene cluster. Thus, GTA particles will only occasionally contain GTA-encoding genes, and as these do not encode the complete gene cluster they cannot confer the ability to produce more GTA. This is in direct contrast to phage particles, which contain the complete phage genome and only the occasional fragment of host DNA. All known GTAs resemble tailed-phages, and are presumed to exit the cell by lysis of the donor (Lang, Zhaxybayeva et al. 2012).  1.4 RcGTA Figure 1.2 shows the 14 kb RcGTA gene cluster, which consists of a collection of 15 open reading frames (ORFs) as well as two genes located ~670 kb away. Many of these genes have sequence similarity to phage genes such as a terminase, a prohead protease, a portal protein, a capsid protein, and a major tail protein (Lang and Beatty 2000; Lang and Beatty 2007). The two accessory genes are thought to be tail fiber proteins that while not necessary for RcGTA function, increase the ability of RcGTA to bind to recipient cells and transfer DNA (Andrew Lang, unpublished observations). Many of the genes found in this cluster were shown to be a part of the RcGTA particle using a proteomics approach (Chen, Spano et al. 2009). The tailed, phage- 4   Figure 1.1 Comparison of gene transfer by GTAs and transducing phage. A) GTA particles contain random fragments of host genome, with only occasional GTA genes. B) Phages produced during infection contain mainly phage genome, and only occasional fragments of host genome. Adapted from (Lang, Zhaxybayeva et al. 2012).       Figure 1.2 Representation of the RcGTA gene cluster and neighbouring genes. Protein functions predicted by homology to known phage genes are indicated above, and ORF locus tags are specified below. RcGTA genes are shown in blue, while non-RcGTA genes are shown in green. Arrows indicate the direction of transcription. Figure adapted from Ref. (Lang, Zhaxybayeva et al. 2012). 5  like particle encoded by this cluster (Figure 1.3A) contains 4.5 kb of random double-stranded genomic DNA, which would not allow it to transfer a complete copy of its own gene cluster. Additionally, two overlapping genes found to be essential for GTA release have been found elsewhere in the genome. It is thought that these encode an N-acetylmuramidase lysozyme protein and a holin protein (Hynes, Mercer et al. 2012). The mechanism of RcGTA release has long been a topic of debate. Escape of a tailed phage from cells without lysis has never been reported, and yet despite the detectable presence of RcGTA in culture supernatants, no lysis was observed. Recently, however, it was found that RcGTA is not expressed equally within a population. Instead, a small subset of cells (~3%) expresses the RcGTA genes at a higher level than the remainder of the population (~9-fold higher expression). It is this subset that lyses and releases RcGTA with the action of the endolysin and holin proteins. The small size of the subset explains the lack of detectable lysis in cell cultures (Fogg, Westbye et al. 2012; Hynes, Mercer et al. 2012). GTAs are thought to bind to recipient cells by specific tail-receptor interactions, although a GTA receptor has not yet been identified for any of the GTAs. Once the DNA enters the cell, it needs to be integrated into the host genome in order to survive degradation by cellular nucleases to be maintained in the cell’s progeny. For RcGTA, it is thought that DNA recombinase A (RecA) is responsible for incorporation of DNA from RcGTA particles into the recipient chromosome by homologous recombination (Genthner and Wall 1984). This is supported by the fact that use of RcGTA to replace a chromosomal allele with a gene disrupted by a marker always results in integration at the expected location (Lang, Zhaxybayeva et al. 2012).  6   Figure 1.3 Electron micrographs of GTA particles. A) RcGTAs resemble phage particles with a head diameter of ~30 nm and a tail length of ~50 nm. B) Dd1 particles from Desulfovibrio desulfuricans have a head diameter of ~43 nm and a tail length of ~7 nm. C) VSH-1 particles from Brachyspira hyodysenteriae have a head diameter of ~45 nm and a tail length of ~64 nm. D) VTA particles from Methanococcus voltae have a head diameter of ~40 nm and a tail length of ~61 nm. Figure adapted from Ref. (Lang, Zhaxybayeva et al. 2012).   1.5 RcGTA-like genes in the α-proteobacteria Homologues of genes found in the RcGTA gene cluster are widespread within the α- proteobacteria, and the cluster itself is widely and highly conserved within the taxonomic order Rhodobacterales, suggesting that RcGTA-like gene exchange may be a common feature in these bacteria (Lang and Beatty 2007). Figure 1.4 shows the prevalence and completeness of the RcGTA-like gene cluster in the α-proteobacteria as of 2007. The presence of RcGTA-like genes ranges from a complete cluster (ORFs 1-15) to a single RcGTA-like gene located in a RcGTA- 7    Figure 1.4 A 16S phylogenetic tree of α-proteobacteria. Species have been colour-coded to indicate the presence and distribution of RcGTA-like genes. Pink shows a complete RcGTA-like cluster, purple indicates the presence of all RcGTA-like genes but spread across two locations, light blue shows a partial and/or rearranged RcGTA-like cluster, green indicates the presence of RcGTA-like genes in multiple locations in the genome, yellow indicates the presence of a single RcGTA-like gene in a RcGTA-unlike prophage, and orange indicates the presence of several RcGTA-like genes in a lytic phage that infects this bacterium. (Lang and Beatty 2007)  8  unlike prophage. The order Rhodobacterales includes the Roseobacter group, which makes up as much as 20% of total microbial populations in some marine waters (Buchan, Gonzalez et al. 2005), and so RcGTA-like gene exchange may be prevalent in natural environments. Three members of the Roseobacter group, Ruegeria (formerly Silicibacter) pomeroyi (Biers, Wang et al. 2008), Ruegeria mobilis, and Roseovarius nubinhibens (McDaniel, Young et al. 2010) have been experimentally shown to produce GTA particles capable of transferring genes.  1.6 Expression and regulation of RcGTA 1.6.1 Nutrient availability and growth conditions  R. capsulatus is usually grown either in YPS, a complex medium containing yeast extract and peptone, or RCV, a defined minimal medium. It may be grown phototrophically (anaerobically in the presence of light), or chemotrophically (aerobically in the absence of light). These conditions cause different levels of RcGTA gene expression, with YPS photosynthetic and RCV aerobic growth giving the highest levels, and RCV photosynthetic giving the lowest levels (Leung 2010).  Under photosynthetic conditions, nutrient limitation also plays an important role in RcGTA production and release (Taylor 2004). In the RCV defined medium, cells produce RcGTA, but very little is released into the environment. In contrast, when cells are grown in YPS medium or in RCV medium with limited phosphate, a significant amount of RcGTA is released. When cells are grown in RCV with limited carbon, there is an increase in intracellular RcGTA, but no detectable increase in extracellular RcGTA (Taylor 2004) (Figure 1.5).  9    Figure 1.5 Western blot of RcGTA capsid protein in intracellular and extracellular fractions. Cultures were grown in YPS medium, RCV medium, or nutrient-limited variants of RCV. The limited nutrients were phosphate (P) and carbon (C). Adapted from (Taylor 2004).   1.6.2 CtrA and CckA Signal transduction in bacteria is often achieved via a phosphorelay system. A well characterized system is that of CtrA, ChpT, and CckA of Caulobacter crescentus. CckA acts as the sensor histidine kinase, ChpT is a histidine phosphotransferase, and CtrA is the response regulator that affects the expression of many downstream genes (Brown, Hardy et al. 2008). R. capsulatus possesses homologues of these three genes, which, unlike in C. crescentus, are not essential for cell survival. Mutants lacking any of these three genes are lacking in RcGTA production, although in the CtrA knockout this is due to decreased RcGTA gene transcription, whereas in the other two knockouts it is due to reduced release of RcGTA from cells (Lang and Beatty 2000; Mercer, Quinlan et al. 2012). The R. capsulatus homologues of CtrA, ChpT, and CckA genes have also been found to be involved in the regulation of R. capsulatus motility (Lang and Beatty 2002; Mercer, Quinlan et al. 2012). Figure 1.6 shows a proposed model of this 10  signal transduction system wherein ChpT and CckA both affect motility and RcGTA release directly, and are also thought to act on RcGTA expression indirectly via CtrA.    Figure 1.6 Proposed model for the R. capsulatus CtrA signal transduction system. (Mercer, Quinlan et al. 2012)  1.6.3 Quorum sensing It has been shown that RcGTA production begins in early stationary phase and is released into the environment shortly afterwards (Solioz, Yen et al. 1975; Florizone 2006). This growth phase-dependent regulation is thought to be due (at least in part) to a long-chain acyl-homoserine lactone (acyl-HSL) quorum sensing system (Schaefer, Taylor et al. 2002). This is a two- component system consisting of the gtaI and gtaR genes. GtaI synthesizes the acyl-HSL signaling molecule (Schaefer, Taylor et al. 2002), while GtaR is a receptor protein that modulates transcription of a set of genes in response to a threshold concentration of acyl-HSL (Leung, Brimacombe et al. 2012). In the absence of acyl-HSL, GtaR appears to negatively regulate RcGTA production via an intermediate, because although the GtaR protein regulates transcription of the gtaRI operon and was found to bind to an inverted repeat sequence in the gtaRI promoter region, the GtaR protein did not bind to the RcGTA promoter region (Leung 11  2010). Both endogenous and exogenous acyl-HSLs stimulate GTA production (Leung, Brimacombe et al. 2012).  1.6.4 Subinhibitory concentrations of antibiotics  Subinhibitory concentrations of antibiotics (concentrations that allow growth to occur at the same or similar rate to growth without antibiotic) have been shown to have profound effects on bacterial transcription profiles. In Salmonella typhimurium, as many as 5% of genes were affected (activated or repressed) by the presence of sub-lethal amounts of erythromycin and rifampicin (Goh, Yim et al. 2002). Low levels of antibiotics have been found to induce the SOS response and stimulate HGT in several organisms. Ciprofloxacin and trimethoprim were shown to induce conjugation-mediated transfer of antibiotic resistance genes via the SOS response in Vibrio cholerae (Beaber, Hochhut et al. 2004), and both floroquinolones and beta-lactam antibiotics were shown to induce the SOS response in Staphylococcus aureus (Mesak, Miao et al. 2008), leading to phage induction and transduction of pathogenicity islands (Ubeda, Maiques et al. 2005; Maiques, Ubeda et al. 2006). In Shiga toxin-producing Escherichia coli, floroquinolones such as ciprofloxacin (Zhang, McDaniel et al. 2000) and norfloxacin (Herold, Siebert et al. 2005) were shown to induce bacteriophage production and horizontal transfer of virulence factors in an SOS-dependent manner. In a uropathogenic strain of E. coli, certain beta- lactam antibiotics stimulated host cell production of phage in an SOS-independent manner as well (Comeau, Tetart et al. 2007).  A recent metagenomic study of swine intestinal microbiomes showed that in-feed antibiotics (carbadox and ASP250, a mixture of chlortetracycline, sulfamethazine, and penicillin) induce phages in the pig gut and significantly affect both phage and bacterial community 12  structure (Allen, Looft et al. 2011). Interestingly, Brachyspira hyodysenteriae, the agent of swine dysentery, contains a GTA called VSH-1. Both carbadox and metronidazole have been shown to induce VSH-1 (Stanton, Humphrey et al. 2008).  1.7 Type II DNA topoisomerases 1.7.1 DNA gyrase and topoisomerase IV DNA topoisomerases are enzymes that catalyze the interconversion of relaxed and supercoiled forms of DNA, as well as introduce and remove catanenes and knots. These changes in DNA topology are essential for cell survival, and thus these enzymes are found in all cell types. There are two main categories of topoisomerases, depending on whether the reactions they catalyze involve transient breakage of one (type I) or two (type II) strands of DNA (Champoux 2001). DNA gyrase, a type II enzyme, is able to catalyze both the introduction of negative supercoils to and the relaxation of DNA, while all other topoisomerases can only catalyze the relaxation. DNA topoisomerase IV (topoIV), another type II topoisomerase thought to have arisen due to gene duplication, specifically catalyzes the decatenation of daughter chromosomes following replication. DNA gyrase and topoIV share significant sequence similarity. In prokaryotes, DNA gyrase consists of two subunits, GyrA and GyrB, which associate to form an A2B2 complex in the active enzyme, while topoIV consists of ParC and ParE in an equivalent C2E2 complex. The A and C subunits are involved in interactions with DNA while the B and E subunits contain the ATPase active site. GyrB is highly conserved across all bacteria, with approximately 60% sequence similarity across eight representatives of the major classes of prokaryotes (Huang 1996). 13  All known proteobacterial GyrBs have a similar, approximately 800 amino acid sequence, while most other GyrBs have a shorter, 650 amino acid sequence. The extra 150 amino acids occur as a conserved block in domain III of the protein. This 150-residue insertion is universally absent in ParE homologues. There are no large length differences across the GyrA and ParC proteins, although ParC is generally shorter than the homologous GyrA. R. capsulatus contains both DNA gyrase and topoIV. There is 34.5% sequence identity between R. capsulatus GyrB and ParE, and 30.9% sequence identity between GyrA and ParC.  1.7.2 DNA gyrase inhibitors Drugs that target DNA gyrase usually act via one of two mechanisms. Either the enzymatic activity of gyrase is inhibited, or the covalent enzyme-DNA complex is stabilized. Aminocoumarins such as novobiocin (Nb) and clorobiocin (Cb) inhibit the ATPase activity of gyrase (subunit B), while ciprofloxacin (Cip), a fluoroquinolone, interrupts religation of cleaved DNA and stabilizes the cleavage complex (subunit A). Generally, the most effective gyrase inhibitors are the cleavage complex stabilizers, as a relatively low amount of an inhibitor bound to its target can lead to sufficient stabilized DNA breaks to initiate processes leading to cell death (Collin, Karkare et al. 2011). Given the similarity between GyrB and ParE, it is not surprising that aminocoumarins have also been found to act on ParE (Hardy and Cozzarelli 2003). However, several studies have shown that this inhibition is secondary to GyrB inhibition, with antibiotic concentrations that inhibit 50% of enzyme activity being one or two orders of magnitude higher for topoIV compared to DNA gyrase (Flatman, Eustaquio et al. 2006; Alt, Mitchenall et al. 2011). This is supported by sequential selection of aminocoumarin-resistant mutants in E. coli, where first-step 14  mutants all contained a point mutation in GyrB. Second-step mutants mostly contained a second point mutation in GyrB, although a few contained point mutations to homologous resistance- conferring residues in ParE (Fujimoto-Nakamura, Ito et al. 2005). Fluoroquinolones have similar activities, in that they act primarily on DNA gyrase (GyrA), but have secondary activity against topoIV (ParC) (Belland, Morrison et al. 1994; Khodursky, Zechiedrich et al. 1995).  1.7.3 Effects of DNA gyrase inhibitors on gene expression DNA supercoiling has been shown to have an effect on the transcription of many E. coli genes in vitro (Wood and Lebowitz 1984; Borowiec and Gralla 1985; Brahms, Dargouge et al. 1985; Wang and Syvanen 1992). It is not surprising, therefore, that DNA gyrase inhibitors modulate transcription through their effects on DNA supercoiling. Aminocoumarins have been shown to affect the transcription of many genes belonging to various functional categories in both Bacillus subtilis (Sioud, Boudabous et al. 2009) and Salmonella typhimurium (Jovanovich and Lebowitz 1987). In B. subtilis, the regulation pattern was indicative of an SOS response, although recA was not induced. In B. subtilis as well as in other organisms, the superhelical density of the DNA of the gyrase promoters affects the transcription of the gyrase genes, with a high level of supercoiling inhibiting transcription and a low level stimulating transcription. In E. coli, both gyrase subunits are under tight homeostatic control (Menzel and Gellert 1983). In Streptomyces sphaeroides, a strain that synthesizes Nb, an alternative, Nb-resistant gyrB is transcribed upon relaxation of its promoter (Thiara and Cundliffe 1989; Dangel, Harle et al. 2009). The differential expression of R. capsulatus photosynthesis genes in response to oxygen is thought to be mediated by the supercoiling of DNA, due to the kinetics of specific mRNA 15  changes in response to DNA gyrase inhibitors (Zhu and Hearst 1988). In E. coli and S. typhimurium, a change in extracellular osmolarity was linked to a change in DNA supercoiling, which has downstream effects on transcription of a specific osmotic stress adaptation locus (Higgins, Dorman et al. 1988). These examples indicate ways in which environmental conditions can modulate gene expression via DNA topology.  1.8 Goals of my research Many genes involved in regulation of RcGTA are known, however the environmental signals stimulating RcGTA production and transfer have not been fully explored. The focus of my thesis was to investigate environmental conditions affecting gene transfer by RcGTA. In particular, I wished to examine the effects of carbon limitation and sub-inhibitory concentrations of antibiotics. I had several specific aims for the investigation of subinhibitory concentrations of antibiotics. I wanted to develop a new transduction assay that could be used to determine the effects of specific substances on gene transfer. I used this assay to evaluate a variety of substances, and identified three DNA gyrase inhibitors as agents that increase gene transfer. My goal was to determine the mechanism by which these antibiotics affected gene transfer. In my research on carbon limitation, I aimed to determine whether the effect of increased RcGTA production in response to decreased carbon was a general response to carbon limitation or specific to one carbon source. Additionally, I investigated whether the effect seen on Western blots corresponded to an increase in gene transfer. I also examined the relationship between carbon availability, growth phase, and RcGTA production. 16  Both carbon limitation and subinhibitory levels of DNA gyrase inhibitors were found to increase the frequency of RcGTA-mediated gene transfer between R. capsulatus strains. It was also shown that carbon-limitation increases RcGTA production and release. However, unexpectedly, gyrase inhibitors did not seem to affect production or release of RcGTA. The effect of Nb, an inhibitor of the B subunit of DNA gyrase, on gene transfer was diminished by overexpression of GyrB in either strain in the transduction assay, and completely reversed by simultaneous GyrB overexpression in both strains. These results are interesting not only in that they show yet another way in which antibiotics can affect HGT, but they also show that environmental conditions affect more than one aspect of RcGTA-mediated gene transfer.  17  2.0 MATERIALS AND METHODS  2.1 Bacterial strains and growth conditions The E. coli strain used for cloning was DH5α, and the strains S17-1 and C600(pDPT51) were used to conjugate plasmids into R. capsulatus. The E. coli strains were grown in lysogeny broth (LB) medium (Sambrook, Fritsch et al. 1989) supplemented with antibiotics as needed at the following working concentrations (in µg/mL): ampicillin, 150; kanamycin, 50; spectinomycin, 100; gentamicin,10. The R. capsulatus strains used are described in Table 2.1. R. capsulatus cultures were grown chemotrophically or phototrophically in RCV minimal medium (Wall, Weaver et al. 1975) or YPS complex medium (Beatty and Gest 1981) and supplemented with appropriate antibiotics at the following working concentrations: kanamycin, 10; spectinomycin, 10; gentamicin, 5; rifampicin, 80. DNase I in culture was used at a concentration of 90 units/mL. RCV medium usually contains 29.8 mM malic acid as the carbon source, however variations of this medium using different carbon sources at equivalent carbon atom concentrations were also used (lactate at 39.8 mM and glutamate at 23.9 mM). Culture density was monitored by measuring absorbance at 660 nm or by measuring light scattering with a Klett-Summerson photometer (filter #66; red). An absorbance of 1.0 is approximately equal to 130 Klett units (KU), or 4.8x10 8  cfu/mL.  18  2.2 Recombinant DNA techniques, plasmids, and primers Standard methods of DNA isolation, analysis, modification and cloning were used (Sambrook, Fritsch et al. 1989). Plasmids used are described in Table 2.2. Primers used are described in Table 2.3.   Table 2.1 R. capsulatus strains used in this study Strain Source Description B10 (Marrs 1974) wild type SB1003 (Yen and Marrs 1976) B10 derivative, wild type with Rif R  Y262 (Yen and Marrs 1976) GTA overproducing strain ΔRC6 (Chen, Beatty et al. 1988) B10 background, puf operon knockout, Kan R  DW5 (Wong, Collins et al. 1996) SB1003 background, translationally in-frame puhA deletion SBΔRC6 this study transduction of ΔRC6 puf operon knockout into SB1003, Kan R ΔRC6_Δg4 this study ΔRC6 with g4 (GTA protease gene) disruption, KanR, SpcR DW5_Δg4 this study DW5 with g4 (GTA protease gene) disruption, KanR ΔLHII (LeBlanc and Beatty 1993) SB1003 background, puc operon knockout, Spc R  SM05 (Masuda and Bauer 2004) SB1003 background, spoT and hvrA knockout, Kan R , Tc R JS9 (Mosley, Suzuki et al. 1994) spontaneous point mutation of SB1003 regB gene MS01 (Sganga and Bauer 1992) SB1003 background, regA knockout, Kan R IKOI (Schaefer, Taylor et al. 2002) SB1003 background, gtaI knockout, Spc R B10S-T7 (Katzke, Arvani et al. 2010) B10S-derived strain where a cassette containing T7 polymerase under the control of a fructose-inducible promoter is inserted into recA, Gm R , Spc R   19  Table 2.2 Plasmids used in this study Plasmid Source Description p9H54 (Lang 2000) 3.7 kb EcoRI fragment containing the orfg2-orfg5 region of the GTA gene cluster in pUC13, Amp R  pDPT51 (Taylor, Cohen et al. 1983) mobilizing vector, Amp R , Tmp R  p54KIXF3 (Lang 2000) KIXX cartridge inserted in the EcoRV site of p9H54, in the g4 (protease) gene, Amp R , Kan R  pHP45Ω (Fellay, Frey et al. 1987) vector containing Ω fragment, AmpR, SpcR p9H54Ω this study Ω fragment inserted in the EcoRV site of p9H54, in the g4 (protease) gene, Amp R , Spc R  pZJD29A (Masuda and Bauer 2004) suicide vector, sacB, Gm R  pZJD29A KOB lab strain collection derivative of pZDJ29A containing phoB knock-out sequences, sacB, Gm R  pUC19 (Norrander, Kempe et al. 1983) Amp R pUC19gyrB this study pUC19 with gyrB inserted between NdeI and BamHI, Amp R  pUC19R147S this study pUC19 with site-directed mutagenesis at Arg147 to create a serine (CGC→AGC), AmpR pRhoTHi-6 (Katzke, Arvani et al. 2010) Inducible expression plasmid with T7 promoter, Kan R , Spc R pRhoKHi-6 (Katzke, Arvani et al. 2010) Constitutive expression plasmid with T7 promoter, Kan R , Spc R pRhoTgyrB this study pRhoTHi-6 with wild type gyrB inserted between NdeI and BamHI, Kan R , Spc R pRhoTR147S this study pRhoTHi-6 with R147S gyrB inserted between NdeI and BamHI, Kan R , Spc R  pRhoKgyrB this study pRhoKHi-6 with wild type gyrB inserted between NdeI and BamHI, Kan R , Spc R  pRhoTR147S this study pRhoKHi-6 with R147S gyrB inserted between NdeI and BamHI, Kan R , Spc R     20  Table 2.3 Primers used in this study Primer  Description Sequence (5ʹ-3ʹ) GyrB_F Used for sequencing gyrB in R. capsulatus. The numbers indicate the approximate position of the primer within the gyrB coding sequence. TCCGCAACGAAAAGCAGGAT 820_R ACCACATCGCCACTTCGAC 820_F GTCGAAGTGGCGATGTGGT 1640_R GCGGCTGCGCGATATAAAG 1640_F CTTTATATCGCGCAGCCGC GyrB_R TGATAATCCCCTTGCGTCGC GyrB_F_NdeI Used to amplify the R. capsulatus gyrB gene with addition of NdeI and BamHI cut sites at the 5ʹ and 3ʹ ends, respectively. GCCTATCATATGACCGAAACC CCGAAGAA GyrB_R_BamHI ATATAGGGATCCTCAGAAATC GAGGTTTTCCACG GyrB Mut_F Used for site-directed mutagenesis of gyrB, changing arginine 147 to serine. GGCTGGAGCTGAAGGTCTGG AGCAATGACAAGGTCTATTTC GyrB Mut_R GAAATAGACCTTGTCATTGCT CCAGACCTTCAGCTCCAGCC SmaI Omega Used to amplify the omega fragment of plasmid pHP45Ω. GGGGATCCGGTGATTGATTGA GCAA T7 F Used for amplifying inserts in the pRho plasmids TAATACGACTCACTATAGGG -76 M13 Used for amplifying inserts in pUC19 TCCGGCCTCGTATGTTGTGTGGA AT  2.3 Transduction assay Strains were grown in 25 mL YPS medium overnight, then spun down and resuspended in 10 mL YPS. Strains were inoculated in YPS at a density of 20 KU either alone as controls or mixed in a 1:1 ratio. These were incubated at 30°C for 24 hours. 10 9  cells were plated out onto YPS agar plates in 3 mL of molten YPS soft agar (0.4%) and incubated under photosynthetic conditions in anaerobic Gas-Pak jars for 2-3 days (figure 2.1A).  2.4 RcGTA bioassay Bioassays were performed as described (Donohue, Kaplan et al. 1991) with slight modifications. Donor cultures were grown aerobically instead of photosynthetically. After incubation, the transduction mixtures were plated onto YPS agar plates in 3 mL of molten YPS 21  soft agar (0.4%), and recipients were selected either by antibiotic resistance or restoration of photosynthetic growth (figure 2.1B).    Figure 2.1 Schematic of transduction assay (A) and bioassay (B). Antibiotics may be added to the steps in bold to determine their effect on the various stages of gene transfer. The * indicates a phenotypically different allele of a gene.  22  2.5 RcGTA attachment assay   Bioassays were performed as above, with modifications. Supernatant from a single RcGTA donor was incubated with recipient cells grown under different conditions. After a one- hour incubation, these cells, along with any RcGTA attached to their outer membranes, were filtered out using a 0.2 μm filter. The amount of RcGTA remaining in these filtrates was assayed by bioassay using a single recipient strain for all samples. A control sample wherein donor supernatant was not incubated with recipient cells in the initial incubation was used to determine the amount of RcGTA adsorbed by initial recipient strains.  2.6 SDS-PAGE and Western blot R. capsulatus cultures were grown for 24 hours from a starting density of 20 KU and then harvested. Cellular and extracellular samples were separated by centrifugation. Cell-free culture supernatant was concentrated by SpeedVac. Samples were lysed and denatured by boiling for 10 minutes. Aliquots of sample from equivalent amounts of cells, determined by culture absorbance at 660 nm, were separated on 12% SDS-PAGE gels and blotted onto nitrocellulose membranes. Blotting was performed with a Mini Trans-Blot apparatus (BioRad) in Electroblot Buffer [27.5 nM Tris-Base, 192 mM glycine, 20% methanol] at 100V for 1.5 hours. The primary antibody was a rabbit antibody raised against the R. capsulatus RcGTA capsid protein (Taylor 2004). The primary antibody was bound by donkey anti-rabbit Ig secondary antibody linked to peroxidase (Amersham). This was detected by the electrochemiluminescence (ECL) kit according to manufacturer’s instructions (Amersham).  23  2.7 Construction of g4 gene disruption strains The spectinomycin resistance-containing omega fragment (Ω) was excised from pHP45 by SmaI digestion and inserted into the EcoRV site of the g4 gene on p9H54. This resulting construct (p9H54Ω) was then recombined with pDPT51 in C600 and conjugated into Y262. This was used as a source of GTA-containing supernatant that was used to transduce the recipient strain, ΔRC6. Recipients with a disrupted g4 gene were selected for by spectinomycin resistance. The DW5 g4 disruption was created in a similar manner, except the g4 gene in p9H54 was disrupted with a KIXX cartridge, and the final mutants were selected for by kanamycin resistance. Inability to produce GTA in both mutants was confirmed by lack of mature capsid protein on a Western blot.  2.8 Conjugation E. coli donor cells were grown up overnight in LB with the necessary selection, while R. capsulatus recipient cells were grown up overnight in RCV medium. 200 µL of donor cells were washed in RCV and then mixed with 500 µL of recipients. The conjugation mixture was spotted onto a 0.2 µm filter on an RCV agar plate and incubated at 30ºC overnight. This mixture was then resuspended in RCV and plated on RCV with selection.  2.8.1 Conjugation efficiency assay In order to determine recombination efficiency under different conditions, pZJD29A KOB was used as a donor plasmid and SB1003 was used as a recipient strain. Equal amounts of the conjugation mixture were spotted onto 0.2 µm filters on an RCV agar plate. These were incubated at 30°C for two hours to allow conjugation to occur, at which point the cell mixtures 24  were resuspended in 5 mL of RCV with or without Nb at 0.2 µ/mL and incubated for 24 hrs at 30°C to allow recombination to occur. The incubation in liquid removed the effect of Nb on the E. coli donor, as the volume of the culture was large enough that cell-cell contact was essentially eliminated. Cells were then plated on selective plates (to obtain the total number of conjugants) or on plates without selection (to obtain the total number of viable cells) in 3 mL of molten RCV soft agar (0.4%). Conjugation efficiency was then calculated from those values.  2.9 UV mutagenesis for isolation of Nb R  mutants Strains to be mutagenized were grown aerobically to stationary phase in 25 mL YPS medium. Cultures were spun down and resuspended in 10 mL of cold 0.1 M MgSO4, spun down again, and resuspended in a fresh 15 mL of cold 0.1 M MgSO4. Cells were then chilled on ice for 10-15 minutes, after which the culture was split into three plastic Petri dishes that were UV- irradiated for 0, 5 or 10 seconds at the ‘sterilize’ setting of a Bio Rad GS Gene Linker™. 2 mL of irradiated culture was then allowed to recover for 4 hours in foil-wrapped culture tubes with 2 mL of YPS medium. At this point, cells were either plated out onto YPS agar to determine the survival rates of the irradiated cultures (see Appendix A), or were inoculated into liquid YPS and allowed to grow to stationary phase. Once in stationary phase, cultures were plated out onto YPS with Nb at 40 µg/mL to select for Nb-resistant (Nb R ) mutants.  2.10 DNA sequencing Sequencing was performed by Genewiz.  25  2.11 Site-directed mutagenesis  The construction of the mutated GyrB expression plasmids used site-directed mutagenesis to replace Arg147 with a serine residue. The gyrB gene was amplified by PCR with primers GyrB_F_NdeI and GyrB_R_BamHI using SB1003 chromosomal DNA as the template. The resulting PCR product was gel purified and digested with NdeI and BamHI, and inserted into the pUC19 NdeI and BamHI cut sites to create pUC19gyrB. This construct was used as a template for site-directed mutagenesis with the primer set GyrB Mut_F and GyrB Mut_R, resulting in the plasmid pUC19R147S.  2.12 Construction of Rho expression constructs  Wild type gyrB amplified by PCR and mutated gyrB excised from pUC19gyrB-R147S were each ligated into the expression vectors pRhoTHi-6 (inducible) and pRhoKHi-6 (constitutive) between the BamHI and NdeI restriction sites, giving four new expression constructs: pRhoTgyrB, pRhoTR147S, pRhoKgyrB, and pRhoKR147S.  2.13 Sequence alignment Global sequence alignments were performed using Needleman-Wunsch global alignment.  2.14 Statistical analysis Statistical significance was determined using the unpaired, two-tailed student’s t-test.  26  3.0 RESULTS  3.1 Development of a new transduction assay A new assay to examine levels of gene transfer between two strains was developed. Two non-photosynthetic strains, DW5 and ΔRC6, each containing mutations in different photosynthesis genes, were allowed to grow together in a mixed culture for 24 hours. Gene transfer could occur in either direction, and restoration of the ability to grow photosynthetically was used as an indicator of gene transfer. After plating the mixture on agar medium and selecting for photosynthetic growth, the number of colonies yielded a quantitative measurement of the frequency of gene transfer events. The effect of any substance on gene transfer can be determined by adding it to the mixture of cultures and incubating, then comparing the number of photosynthetic colonies obtained with that of a control with no substance added.  3.2 Subinhibitory concentrations of antibiotics 3.2.1 Effects of subinhibitory concentrations of antibiotics on gene transfer Several antibiotics were screened at subinhibitory concentrations using the transduction assay for their effects on gene transfer. The most striking results were found when using low concentrations of DNA gyrase inhibitors. Inhibitors of gyrase subunit A (Cip) and gyrase subunit B (Nb and Cb) were found to cause a significant increase in the frequency of gene transfer compared to cultures without antibiotics (Figures 3.1, 3.2). Interestingly, all concentrations of coumermycin (another gyrase inhibitor) that were tested completely blocked gene transfer, although this effect was not followed up on. These results, as well as those of other compounds that were not followed up on are shown in Table 3.1. 27   Figure 3.1 Representative plates from a transduction assay. Left, mixed culture of strain DW5 and ∆RC6 grown in the absence of antibiotic. Right, mixed culture grown in the presence of Nb at 2 μg/mL, showing an approximately 30-fold increase in number of colonies and therefore gene transfer.   Figure 3.2 Representative results from selected transduction assays with DNA gyrase inhibitors. Cultures (mixture of DW5 and ∆RC6) were grown in triplicate with or without antibiotics. Concentrations in μg/mL, * p < 0.01 compared to cultures grown without antibiotic.  28  Table 3.1 A variety of substances tested in the transduction assay, using strains DW5 and ∆RC6. These substances gave inconsistent or negative results, or were not followed up on for other reasons. Antibiotic Trial # Concentration (µg/mL) # of colonies Fold change Streptomycin 1 0 69 (52, 76, 81)   .5 166 (178, 161, 159) 2.4  2 0 440 (336, 394, 589)   .5 135 (133, 122, 151) 0.31  3 0 140 (112, 243, 64)   .5 17 (13, 11, 26) 0.12  4 0 87 (68, 86, 107)   .5 0.3 (0, 0, 1) 0 Rifampicin 1 0 69 (52, 76, 81)   1 319 4.6   2 427 6.2  2 0 440 (336, 394, 589)   2 122 (140, 138, 87) 0.28  3 0 156 (90, 195, 182)   2 25 (19, 34, 21) .16   4 30 (34, 27, 28)  .19 Erythromycin 1 0 440 (336, 394, 589)   .5 64 (61, 72, 59) .15 Bacitracin 1 0 11 (9, 13, 10)   .5 7 .64   2 14 1.3   5 11 1   10 8 .72 Trimethoprim 1 0 11 (9, 13, 10)   .5 12 1.1   2 9 .82   5 6 .55   10 5 .45 Coumermycin 1 0 11 (9, 13, 10)   .5 0 0   2 0 0   5 0 0   10 0 0 HSLs 1 0 87 (68, 86, 107)   -C12  2 μM 120 (90, 150) 1.4   -C16C  2 μM 61 (54, 68) .70   -C16  2 μM 44 (30, 57) .51   -C18  2 μM 118 (124, 111) 1.4 Indole acetic acid  1 0 46 (66, 64, 9) (IAA)  2 75 1.6   5 105 2.3   10 255 5.5  2 0 190 (233, 197, 141)   10 410 (436, 411, 382) 2.2  29  The effect of Nb on gene transfer was due to transduction by RcGTA, because disruption of the g4 gene of the RcGTA gene cluster (encoding a protease needed for capsid maturation) in both DW5 and ΔRC6 resulted in the absence of recombinants. Additionally, a transduction assay using DW5 and ΔRC6 was performed in the presence of DNase I to examine whether any gene transfer was due to the presence of free DNA. DNase I was found to increase the frequency of gene transfer, indicating that free DNA does not contribute to gene transfer in R. capsulatus (Figure 3.3A). A Western blot was also used to evaluate the amount of intracellular and extracellular capsid protein as a measure of the amount of RcGTA. The intensity of the intracellular bands indicates that DNase I may decrease the amount of RcGTA in the cell despite the observed increase in gene transfer (Figure 3.3B). No extracellular bands were detected on this blot.  3.2.2 Effects of subinhibitory concentrations of novobiocin on RcGTA production and release Four possible ways in which Nb could affect gene transfer were identified: production, release, and uptake of RcGTA, and genomic integration of DNA from RcGTA. Production and release were analyzed first. Both of these can be analyzed by Western blot, and release can also be evaluated by bioassay. Cultures used in a transduction assay of the effect of Nb on gene transfer were analyzed by Western blot to determine how this antibiotic affected the production of the RcGTA capsid protein. Intracellular and extracellular samples were analyzed to assess if the presence of Nb caused an increase in RcGTA production or release. It was found that there was no difference between the band intensity in the presence or absence of Nb both intracellularly and 30  extracellularly, indicating that these subinhibitory levels of Nb did not affect RcGTA production or release (Figure 3.4B). Although extracellular RcGTA capsid protein was not always detectable on Western blots, possibly due to limited detection by the primary antibody, when detected, levels were unchanged in the presence or absence of Nb. This finding was supported by a bioassay using supernatants from the same transduction assay as GTA donors. Again, no increase in gene transfer occurred (Figure 3.4A). This suggested that Nb may affect the ability of the recipient strain to take up or integrate the foreign DNA into the genome.    Figure 3.3 Transduction assay in the presence of Nb and/or DNase I. Cultures (mixture of DW5 and ∆RC6) were grown in triplicate in Nb (2 μg/mL), DNase I (90 Kunitz units/mL), or a combination of Nb and DNase I. A) Results of transduction assay. * p < 0.005 compared to cultures grown without antibiotic. B) Western blot of each of the triplicate transduction assay cultures, probed with RcGTA capsid antiserum.  31   32    Figure 3.4 Production and release of RcGTA in the presence or absence of novobiocin. A) Comparison of gene transfer in a transduction assay and bioassay. Transduction assay cultures (mixture of strains DW5 and ∆RC6) were grown in duplicate and used for a transduction assay as well as for supernatant donors in a bioassay. * p < 0.05 compared to cultures grown without antibiotic. B) Intracellular and extracellular fractions of each of the duplicate transduction assay cultures were also examined by Western blot, probed with RcGTA capsid antiserum.  3.2.3 Effect of a subinhibitory concentration of novobiocin on DNA uptake and recombination Because production and release of RcGTA did not seem to be affected by Nb, uptake and integration were evaluated next. The effect of Nb at a concentration of 2 µg/mL on the uptake and genomic integration of DNA was tested by bioassay. In this bioassay, RcGTA-containing cell-free supernatant from a donor strain was incubated with the recipient strain in the presence 33  or absence of Nb. The presence of this antibiotic did not seem to affect gene transfer because there was an average of 2796 (n = 2) colonies in the absence of Nb compared to an average of 2836 (n = 3) colonies in the presence of this subinhibitory concentration of Nb. These results indicate that RcGTA-transduced DNA uptake and genomic integration in the recipient strain were not affected by this concentration of Nb. The possibility that Nb ‘primes’ recipient cells for uptake of GTA or integration of DNA was also examined by a bioassay, wherein recipient cells were grown in the presence of Nb before being incubated with donor supernatant. Additionally, a bioassay was performed where both the donor and the recipient were grown in Nb. For both conditions, little or no change in frequency of gene transfer was observed in comparison with the untreated control (Figure 3.5). To further explore the general effect of Nb on integration of DNA while removing any possible role of RcGTA, I investigated the frequency of transfer of a suicide plasmid that conatins an R. capsulatus gene by conjugation from an E. coli donor strain to an R. capsulatus recipient strain. This experiment compared the frequency of recombination of a plasmid-borne antibiotic resistance gene into the R. capsulatus genome in the presence or absence of a subinhibitory concentration of Nb. Donor and recipient cells were incubated together on an agar medium in the absence of any antibiotic in order for conjugation to occur, and then diluted into a larger volume of liquid medium where recombination of donor DNA could occur without any further DNA being received from donor cells. Recombination in the liquid media took place in the presence or absence of Nb, and it was found that there was no increase in the rate of recombination in its presence (Figure 3.6).  34   Figure 3.5 Bioassay of the effects of novobiocin on gene transfer. Donors (strain ∆RC6) were either untreated (D 0) or grown in Nb at 2 μg/mL (D 2), as were the strain DW5 recipients (R 0 and R 2, respectively). n = 2 for each mixture.    Figure 3.6 Conjugation assay of the effect of novobiocin on recombination. An auxotrophic E. coli donor strain containing a Gm resistance-encoding suicide plasmid and R. capsulatus recipient strain SB1003 cells were allowed to conjugate for two hours on a solid surface and then were transferred to liquid culture and incubated in RCV medium (Nb 0) or in RCV plus Nb at 0.2 μg/mL (Nb 0.2). n = 3 for both conditions. The recombination frequency was determined by dividing the number of recombinants (measured by Gm resistance) by the number of viable cells. 35     Figure 3.7 Effect of novobiocin on the attachment of RcGTA to strains DW5 and ∆RC6. Filtered supernatant from GTA over-producer DE442 was incubated for 1 hour in the absence of cells, or with DW5 or ∆RC6 grown in the presence of Nb 0, 0.5 or 2 μg/mL. These mixtures were then filtered and used as donor supernatants in a bioassay using B10 as a recipient and selecting for rifampicin resistance as a marker. Adsorbed GTA was calculated by subtracting the number of colonies for each condition from the number of colonies obtained with the no-cell control.  3.2.4 Effect of novobiocin on RcGTA attachment to recipient cells A modified bioassay was used to detect the effect of Nb on the ability of strains to bind RcGTA. Both DW5 and ∆RC6 were grown in varying concentrations of Nb and tested for their ability to bind RcGTA. A very slight (on the order of 2-3%) increase in adsorption was observed in the presence of Nb (Figure 3.7). However, because the experiment had only a single sample for each condition, this change is not statistically significant. Regardless, the possible change in RcGTA adsorption due to the presence of Nb is far from the magnitude of the increase in gene transfer under the same conditions. Therefore, the effect of these subinhibitory concentrations of Nb on genetic exchange does not appear to result from changes in the binding of RcGTA particles to recipient cells.  36  3.2.5 Direction of gene transfer between the strains DW5 and ΔRC6 Because Nb did not seem to affect RcGTA production, release, uptake, or integration, other aspects of gene transfer in the transduction assay were examined. The direction of gene transfer was tested by patching cells in colonies resulting from transduction assays onto media containing rifampicin. Strain DW5 contains a rifampicin resistance gene whereas ∆RC6 contains a kanamycin resistance gene. Therefore, only those colonies that grew as a result of transfer of a photosynthesis gene from strain ΔRC6 to DW5 would be able to subsequently grow in the presence of rifampicin. It was unlikely that a rifampicin resistant colony could have arisen due to RcGTA-mediated transfer of both the required photosynthesis gene and the rifampicin resistance gene from DW5 because the probability of a double transfer event is extremely low. Gene transfer was found to occur only from ΔRC6 to DW5, because out of 30 photosynthetically competent transductants, 30 were found to be resistant to rifampicin. These results seemed to indicate that subinhibitory levels of Nb may mediate a strain-dependent genetic exchange from ΔRC6 to DW5. To address the question of strain-dependence, the ΔRC6 mutation was RcGTA-transduced into the SB1003 parental strain of DW5, such that both strains were isogenic except for the photosynthesis mutations, and the transduction assay was performed with these strains. Although the effect was not as strong as seen previously, subinhibitory levels of Nb were found to increase gene transfer between strains up to 5-fold (data not shown), indicating that the effect of Nb is not entirely due to differences in the background strains. The difference between this 5-fold increase in transfer and the 30-fold increase shown in Figure 3.2 using the original transduction assay strains may be partially due to variation in the assay. Numbers of transductants as well as the fold-change in gene transfer frequency due to Nb do change from 37  assay to assay, although within each assay the numbers of transductants are consistent for replicates of each condition. Additionally, the transduction assay was performed using ΔLHII (which contains a spectinomycin resistance cartridge inserted into a non-essential LHII gene as well as a rifampicin resistance gene from its SB1003 parent strain) instead of DW5, and recombinants were selected for by the presence of both kanamycin resistance (from ΔRC6) and rifampicin resistance (from ΔLHII) instead of photosynthetic growth. Direction of transfer could be determined by testing the doubly resistant transductants for spectinomycin resistance. Although an increase in gene transfer was still observed in the presence of Nb (Figure 3.8), the transfer was found to occur mostly but not entirely from ΔLHII to ΔRC6 (4 out of 52 transductants tested were resistant to spectinomycin). This indicates that the single direction of transfer seen earlier is probably DW5- specific rather than due to differences in background strains.  Figure 3.8 Transduction assay with alternative strains (∆RC6 and ∆LHII instead of ∆RC6 and DW5) in the presence of Nb at 0, 0.5 or 2 μg/mL. Cultures were grown in triplicate. * p < 0.005 compared to cultures grown without antibiotic.  38   Figure 3.9 Effects of cell-cell contact on gene transfer in a bioassay. Donors of strain ∆RC6 were grown in 0, 0.5 or 2 μg/mL Nb and either removed by filtration (dark grey) or retained without filtration of the culture (light grey) before being incubated with recipient cells of strain DW5. Experiments were performed in duplicate.  3.2.6 Cell-to-cell contact is not involved in the novobiocin-induced increase in gene transfer Because Nb was seen to have an effect on gene transfer in the transduction assay, where cells are directly mixed, but not in the bioassay where recipients are incubated with cell-free donor culture supernatants, it was thought that cell-cell contact may be necessary for Nb to have its effect on gene transfer. In order to test the need for cell-to-cell contact, a modified bioassay was performed. In this modified bioassay, a portion of the donor culture was incubated with recipient cells in parallel with the usual method of incubation with filtered (cell-free) donor supernatant. Because gene transfer had previously been observed only from the donor (ΔRC6) to the recipient (DW5), it was thought that any difference between the two conditions in the frequency of gene transfer would not be due to gene transfer from the recipient to the donor. Using donor cells that had been grown alone or with Nb at 0.5 or 2µg/mL, no great difference was observed between any of the conditions (Figure 3.9). Because this modified bioassay was 39  essentially the same as a transduction assay, except on a shorter time scale, this led to the idea that Nb may affect the timing of GTA production and release.  3.2.7 Effect of a subinhibitory concentration of novobiocin on the kinetics of RcGTA production It was possible that Nb causes an increase in the production of RcGTA at an early phase of growth, such that the stationary phase samples studied above did not reveal the genuine difference between +/- Nb cultures.  Therefore, early time points of a transduction assay were analyzed by Western blot for the presence of the capsid protein intracellularly and extracellularly. In a separate experiment, the Nb-mediated increase in gene-transfer was seen as early as 12 hours after culture inoculation (Figure 3.10A). However, Western blots indicated that RcGTA production by cultures grown in the presence of Nb is detectable at the same time or later than that of cultures grown without Nb (Figure 3.10B). Therefore, it appears that this subinhibitory concentration of Nb does not change the kinetics of RcGTA production.  3.2.8 Creation of Nb R  mutants and characterization in the transduction assay In order to elucidate the link between Nb and gene transfer, an attempt was made to isolate Nb-resistant (Nb R ) mutants of DW5 and ΔRC6  that were resistant due to a mutation in GyrB, the target of Nb. These strains were subjected to UV mutagenesis and seven Nb R  mutants of ΔRC6 were isolated on Nb at 40 µg/mL from cultures that had been UV-irradiated for 10 seconds. The gyrB gene from these mutants was sequenced, however no mutations were observed in the coding sequence of the gene. As no mutants had been isolated in DW5, we attempted to RcGTA-transduce the Nb R  phenotype from a ΔRC6 NbR strain to DW5. No NbR 40  mutants were isolated, indicating that the genetic changes that induced resistance were either too large to be transferred by RcGTA, or located at sites separated by more than 4.5 kb.     Figure 3.10 Time course of transduction assays in the presence or absence of 2 µg/ml Nb. A) Transduction assay of strains ∆RC6 and ∆LHII with time points at 12, 24, 48 and 72 hours, yielding 5.1-, 7.3-, 27.3-, and 11.5-fold increases in gene transfer, respectively, compared to a culture grown without antibiotic at the same time point. * p < 0.05, ** p < 0.1. B) Two repeats of a Western blot of intra- and extracellular fractions of a transduction assay from 14 to 24 hours, probed with RcGTA capsid antiserum.  41  In an attempt to obtain a Nb R  mutant that is resistant due to a mutation in the gyrB gene, site-directed mutagenesis was performed. The goal of this experiment was to change the GyrB protein Arg-147 to Ser, based on evidence that this residue is important in conferring resistance to Nb and other coumarins in other species (del Castillo, Vizan et al. 1991; Contreras and Maxwell 1992; Fujimoto-Nakamura, Ito et al. 2005; Garrido, Scatigno et al. 2005). An alignment of the R. capsulatus GyrB protein with the proteins in which the Arg-147 to Ser mutation leads to resistance is shown in Figure 3.11. The modified gyrB was inserted into the constitutive expression plasmid pRhoK (Katzke, Arvani et al. 2010) to give the construct pRhoKR147S, which was conjugated into the RcGTA over-producer strain DE442. Because a high copy number of a wild type gyrB gene may induce resistance to Nb (del Castillo, Vizan et al. 1991), this strain was compared to DE442 that contains the wild type gyrB gene on the same expression plasmid (pRhoKGyrB) rather than to DE442 without plasmid or the plasmid without insert. Strains were grown in the presence of varying amounts of Nb (0, 5, 10, 20, and 40μg/mL). Although the initiation of growth of DE442 strains containing plasmids that constitutively express either the wild type or mutant gyrB was delayed in proportion to the concentration of Nb, all cultures eventually plateaued at a similar density (Figure 3.12). Although the resistance to Nb of the DE442 strains carrying either the Arg-147 to Ser mutation or wild type gyrB was likely due to over-expression of the gene from the high copy number plasmid, an attempt was made to transduce resistance via RcGTA to DW5 and ∆RC6. Because RcGTA-transduction recipients integrate the DNA they receive by homologous recombination, only the base pair change in the modified gyrB would be transferred, rather than the high copy number of the gene. Unfortunately, no Nb-resistant strains were isolated (at 40  42    R.c  ---MTETPKNKAEYGAESIKVLKGLEAVRKRPGMYIGDTDDGSGLHHMVYEVVDNGIDEALAGHANYVAVKIHADSSVSVRDNGRGIPVDIHPEEGVSAAEVIMTQLHAGGKFNNTDEGG 117 E.c  ---MSNS------YDSSSIKVLKGLDAVRKRPGMYIGDTDDGTGLHHMVFEVVDNAIDEALAGHCKEIIVTIHADNSVSVQDDGRGIPTGIHPEEGVSAAEVIMTVLHAGGKFD-----D 106 S.a  MVTALSDVNNTDNYGAGQIQVLEGLEAVRKRPGMYIGSTSE-RGLHHLVWEIVDNSIDEALAGYANQIEVVIEKDNWIKVTDNGRGIPVDIQEKMGRPAVEVILTVLHAGGKFG-----G 114           .       *.: .*:**:**:***********.*.:  ****:*:*:***.*******:.: : * *. *. :.* *:*****..*: : * .*.***:* *******.     .  R.c  NAYKVSGGLHGVGVSVVNALSDWLELKVWRNDKVYFARFEKGDCVVHVHEIGEAPGEKGTEVRFMASAKTTDPEGTFSNLDYVFKTLETRLRELAFLNSGVRIILEDERPAEPL-RSELH 236 E.c  NSYKVSGGLHGVGVSVVNALSQKLELVIQREGKIHRQIYEHGVPQAPLAVTGET-EKTGTMVRFWPSLETFT-----NVTEFEYEILAKRLRELSFLNSGVSIRLRDKRDGK---EDHFH 217 S.a  GGYKVSGGLHGVGSSVVNALSQDLEVYVHRNETIYHQAYKKGVPQFDLKEVGTT-DKTGTVIRFKADGEIFT-----ETTVYNYETLQQRIRELAFLNKGIQITLRDERDEENVREDSYH 228      ..*********** *******: **: : *: .::   :::*     :   * :  :.** :** .. :        .   : :: *  *:***:***.*: * *.*:*  :   ..  *  R.c  YEGGVREFVKYLDRSKTAVMPDPIYMVGEVRGIGVEVAMWWNDSYHETVLPFTNNIPQRDGGTHLAGFRGALTRTITKYAQDSGIAKREKIDFTGDDAREGLTCVLSVKVPDPKFSSQTK 356 E.c  YEGGIKAFVEYLNKNKTPIHPNIFYFSTEKDGIGVEVALQWNDGFQENIYCFTNNIPQRDGGTHLAGFRAAMTRTLNAYMDKEGYSKKAKVSATGDDAREGLIAVVSVKVPDPKFSSQTK 337 S.a  YEGGIKSYVELLNENKEPIHDEPIYIHQSKDDIEVEIAIQYNSGYATNLLTYANNIHTYEGGTHEDGFKRALTRVLNSYGLSSKIMKEEKDRLSGEDTREGMTAIISIKHGDPQFEGQTK 348      ****:: :*: *:..* .:  : :*:  .  .* **:*: :*..:  .:  ::***   :****  **: *:**.:. *  ..   *. *   :*:*:***: .::*:*  **:*..***  R.c  DKLVSSEVRPAVENLVNEKLAEWFEEHPTEAKGIVGKIIEAALAREAARKARELTRRKTAMDVASLPGKLADCQEKDPALSEVFLVEGDSAGGSAKQGRERKNQAVLPLRGKILNVERAR 476 E.c  DKLVSSEVKSAVEQQMNELLAEYLLENPTDAKIVVGKIIDAARAREAARRAREMTRRKGALDLAGLPGKLADCQERDPALSELYLVEGDSAGGSAKQGRNRKNQAILPLKGKILNVEKAR 457 S.a  TKLGNSEVRQVVDKLFSEHFERFLYENPQVARTVVEKGIMAARARVAAKKAREVTRRKSALDVASLPGKLADCSSKSPEECEIFLVEGDSAGGSTKSGRDSRTQAILPLRGKILNVEKAR 468       ** .***: .*:: ..* : .:: *:*  *: :* * * ** ** **::***:**** *:*:*.********..:.*  .*::**********:*.**: :.**:***:*******:**  R.c  FDRMLSSDQIGTLITALGTGIGRDEFNIAKLRYHKIVIMTDADVDGAHIRTLLLTFFFRQMPELIEHGYLYIAQPPLYKVARGKSEVYLKDQAALEDYLVHQGVEGAILRLGDGT-DIIG 595 E.c  FDKMLSSQEVATLITALGCGIGRDEYNPDKLRYHSIIIMTDADVDGSHIRTLLLTFFYRQMPEIVERGHVYIAQPPLYKVKKGKQEQYIKDDEAMDQYQISIALDGATLHTNASAPALAG 577 S.a  LDRILNNNEIRQMITAFGTGIG-GDFDLAKARYHKIVIMTDADVDGAHIRTLLLTFFYRFMRPLIEAGYVYIAQPPLYKLTQGKQKYYVYND---------------------------- 559      :*::*..:::  :***:* *** .:::  * ***.*:*********:**********:* *  ::* *::*********: :**.: *: ::  R.c  ADLTRLVEEARQVRRILQAFPTHYPQRILEQAAIAGALLPGRIDADAQGVADELSARLDLIALEYERG---WMGRPTQDHGLRMSRILR----GVEEVRTLDGPVLRSGEARRLSQFTTS 708 E.c  EALEKLVSEYNATQKMINRMERRYPKAMLKELIYQPTLTEADLS-DEQTVTRWVNALVSELNDKEQHGSQWKFDVHTNAEQNLFEPIVRVRTHGVDTDYPLDHEFITGGEYRRICTLGEK 696 S.a  RELDKLKSELNPTP----------------------------------------------------------------------------------------------------------        * :* .* . .  R.c  LQESYRAPARLIRKDREQFIHGPLDLLTAILQEGEKGLTLQRYKGLGEMNPEQLWETTLDPAARTLLQVRVDDVAEAEDIFSKLMGDVVEPRREFIQQNALSVENLDF 816 E.c  LRGLLEEDAFIERGERRQPVASFEQALDWLVKESRRGLSIQRYKGLGEMNPEQLWETTMDPESRRMLRVTVKDAIAADQLFTTLMGDAVEPRRAFIEENALKAANIDI 804 S.a  ------------------------------------KWSIARYKGLGEMNADQLWETTMNPEHRALLQVKLEDAIEADQTFEMLMGDVVENRRQFIEDNAVYAN-LDF 644                                            :: *********.:******::*  * :*:* :.*.  *:: *  ****.** ** **::**: .  :*:   Figure 3.11 Alignment of the GyrB proteins from R. capsulatus (R.c), E. coli (E.c), and S. aureus (S.a). The Arg residue shown to yield Nb R  in E. coli and S. aureus and equivalent to R. capsulatus R147 is highlighted in grey. * = identical residue, : = conserved substitution, . = semi-conserved substitution. Sequences were obtained from NCBI (YP_003576176.1, BAA20341.1, and BAA01369.1).   43     Figure 3.12 Growth of strain DE442 containing pRhoK constructs in the presence of varying amounts of Nb. Cultures were grown in 0, 5, 10, 20, or 40 μg/mL Nb. KG = pRhoKGyrB, KM = pRhoKR147S   44  µg/mL) by this technique. This was not due to defective gene transfer by the DE442 pRhoKR147S donor strain, because transfer of rifampicin resistance, a different single base pair modification, occurred at a frequency yielding 543 colonies in excess of the spontaneous mutation frequency. The mutant and wild type gyrB genes were also inserted into the fructose-inducible expression plasmid pRhoT (Katzke, Arvani et al. 2010) to yield the constructs pRhoTR147S and pRhoTGyrB, which were conjugated into the expression strain B10S-T7. No particular induced protein was visible in either strain in an SDS-PAGE gel of whole-cell cultures induced with concentrations of fructose up to 16mM (data not shown). Nevertheless, Nb resistance was evaluated in both fructose-induced and uninduced cultures of R. capsulatus strains B10S-T7 and B10S-T7 containing either pRhoTGyrB or pRhoTR147S. All uninduced cultures grew to the same density in the absence of Nb (~280 KU); however uninduced cultures grown in the presence of 40 µg/mL Nb differed. Both B10S-T7 pRhoTGyrB and B10S-T7 lacking the expression plasmid seemed to be moderately resistant to Nb (~160 KU), although they did not grow to as high a density as the cultures without Nb. Growth of B10S-T7 pRhoTR147S, however, was almost completely inhibited by the addition of Nb (~75 KU). In the induced cultures, all strains grew to the same density without Nb (~450 KU), but only B10S-T7 pRhoTGyrB showed resistance to Nb (~430 KU). It grew to almost the same density as its antibiotic-untreated counterpart, although it took much longer to get to the same density. Both B10S-T7 and B10S-T7 pRhoTR147S induced cultures were almost entirely inhibited (~60 KU) by the addition of Nb (Fig 3.13).  45     Figure 3.13 Induction of gyrB and growth curve of strain B10S-T7 containing pRhoT constructs compared to B10S-T7. Cultures were either induced with 20 mM fructose or untreated for 3 hours, and then further divided into Nb-treated (40 μg/mL) or untreated. TG = pRhoTGyrB, TM = pRhoTR147S, (0, 0) = untreated, (0, Nb) = uninduced plus Nb, (F, 0) = induced, no antibiotic, (F, Nb) = induced plus Nb   46  Although Nb resistance was not transferable from DE442 pRhoKR147S by RcGTA transduction, Nb resistance was conferred to DW5 and ∆RC6 by conjugation of the constitutive pRhoK vector containing the wild type gyrB gene. These Nb R  strains were used in a transduction assay where cultures were grown in 0 or 2 μg/mL Nb, and the numbers of phototrophic colonies were compared with a transduction assay using the Nb-sensitive (Nb S ) counterparts. Additionally, transduction assays were performed where only one of the two strains was Nb R . For both Nb S /Nb R  mixtures, the Nb-induced increase in gene transfer was significantly diminished compared to the assay using both Nb S  parental strains, although the frequency of gene transfer was significantly higher in the presence of Nb than in the absence. However, the presence of Nb completely abrogated gene transfer in the mixture of Nb R  strains (Figure 3.14). The direction of transfer was found to be entirely from strain ∆RC6 (with or without plasmid) to strain DW5 (with or without plasmid) in the presence or absence of Nb, measured by rifampicin resistance of the resulting phototrophic colonies. Approximately 35 colonies were tested for each condition, and all were resistant to rifampicin. These results indicate that the effect of Nb with respect to gene transfer on both RcGTA donors and recipients is affected by the presence of excess GyrB.  47   Figure 3.14 Transduction assays with Nb R  strains. Strains DW5, ∆RC6, DW5 pRhoKGyrB (DW5 KG), and ∆RC6 pRhoKGyrB (∆RC6 KG) were used. All combinations were grown with or without Nb at 2 µg/mL. In the order shown above, the four different mixtures yielded 73-, 6.7- , 44-, and 0-fold increases in gene transfer frequency in the presence of Nb. * p < 0.01 compared to the same mixture of strains grown without antibiotic.   3.3 Effect of novobiocin on mutation rate It was of interest to determine whether some colonies attributed to gene transduction could have arisen by spontaneous mutation that was stimulated by the presence of Nb. Therefore, the effect of Nb on the basal mutation rate was evaluated using B10 cultures grown in the presence of varying concentrations of Nb and plated on rifampicin in order to count the number of rif-resistant mutants. Rifampicin resistance is most often caused by a single base pair mutation in the RNA polymerase gene rpoB (Campbell, Korzheva et al. 2001) and therefore is a good indicator of the mutation rate of a culture. It was found that Nb had no significant effect on the mutation rate at either 0.5 or 2μg/mL (Figure 3.15). 48   Figure 3.15 Effect of Nb on the mutation rate. Cultures of strain B10 were grown in 0, 0.5, or 2 μg/mL Nb (n = 2 for each condition) and then plated on RCV medium supplemented with rifampicin. p > 0.2 for both sets of cultures grown in the presence of Nb compared to the untreated cultures.  3.4 Effects of DNase on RcGTA production, release, uptake and integration In the course of experiments looking at the effect of subinhibitory concentrations of Nb on gene transfer, it was observed that DNase I caused a significant increase in gene transfer. To further examine this effect, a bioassay was performed where either the donors or the recipients were grown in the presence or absence of DNase I at 90 units/mL. No statistically significant change in gene transfer was observed between conditions (Figure 3.16). This was congruent with the results of Western blots probed with RcGTA capsid antiserum, where cultures grown in the presence of DNase I showed no increase in either intracellular or extracellular levels of GTA compared to those grown without (Figure 3.3B).  49   Figure 3.16 Effect of DNase I on RcGTA production and release. A bioassay was performed where either the donor (strain ∆RC6) or recipient (strain DW5) was grown in the presence of DNase I at 90 Kunitz units/ml. (D Ø) = donor untreated, (R Ø) = recipient untreated, (D D) = donor treated with DNase, (R D) = recipient treated with DNase. p > 0.1 for both conditions compared to untreated sample.  3.5 Carbon limitation 3.5.1 Effects of carbon limitation on production of RcGTA Cultures of the R. capsulatus wild type strain SB1003 grown in the minimal medium RCV containing varying concentrations of malic acid as the sole carbon source were analyzed by Western blot, probing for the presence of the RcGTA capsid protein. It was found that a decrease in malic acid concentration resulted in increased production of the RcGTA capsid protein within cells, although possible changes in the release of RcGTA could not be determined because the RcGTA capsid protein was not detected in the extracellular fraction of these cultures (Figure 3.17). Cultures were also grown in modified minimal media, where the carbon source malic acid was replaced with lactate or glutamate at full or decreased concentrations. The purpose of these experiments was to investigate whether there is a general carbon availability effect or 50  whether the effect is malate-specific. It was found that although overall RcGTA production was diminished in all of these different carbon sources (relative to growth in malic acid), more RcGTA was produced in cultures with decreased carbon content compared to their respective replete counterparts (Figure 3.18).  Therefore, it appears that the effect of increased intracellular RcGTA capsid protein is a general effect rather than malate-specific.   Figure 3.17 Western blot of RcGTA capsid protein production by strain SB1003 grown in varying amounts of malate (carbon source). Cultures were grown in RCV medium (1C) or RCV with malate concentrations at 2/3, 1/2, 1/3, and 1/6 that of replete RCV. Intracellular and extracellular fractions are shown. Samples were normalized such that equal amounts of cells and corresponding volumes of cell-free culture medium were loaded in each lane.   Figure 3.18 Western blot of SB1003 grown in RCV  medium with alternative carbon sources with replete and depleted carbon concentrations. Cells were grown in malate (the usual carbon component of RCV), lactate, or glutamate at equivalent carbon atom concentrations. Media variants contained either full or 1/6 carbon content. 51   3.5.2 Effect of carbon limitation on gene transfer The lack of detectable extracellular RcGTA capsid protein in Western blots led to the question of whether carbon limitation affects RcGTA-mediated gene transfer. To test this, a bioassay was performed using cultures grown in replete and malate-depleted RCV medium as GTA donors. Cultures grown in the depleted medium showed an increase in gene transfer compared to those grown in replete RCV medium (Figure 3.19). Additionally, the transduction assay described above was used to test the effect of carbon (malic acid) concentration on gene transfer, and it was found that under carbon-depleted conditions, gene transfer between strains DW5 and ΔRC6 was increased 6-10 fold (Figure 3.20). These results indicate that the increased amount of intracellular capsid protein in carbon-depleted media visible on Western blots correlates with an increase in gene transfer.   Figure 3.19 Bioassay examining the effect of culture carbon source depletion on gene transfer. Strain SB1003 donor cultures (n = 3) were grown in RCV medium (1 C) or carbon-depleted RCV (1/6 C). Strain DW5 was the recipient, and restoration of photosynthetic growth was the phenotype marker. * p < 0.01  52    Figure 3.20 Effect of carbon depletion on gene transfer and RcGTA production. A) A transduction assay was performed where mixed strain DW5 and ∆RC6 cultures (n = 3) were grown in replete (1 C) or depleted (1/6 C) RCV media. * p < 0.01 B) Western blot of transduction assay culture supernatant or cellular fractions, probed with capsid antiserum.  3.5.3 Production of RcGTA in regulatory gene mutants subjected to carbon deprivation In an attempt to identify genes involved in signaling carbon availability to the cell, strains with mutations in the global regulatory genes regA, regB, and the homologue of spoT, the quorum-sensing gene gtaI, and the polyphosphate kinase ppk1 were tested by the same western blotting method. All of these mutant strains exhibited increased RcGTA capsid production with decreased carbon concentration (data not shown), indicating that these genes are not involved in a signaling pathway that connects carbon availability to RcGTA production. 53   3.5.4 Effects of culture growth phase on stimulation of RcGTA production by carbon deprivation It was observed that cultures grown in carbon- (malate-) depleted RCV medium reached stationary phase sooner and at a lower density than those grown in replete carbon. In order to investigate the importance of growth phase on the carbon effect, I measured RcGTA production over time in cultures grown in malate-replete and depleted RCV medium. Because cultures in depleted carbon entered stationary phase earlier than cultures grown in malate-replete medium (Figure 3.21A), it was possible that the increased RcGTA capsid protein visible in Western blots at 24 hours was simply due to the cultures beginning to produce RcGTA earlier, and thus having accumulated a larger amount in the same time period than cultures grown in replete medium and existing in the stationary phase for a shorter period of time. However, Western blots revealed that although cultures grown in reduced carbon do indeed begin producing GTA earlier, cultures grown in the replete medium did not accumulate as much intracellular RcGTA in the 40-hour course of this experiment (Figure 3.21B).   54    Figure 3.21 Time course of RcGTA production in RCV medium and carbon limited culture. A) Growth curve of cultures grown in replete (1 C) and carbon-depleted (1/6 C) RCV media. B) Western blots were performed on intact cell samples taken over two days (up to 40 hours after inoculation) from growth curve cultures, probed with RcGTA capsid antiserum.  A B 55  4.0 DISCUSSION  4.1 A new transduction assay In this study, a new transduction assay is introduced to study genetic exchange mediated by the R. capsulatus gene transfer agent RcGTA. Previously, assays examining the effects of various substances on R. capsulatus gene transfer by RcGTA involved unidirectional transfer from a donor strain to a recipient strain. These previous assays are referred to as “bioassays,” in which strains are grown separately, and each may be treated separately. Although the bioassay is useful for examining particular aspects of gene transfer, such as RcGTA production and release, it does not show the effect of a given substance on the entire process of gene transfer. In the transduction assay two strains are mixed together, gene transfer can proceed in either direction, and the substance being tested can act on any (or more than one) stage of gene transfer. Thus the transduction assay may be a good representation of gene transfer in a natural environment, where bacterial strains are intermingled and the conditions of the environment affect both RcGTA donors and recipients. The transduction assay described here can be used to determine the effect of a wide range of variables on gene transfer. In this study it is used to examine both the effect of subinhibitory concentrations of antibiotics and carbon limitation, but the substances that could be tested vary widely. The effects of excess or limitation of any nutrient could be assayed, as could the effects of bacterial signaling molecules, subinhibitory concentrations of any anti-bacterial compounds, or other molecules of interest. Additionally, other factors such as pH and temperature could be examined for their effects on gene transfer. 56  Also, although this work relied largely on the transduction of photosynthesis genes between the strains DW5 and ∆RC6, the assay is applicable to any combination of strains that each have a distinct marker. This was shown by the use of Rif R  strain ∆LHII and KanR strain ∆RC6 in an assay where double antibiotic resistance (RifR/KanR) was used to indicate whether gene transfer had occurred (Figure 3.8).  4.2 Subinhibitory concentrations of antibiotics Antimicrobials have been shown to affect bacteria in more than just bactericidal or bacteriostatic effects. At subinhibitory concentrations, the effects of antibiotics include large- scale transcriptional changes in bacteria such as S. typhimurium (Goh, Yim et al. 2002) as well as in complex microbial communities (Yergeau, Lawrence et al. 2010). Subinhibitory concentrations of antibiotics also affect specific bacterial pathways such as in virulence gene expression (Weir, Martin et al. 2008; Knudsen, Holch et al. 2012) and biofilm formation (Hoffman, D'Argenio et al. 2005). Furthermore, antibiotics stimulate horizontal gene transfer by a variety of mechanisms. In Streptococcus pneumonia, Legionella pneumophila and Helicobacter pylori, subinhibitory levels of fluoroquinolones increase HGT (by transformation) by inducing competence genes (Prudhomme, Attaiech et al. 2006; Dorer, Fero et al. 2010; Charpentier, Kay et al. 2011). The transfer of conjugative transposons is increased by certain antibiotics in Vibrio cholerae (Beaber, Hochhut et al. 2004) and the Bacteroides (Whittle, Shoemaker et al. 2002). Transduction by phage is stimulated by low levels of antibiotics in a variety of bacteria (Karaolis, Somara et al. 1999; Herold, Siebert et al. 2005; Maiques, Ubeda et al. 2006; Comeau, Tetart et al. 2007; Allen, Looft et al. 2011). 57  Because of discoveries in other experimental systems prior to my research, summarized above, I decided to investigate whether subinhibitory concentrations of antibiotics might affect the gene transfer agent of R. capsulatus.  4.2.1 Substances affecting gene transfer in R. capsulatus In this thesis research, I demonstrated that subinhibitory levels of DNA gyrase inhibitors cause an increase in gene transfer frequency in R. capsulatus. The gyrase inhibitors that showed this effect were Nb and Cb, both aminocoumarins that affect the B subunit of gyrase, and Cip, a fluoroquinolone that targets the A subunit of gyrase. Surprisingly, coumermycin, another aminocoumarin, appeared to completely abolish gene transfer, although coumermycin was only tested once, and so it is unclear whether this is a genuine effect. It would be interesting to explore other gyrase subunit A or B inhibitors to determine whether the effects observed with Nb, Cb, and Cip are characteristic of all gyrase inhibitors. Of particular interest would be the simocyclinones, a class of gyrase inhibitors that affect the A subunit of gyrase, but with a different mechanism than fluoroquinolones (Flatman, Howells et al. 2005; Edwards, Flatman et al. 2009). Although fluoroquinolones stabilize the GyrA-DNA complex, simocyclinones prevent binding of GyrA to DNA. Other substances were tested for their effects on gene transfer, and although many of these were not reproduced enough times to be statistically significant, none of them showed the same large increases in gene transfer frequency that were seen with Nb, Cb, and Cip (Table 3.1). Two inhibitors of protein synthesis, streptomycin and erythromycin, were tested. Streptomycin gave results that varied widely, initially appearing to increase the frequency of 58  gene transfer, but later having the opposite effect, and erythromycin seemed to decrease gene transfer.  Rifampicin, an RNA polymerase inhibitor, initially seemed to increase gene transfer, but later had the opposite effect. Bacitracin and trimethoprim, substances that interfere with peptidoglycan synthesis and tetrahydrofolic acid synthesis respectively, both seemed to have no effect on the frequency of gene transfer. These results indicate that increased gene transfer is not the default response of R. capsulatus to any antibiotic, but instead may be due to the specific mechanism of action of the gyrase inhibitors. Another substance tested was indole acetic acid (IAA), which is a plant hormone that regulates plant development and cellular processes, but is also synthesized by a wide variety of bacteria (Tsavkelova, Cherdyntseva et al. 2007; Sudha, Gowri et al. 2012) and is found in many environments. In E. coli, IAA was found to upregulate several genes involved in adaptation to stress, and to improve the cellular response to stresses such as antibiotics, acid, heat, and osmotic shock, UV irradiation, and oxidative stress (Bianco, Imperlini et al. 2006). An increase in gene transfer frequency was observed in R. capsulatus in the presence of IAA (Table 3.1), suggesting that perhaps upregulation of RcGTA-mediated gene transfer in R. capsulatus is an adaption to stress. It would be interesting to look further into the effect of IAA on R. capsulatus stress responses.  4.2.2 Effects of novobiocin on different aspects of gene transfer The effect of Nb on gene transfer was shown not to be due to an increase in production or release of RcGTA, on the basis of bioassays and Western blots probed with capsid antiserum (Figure 3.4). This is unexpected, as antibiotic-induced transduction by prophages is typically due 59  to increased phage induction. Additionally, the non-homologous GTA found in Brachyspira hyodysenteriae, VSH-1, mediates increased gene transfer in the presence of subinhibitory levels of carbadox and metronidazole through increased transcription of VSH-1 genes (Stanton, Humphrey et al. 2008). Both carbadox and metronidazole are metabolized to products that interact with bacterial DNA and cause mutations and DNA strand breaks. The gyrase inhibitors I studied (Nb, Cb and Cip) also cause DNA damage, although they do this through stoppage of the DNA replication fork and deregulation of DNA supercoiling (Collin, Karkare et al. 2011). It is interesting that these two very disparate organisms show an increase in gene transfer by non- homologous GTAs in response to DNA-damaging agents, although the mechanism by which the increase in gene transfer occurs is different for both. Another aspect of RcGTA transduction that I examined was adsorption of RcGTA to recipient cells. The RcGTA ORF14 encodes a protein that is thought to aid in peptidoglycan degradation during infection by RcGTA (Fogg, Westbye et al. 2012). Fluoroquinolones have been shown to profoundly modify peptidoglycan structure (Bryskier 1993), and so it was thought that perhaps gyrase inhibitors caused a modification to the cell wall that facilitated RcGTA attachment or uptake. However, an attachment assay demonstrated that there was no significant difference in RcGTA adsorption by cells grown with or without Nb (Figure 3.7). Additionally, Nb did not ‘prime’ recipient cells in any way, because recipient cells grown in the presence of Nb were transduced by RcGTA at the same frequency as those grown in the absence of Nb (Figure 3.5). When cells in colonies resulting from transduction assays were examined, it was found that all transductants tested resulted from transfer from ∆RC6 to DW5. This suggested a strain- dependent effect, and so a transduction assay was performed using strain ∆LHII instead of strain 60  DW5. Additionally, instead of testing for transfer of photosynthetic genes, two different markers, spectinomycin resistance and kanamycin resistance, were used for selection. An increase in gene transfer frequency was still observed in the presence of Nb (Figure 3.8), indicating that the Nb effect is independent of both strain and marker. This finding was supported by the transduction of the ∆RC6 mutation into the same parental strain, SB1003, as that of DW5. An increase in gene transfer in the presence of Nb was still observed in a transduction assay with both strains (SB∆RC6 and DW5) in the same SB1003 background. There was major difference between the transduction assay where the Nb effect was observed and the bioassay wherein no Nb effect was seen. This difference was the co-incubation of cells in a single, mixed culture in the transduction assay, in contrast with the bioassay where two different cultures are grown followed by mixing of cells from one culture and cell-free filtrate from the other. A modified bioassay was performed to determine whether some aspect of the mixed culture affected gene transfer in the presence of Nb. However, no difference was observed between the typical bioassay, wherein recipients were mixed with filtered, cell-free donor supernatant, and the modified bioassay, wherein recipients were mixed with an equivalent amount of unfiltered donor culture (Figure 3.9). This indicated that the direct mixing of donor and recipient cultures did not influence the frequency of gene transfer. Another major difference between the transduction assay and the bioassay is the amount of time during which the donors and recipients are incubated together. In the transduction assay, cells are started at a low density, but are allowed to grow together for 24 hours before selection for gene transfer. In the bioassay, cultures are at a much higher density when the cell-free donor supernatant is collected and mixed with the recipient strain, but they are only incubated together for one hour, followed by a 1/3 dilution into fresh medium and a four-hour incubation to allow 61  gene expression before selection. This led to the thought that Nb may influence the time-course of RcGTA expression. A transduction assay was performed where cultures were incubated for only 12 hours before selection, and gene transfer frequency had already increased five-fold in the presence of Nb. However, when transduction assay cultures were examined by Western blot (probed with capsid antiserum) at times between 14 and 24 hours, the appearance of RcGTA in the culture supernatants seemed to be unaffected or even delayed in the presence of Nb (Figure 3.10). This result indicated that Nb does not affect the kinetics of RcGTA production. Replication of DNA causes positive supercoils that, without the opposing action of DNA gyrase, lead to stalling of the replication fork. Processing of stalled replication forks involves the action of various recombination proteins (Michel, Grompone et al. 2004), in particular RecA (Maisnier-Patin, Nordstrom et al. 2001). In some cases, replication fork stalling led to increased homologous recombination (Horiuchi, Fujimura et al. 1994). It was possible that the replication fork stalling caused by gyrase inhibitors in R. capsulatus may have led to increased recombination and hence genomic integration of RcGTA-transduced genes on linear DNA fragments. Also, fluoroquinolones were reported to increase the frequency of homologous recombination in E. coli (Lopez and Blazquez 2009). This increased recombination frequency was shown to involve the SOS response, and was entirely RecA dependent. Additionally, the supercoiling state of DNA has been shown to affect the frequency of recombination (Trigueros, Tran et al. 2009). However, Nb was observed to not have an effect on recombination in R. capsulatus. In bioassays where donor supernatant and recipient cells were incubated in the presence or absence of Nb, the effect of Nb on gene transfer may have been masked by an adverse effect on the 62  viability of recipient cells. However, a suicide plasmid conjugation assay was developed that allowed the measurement of recombination frequency per viable cell, and this too showed no increase in recombination frequency in the presence of Nb (Figure 3.6). Because the conjugation assay removed any aspect of RcGTA from the effect of Nb on recombination, the absence of any increase in recombination suggests that the effect of Nb is specific to a particular aspect of transduction by RcGTA. Perhaps Nb affects the integration of linear DNA, as found in RcGTA particles, rather than circular DNA, as in plasmid conjugation.  4.2.3 Novobiocin resistance and DNA gyrase To evaluate whether Nb acts via DNA gyrase to cause increased RcGTA-mediated gene transfer, I attempted to isolate Nb-resistant (Nb R ) mutants of the strains DW5 and ∆RC6 to be used in the transduction assay. UV mutagenesis yielded 7 Nb R  isolates of ∆RC6, but no NbR isolates of DW5. Because the B subunit of DNA gyrase is the main target of aminocoumarins and resistance to these antibiotics is often a result of mutations in gyrB, the gyrB gene from each of these mutants was sequenced. Surprisingly, none of these genes encoded an altered GyrB protein sequence. This result indicated that there is an alternative mechanism for Nb resistance in R. capsulatus. Some possibilities include overexpression of GyrB or modification of a (potential) aminocoumarin efflux pump. Additionally, the Nb R  mutants of ∆RC6 were unable to transfer Nb resistance to DW5 by transduction. This result indicates that either the resistance-conferring mutation was a deletion or insertion too large to be transferred by RcGTA, or that two mutations occurred at sites more than 4.5 kb apart, making transfer by one RcGTA particle impossible. Alternatively, because the 63  ∆RC6 NbR strain was less healthy than the parent strain, perhaps it was not able to produce or release RcGTA. This could be tested by transfer of a different marker such as Kan R . Because of the failure to isolate Nb R  mutants in known locations in both transduction assay strains, a site-directed mutagenesis approach was chosen. The residue R147 in GyrB was chosen as a target because mutations in homologous residues were shown to confer resistance to Nb and other aminocoumarins in E. coli and S. aureus by preventing the binding of the antibiotic (Contreras and Maxwell 1992; Fujimoto-Nakamura, Ito et al. 2005). Both the wild type and mutated gene conferred resistance when constitutively expressed from a neo gene promoter on a plasmid (Figure 3.12). However this is not surprising because a high copy-number of GyrB has been shown to confer resistance to coumermycin by antibiotic sequestration (del Castillo, Vizan et al. 1991). The site-directed mutant R147S of R. capsulatus gyrB was conjugated into the RcGTA overproducer strain DE442 on a plasmid. However, when transduction of the mutated gyrB gene into the strains DW5 and ∆RC6 was attempted using Nb resistance as a selection, no resistant strains were isolated. These results indicate that the residue chosen for mutation does not confer resistance to Nb in R. capsulatus. However it would be surprising if the R147S mutation did not decrease the binding affinity of GyrB for Nb, because the R. capsulatus predicted GyrB sequence is 54% and 56% identical in alignments with the GyrB proteins of S. aureus and E. coli, respectively, which were shown to require the homologous residue to R147 for binding of Nb. Both the mutated and wild type gyrB genes were inserted into an inducible expression plasmid, pRhoT, and placed in the expression strain B10S-T7. Surprisingly, this strain without plasmid showed a moderate level of Nb resistance. When the uninduced plasmid containing the wild type gyrB was introduced, this level of resistance did not change. However, the introduction 64  of the uninduced mutant gene resulted in almost complete inhibition of growth by Nb. Interestingly, when cultures were induced to express gyrB alleles, only the strain containing the wild type gyrB expression plasmid showed high level Nb resistance, whereas the other two strains (B10S-T7 and B10S-T7 pRhoTR147S) were almost completely inhibited (Figure 3.13). These results showed that induction of the wild type gyrB gene conferred resistance, whereas the presence of the R147S mutant version of the gene was neutral or even harmful in the presence of Nb. This conflicts with results from the constitutive expression plasmids, where overexpression of either the wild type or the R147S version of the gene conferred resistance to Nb. The difference may be due to the plasmid host strain. B10S-T7 contains the T7 RNA polymerase under the control of fructose, and expression of genes from pRhoT is under control of the T7 promoter (Katzke, Arvani et al. 2010). Thus induction with fructose induces high levels of T7 RNA polymerase in the cell, and the combination of high amounts of T7 RNA polymerase and Nb may inhibit cell survival. Additionally, B10S-T7 appears to be a recA mutant, because the T7 RNA polymerase gene was integrated into the R. capsulatus recA gene homologue, and therefore the effects of expressing wild type or mutant gyrB may be different than in other strains. The constitutively-expressed pRhoKGyrB plasmid was used to confer Nb resistance to DW5 and ∆RC6. These strains were then used in a transduction assay (Figure 3.14) and compared to the Nb-sensitive parental strains. In transduction assays where only one strain contained pRhoKGyrB, the Nb-induced increase in gene transfer frequency was less than in the transduction assay using the strains without plasmids. These results indicate that an unnaturally high level of the GyrB protein inhibits gene transfer in the presence of Nb. Additionally, the data indicate that both strains used in the transduction assay respond to Nb in a manner that increases gene transfer, despite the fact that gene transfer appears to occur only in one direction. When 65  both strains contained the plasmid, gene transfer was inhibited entirely in the presence of Nb, although gene transfer occurred at a normal frequency in the absence of Nb. These results may be confounded by the fact that the high amounts of GyrB could bind to Nb entering the cell and prevent it from acting through a secondary target. However, the reversal of the Nb effect when both strains produce excess GyrB rather than an unchanged frequency of gene transfer indicates that cells respond to Nb in a manner that is dependent on the supercoiling state of the genome. Therefore, it is still unclear whether the Nb effect is due to a direct interaction with the GyrB subunit of DNA gyrase.  4.3 Effect of carbon limitation on RcGTA expression and gene transfer The concentration of carbon (malate) in the growth medium was initially observed to have an effect on RcGTA in Western blots (Taylor 2004). I found that the intracellular production of RcGTA increased with carbon limitation, but no extracellular RcGTA was observable by Western blot probed with capsid antiserum under either condition. In order to test whether carbon limitation affects extracellular RcGTA and gene transfer, a transduction assay and a bioassay were used. The transduction assay showed that the frequency of gene transfer increased in carbon-limited cultures (Figure 3.20). The bioassay showed that there was indeed more functional RcGTA released into culture supernatants under limited carbon conditions, despite not being detectable by Western blot (Figure 3.19). The carbon-limitation effect was found to be a general response rather than specific to malate, the carbon source tested in initial Western blots. Other carbon sources tested include lactate and glutamate (Figure 3.18). Malate is an intermediate of the citric acid cycle, and glutamate feeds into the citric acid cycle through α-ketoglutarate. Lactate is converted to the 66  glycolytic intermediate pyruvate before entering the citric acid cycle, either by carboxylation to form oxaloacetic acid, or by decarboxylation and activation to form acetyl-CoA. Because I observed the same response to a variety of carbon sources, I suggest that RcGTA production responds to carbon availability in a general sense. Several regulatory mutants were evaluated, including strains with mutations in regA, regB, gtaI, spot, and ppk1, and all of them demonstrated the same effect of increased RcGTA production with decreasing carbon content. These results indicate that none of these genes are involved in the signaling pathway leading from decreased carbon availability to increased RcGTA production. Lastly, the time course of RcGTA production in replete and carbon-depleted RCV minimal media was compared. Previously, it was shown that RcGTA production and release increase upon entry into stationary phase in the complex YPS medium (Florizone 2006). I found that cultures grown in carbon-limited RCV medium reached stationary phase sooner and at a lower density than cultures in replete medium. Under a variety of carbon substrate concentrations, RcGTA production increased upon entry into stationary phase, and perhaps because cultures in carbon-limited medium reached stationary phase sooner, RcGTA was produced sooner (Figure 3.21). These results indicate a quorum-sensing independent mechanism of sensing stationary phase, which is supported by the unchanged response to carbon in the gtaI (acyl-HSL synthase necessary for quorum sensing) knockout. However, production of RcGTA never reached the same levels in cultures grown in replete medium as those in the depleted medium. These results indicate that RcGTA production not only responds to entry into stationary phase, but also specifically to carbon limitation.  67  4.4 Future Research There are several avenues that could be explored following this research. The transduction assay described allows for evaluation of the effects of a wide variety of variables on gene transfer. Further areas to be investigated could include the effects of temperature, pH, signaling molecules, and nutrient deprivation. In examining the effects of Nb on gene transfer, evaluation of the effects on gene transfer of members of different classes of DNA gyrase inhibitors from those examined here could provide insight into the relationship between DNA gyrase and gene transfer. For example, cyclothialidines and simocyclinones are two classes of gyrase inhibitors that were not examined in this study (Yamaji, Masubuchi et al. 1997). The bactericidal toxins CcdB and microcin B17 have also been found to inhibit DNA gyrase (Couturier, Bahassi el et al. 1998), and it would be interesting to see how they affect gene transfer. Additionally, evaluating the effects of other DNA-damaging agents on gene transfer could further our understanding of the linkage between stress due to DNA damage and gene transfer. Gene expression studies could be useful in determining exactly how Nb influences the frequency of gene transfer. In particular, the effects of Nb on SOS genes and on RcGTA structural and regulatory genes could be informative in determining how gyrase inhibitors increase the frequency of gene transfer. Other areas of gene expression to explore could include potential RcGTA receptor genes and other DNA uptake mechanisms. Additionally, elucidation of the exact mechanism of action of low levels of Nb in R. capsulatus may be useful in isolating or creating Nb R  mutants that are resistant by a different mechanism than that of overexpression of GyrB. Understanding how gene transfer is affected in these mutants may reveal the role of DNA gyrase in RcGTA-mediated gene transfer. 68   It would be interesting to know the genes involved in signaling carbon availability to the cell, and how this signaling leads to increased gene transfer under limited carbon conditions. It is possible that the carbon effect is due to change in expression of sigma factors. This could be explored by knocking out alternative sigma factors and examining the effects on RcGTA production under normal and carbon-limited conditions. Additionally, gene expression studies comparing normal and carbon-limited conditions could reveal any changes in the expression of primary and alternative sigma factors.  4.5 Concluding remarks Both carbon limitation and subinhibitory concentrations of DNA gyrase inhibitors were shown to increase the frequency of gene transfer in R. capsulatus, but by different mechanisms. Although carbon limitation increased the production and release of RcGTA, the mechanism by which gyrase inhibitors affected gene transfer was less clear. Several mechanisms of action were eliminated, including production, release, and uptake of RcGTA. It is possible that the response to gyrase inhibitors was dependent on the supercoiling state of the cell genome. 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Proc Natl Acad Sci U S A 85(12): 4209-4213.    75  APPENDIX A Survival of UV-irradiated cells  0.01 0.1 1 10 100 0 2 4 6 8 10 12 %  S u rv iv al  Time of Irradiation (sec) DW5 ∆RC6

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