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Studies in the regulation of the gene transfer agent (GTA) of Rhodobacter capsulatus Florizone, Sarah Marie 2006

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Studies on the Regulation of the Gene Transfer Agent (GTA) of Rhodobacter capsulatus by S A R A H M A R I E F L O R I Z O N E B . S c , University of British Columbia, 2002 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Microbiology and Immunology) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A M a y 2006 © Sarah Marie Florizone, 2006 Abstract: The gene transfer agent (GTA) of Rhodobacter capsulatus acts as a system of genetic exchange in this purple, non-sulphur, photosynthetic bacterium. G T A is a small bacteriophage-like particle that transfers random 4.5 kbp segments of the donor cell's genome to recipient cells where allelic replacement can occur. The structural gene cluster for G T A encodes 15 open reading frames, most of which are homologous to known phage genes. Three proteins have been found that regulate the expression of G T A genes: Gtal, CtrA and C c k A . Gtal is involved in quorum sensing while CtrA and C c k A are thought to be part of a sensor kinase/response regulator signalling pathway. 1 set out to elucidate this poorly understood regulatory pathway. M y initial hypothesis was that there are other regulatory proteins besides Gtal, CtrA and C c k A , as mutations in any of the genes disrupts, but does not completely abolish, G T A structural gene expression. However, during mutant screening, it became apparent that the reporter plasmid used to monitor G T A gene expression (pYP) caused a decrease in G T A transduction, which I termed the pYP effect. Promoter analysis was performed to determine which part of plasmid p Y P was causing this effect and to study m-active regulatory sequences of the G T A structural gene cluster. Sequencing the G T A promoter from 3 strains that exhibit different levels of G T A gene expression and transduction did not find any differences, suggesting a trans-acimg factor causes the observed differences. 5' m R N A mapping was used to find the 5' end of the G T A transcript, which overlaps with the upstream gene. Capsid levels throughout the growth curve were measured for Y262 ( G T A overproducer), BIO (wild type), Y C K F 2 (ctrA~), and Y K K R 2 (cckA). The immunoblots from these strains showed interesting differences in capsid production that give us a better understanding of G T A production and release from the cell in different stages of growth. Although I was unable to provide a clear pathway for the regulation of G T A , the data in this thesis gives us a better understanding of the complexity of G T A production and regulation. n Table of Contents Abstract : i i Lis t of Tables: v L i s t of Figures: v i 1 Introduction 1 1.1 Horizontal gene exchange 1 1.2 Gene transfer agents in prokaryotes 3 1.3 Transduction and gene exchange in R. capsulatus 4 1.4 Structural genes encoding GTA in R. capsulatus 8 1.5 GTA regulatory genes in R. capsulatus 11 1.6 Overview of the thesis 13 2 Mater ia ls and Methods 16 2.1 Bacterial Strains, plasmids and growth conditions 16 2.2 Creation and screening of a transposon mutagenized R. capsulatus Y262 library containing pYP 18 2.3 Methods of measuring GTA 22 2.3.1 (3-galactosidase assays measure transcription from the G T A promoter 22 2.3.2 Bioassays to determine frequency of gene transduction 22 2.3.3 GTA immunoassays of the major capsid protein (orfg5) 23 2.4 Identification of disrupted genes 24 2.5 Promoter deletions 27 2.6 5'R.A. C.E. analysis ofmRNA 29 3 Results 30 3.1 The search for GTA regulatory mutants 30 i n 3.1.1 Screening of the transposon mutagenized library 30 3.1.2 Studies on 16 mutants found to have reduced G T A expression 35 3.2 Western blot analyses of GTA capsid protein levels 37 3.2.1 The pYP effect 39 3.2.2 Time course analysis of capsid production in Y262 and BIO 41 3.2.3 Time course analysis of capsid production in ctrA' and cckA' strains 43 3.2.4 Immunoblots of BIO, SB 1003 and IKOI 45 3.3 GTA promoter region analysis 47 3.3.1 D N A sequencing 47 3.3.2 Softberry's B P R O M analysis 49 3.3.3 5' end mapping of G T A m R N A (R.A.C.E . ) 51 3.3.4 Promoter deletions 51 3.3.5 Summary of data and proposed promoter for G T A structural genes 52 3.3.6 Immunoassays of Y262(pSMF001)and Y262(pSMF002) 53 4 Discussion 55 4.1 The surprising variety of genes needed for maximal production of GTA 55 4.2 The western blot approach 58 4.2.1 The p Y P effect 59 4.2.2 Time course analysis of capsid production in Y262 and BIO 59 4.2.3 Time course analysis of capsid production in ctrA' and cckA' mutants 61 4.2.4 Immunoblots of B10, SB 1003 and IKOI 62 4.3 GTA structural gene cluster promoter analysis 64 4.3.1 Sequence differences between the G T A promoter in Y262, B10 and SB 1003 65 4.3.2 Predicted promoter region for G T A : R . A . C . E . and Softberry analysis 66 4.3.3 Promoter deletions 67 4.3.4 G T A immunoassays of Y262(pSMF001) and Y262(pSMF002) 68 4.4 Future Research 69 5 References: 71 iv List of Tables: page # Table 1: Summary of G T A - l i k e particle properties 5 Table 2: Bacterial strains used in this study 1 7 Table 3: Plasmids used in this study 1 7 Table 4: Primers used in this study 28 Table 5: 12 mutants with reduced G T A gene expression selected for further study 36 v List of Figures: page # Figure 1: Three types of genetic exchange 2 Figure 2: Electron micrograph of G T A particles 9 Figure 3: G T A structural gene cluster 10 Figure 4: Plasmids pYP and pYnP 19 Figure 5: Stab plate of mutants and control strains on X-gal 21 Figure 6: Southern blot to determine size of Tn5 containing fragment 26 Figure 7: P-galactosidase assay results 3 1 Figure 8: Overview chart of G T A transduction activity for 40 mutants 33 Figure 9: G T A transduction bioassay results 34 Figure 10: G T A immunoassays: resolving the extra bands 38 Figure 11: The p Y P effect: G T A immunoassay of Y262, Y262pYP and Y262pYnP 40 Figure 12: Y262 and BIO capsid production over the growth curve 42 Figure 13: Capsid production in two G T A mutants, ctrA' and cckA' 44 Figure 14: BIO capsid production compared to SB1003 and IKOI 46 Figure 15: G T A structural gene cluster promoter: overview 48 Figure 16: G T A structural gene cluster promoter: sequence view 50 Figure 17: G T A immunoassay of pSMFOOl and pSMF002 compared to controls 54 vi List of Abbreviations: aa amino acid A p (Ap R ) ampicillin (ampicillin resistance) B L P bacteriophage-like particle B S A bovine-serum albumin C F U colony fanning units D M S O dimethyl sulphoxide ds double stranded G T A gene transfer agent H S L homoserine lactone kb kilobase pair kDa kilodalton K m ( K m R ) kanamycin (kanamycin resistance) K U Klett unit L B Luria-Bertani medium M b Megabase pair N S P P B Nonsulfur, purple photosynthetic bacteria O D optical density O R F open reading frame phage bacteriophage R C V Rhodobacter capsulatus minimal growth medium S Svedberg unit S D S - P A G E sodium dodecyl sulphate polyacrylamide gel electrophoresis Tc (Tc R ) tetracycline (tetracycline resistance) Tn5 Transposon 5 Y P S yeast-peptone salt (rich media) vi i 1 Introduction 1.1 Horizontal gene exchange Horizontal gene transfer between bacteria is an important mechanism contributing to the evolution of bacterial genomes. It is a critical area of research to the medical community as the number of multidrug resistant bacterial strains continues to increase due to the over prescribing of antibiotics and the prevalence of virulence genes that can be readily transferred between bacteria (Davies, 1994). Horizontal gene transfer is thought to be integral to the evolution of pathogenic enteric bacterial strains as it has been recently found that horizontally acquired 'pathogenicity islands' are important for transferring virulence between pathogenic and non-pathogenic strains (Ochman et al., 2000). There are three main types of horizontal gene exchange in bacteria: transformation, conjugation and transduction, which are summarized in Figure l . Transformation involves a competent (either natural or induced) host cell taking up naked D N A from the environment. D N A from different species can be taken up in this fashion, although a recognition sequence is required, and the D N A can be integrated into the chromosome by homologous recombination i f it contains regions homologous to the hosts' D N A . Natural D N A uptake is thought to be advantageous to the cell on a nutritional basis as it can provide substrates for growth, or at least help to alleviate the demand for new nucleotides to be synthesized when nutrients are low, as opposed to being a mechanism solely for genetic exchange as previously thought (Redfield, 2001). Conjugation involves the transfer of plasmids or mobile elements via cell-to-cell contact. There are various plasmids that carry mobilization elements, such as the F plasmid in Escherichia coli, the T i plasmid in Agrobacterium and broad host-range antibiotic resistance (R) 1 a DNA transfer by transduction Phage-infected donor b DNA transfer by conjugation Common Recipient Donor Recipient Rare H f r \ Donor Recipient c Gene transfer by competence _ ; _ f _ _ • 1 Dead donor Competent recipient Figure 1: Three types of genetic exchange in bacteria: transduction, conjugation and transformation. Transduction is shown in a) in which a phage accidentally packages bacterial D N A and transfers it to a recipient cell upon phage infection. Conjugation is shown in b) in which genetic material is transferred via cell to cell contact. If the mobilization element is integrated into the chromosome, the entire chromosome can be transferred instead of the plasmid by itself. Transformation by competent cells is shown in c) in which a lysed cell's D N A is taken up by a recipient competent cell. This D N A can then be integrated into the chromosome or digested and used for nutrients. Figure taken from (Redfield, 2001). 2 plasmids in a variety of species (Griffiths, 2005). Very rarely, i f the mobile element integrates into the chromosome, the entire chromosome may be transferred from the donor cell into the recipient cell (Campbell, 1996). Transduction is the transfer of host genes between bacteria via a bacteriophage. Bacteriophage usually only transfer their own genes, however bacterial D N A can be accidentally incorporated into the bacteriophage by either specialized or generalized transduction. Specialized transduction occurs when host D N A encoding one or more specific genes is excised with the bacteriophage D N A , because of aberrant excision of a prophage that is integrated into the bacterial chromosome. Generalized transduction occurs when random fragments of host D N A are packaged into the head of the bacteriophage instead of the bacteriophage D N A . The genes that are incorporated or packaged by either method can then be transferred to other cells upon bacteriophage infection, and the D N A can be inserted into the chromosomal D N A by homologous recombination (Campbell, 1996). Bartonella bacilliformis is an example of an organism with virulence genes being transferred between strains by bacteriophage-like particle (BLP) transduction. B. bacilliformis is transmitted by the bite of the female sandfly, and causes Oroya fever (haemolytic type disease) (Barbian and Minnick, 2000). 1.2 Gene transfer agents in prokaryotes A common property to all the B L P s (bacteriophage-like particles or gene transfer agents) described below is that they package an amount of D N A insufficient to encode a complete d s D N A phage, such as X (Campbell, 1996; Hendrix, 1983). Rhodobacter capsulatus G T A packages 4.5 kb of D N A , whereas the gene cluster that encodes its structural components is 15 kb (Lang and Beatty, 2000). It was proposed that the selective advantage afforded to R. capsulatus by evolution of G T A was to prevent damage to the host cell by lysis due to infection 3 by a phage that gave rise to G T A (Redfield, 2001). There are several examples of "constitutive" generalized transduction in distantly related prokaryotes that all engage in genetic exchange between closely related species, so it would seem that there would be a selective advantage for these systems to have evolved. However, it remains to be seen whether the selective pressure was to escape infection and lysis by a phage, or for the purpose of genetic exchange. Several examples of gene transfer agents that exhibit "constitutive" generalized transduction in prokaryotes have been discovered and their properties are listed in Table 1. B. bacilliformis, as mentioned above in Section 1.1, produces B L P s that are 80-nm, tail-less, capsid particles that package 14 kb of linear d sDNA, and is important to the medical community due to the disease Bartonella is responsible for producing. D d l , from Desulfovibrio desulfuricans, packages 13.5 kb fragments of linear d s D N A into a tailed phage structure with head diameter of 43 nm and tail length of 7 nm, and like G T A (see below) is not induced by mitomycin. The spirochete Brachyspira hyodystenteria produces V S H - 1 , which packages 7.5 kb fragments of d s D N A into a tailed phage with a head diameter of 45 nm and a tail length of 64 nm, and is the only gene transfer agent that is inducible by mitomycin (Humphrey, 1997; Matson et al, 2005). Methanococcus voltae, a methanogenic archaebacterium, produces V T A (for voltae transfer agent) which is the most similar bacteriophage-like particle to G T A in that it transfers 4.4 kb of linear d sDNA, is a tailed phage with head diameter of 40 nm and tail length of 61 nm and is not inducible by mitomycin. 1.3 Transduction and gene exchange in R. capsulatus In the early 1970's Barry Marrs was searching for genetic exchange tools for studying photosynthesis in nonsulphur, purple, photosynthetic bacteria (NSPPB) , when he discovered a novel genetic exchange vector that causes generalized transduction between R. capsulatus strains 4 Table 1: Summary of gene transfer agent properties. Agent Host Size and nature of nucleic acid Structure Head diameter, tail length Mi tomyc in induction References G T A Rhodobacter capsulatus 4.5 kb linear d s D N A Tailed phage 30 nm, 50 nm N o (Marrs, 1974; Solioz and Marrs, 1977; Y e n et al., 1979) D d l Desulfovibrio desulfuricans 13.6 kb linear d s D N A Tailed phage 43 nm, 7 nm N o (Rapp and W a l l , 1987) V S H - 1 Brachyspira hyodystenteriae 7.5 kb linear d s D N A Tailed phage 45 nm, 64 nm Yes (Humphrey, 1997) V T A Methanococcus voltae 4.4 kb linear d s D N A Tailed phage 40 nm, 6 1 n m N o (Bertani, 1999; Eiserling et al., 1999) B L P Bartonella bacilliformis 14 kb linear d s D N A Capsid particle 80 nm, N A N o (Barbian and Minnick , 2000) 5 (Marrs, 2002). Rhodobacter species are found in very diverse locations including freshwater, marine, and hypersaline environments as well as soils and paddy fields (Imhoff and Truper, 1989; Imhoff, 1995). They can be grown in the lab under various conditions including anaerobically in the presence of light (photosynthetically), aerobically in the dark, or anaerobically in the dark i f an alternative electron acceptor is present, such as dimethyl sulfoxide ( D M S O ) (Madigan et al., 2003). It is interesting that a bacterium found in such varied environments would have a mechanism for exchanging genetic information, which could further the survival of the population by transfer of an allele that improves fitness when conditions are changed. Marrs was interested in finding tools of genetic exchange to further the study of photosynthesis because the research at that time was limited to studying individual mutants, and so linkage of mutations (genetic maps) could not be obtained (Marrs, 2002). Marrs and his lab members obtained soil and pond samples from a park in Saint Louis from which they isolated N S P P B s by enrichment. From each isolate (i.e. A , B, C, etc.), they selected one spontaneous rifampicin- and one spontaneous streptomycin-resistant mutant, which they mixed in a pair-wise manner (i.e. A r i f + B strep1, A strep1 + B r i f , etc.) and allowed them to grow to stationary phase in liquid culture. They then spread the cultures on plates containing both streptomycin and rifampicin and looked for a double mutant that contained both antibiotic resistance genes. To ensure that the double mutant had not arisen by spontaneous mutation, they also plated each single mutant alone to see how often the second mutation would spontaneously occur. In 1974 MaiTS published the first description of this novel genetic exchange system found in a N S P P B although he had little idea at that time what he had actually discovered (Marrs, 1974). 6 Regardless, this system was soon used to map genes needed for bacteriochorophyll and carotenoid photosynthetic pigment synthesis (Yen and Marrs, 1976). Upon further analysis Marrs found that this system of genetic exchange was not a typical mechanism of D N A exchange as described in Section 1.1 (Marrs, 2002). DNase did not block the genetic transfer, which ruled out transformation of D N A from naturally lysed cells (Marrs, 1974). The genetic material was shed into the medium and the cells did not need to be in contact for the transfer to occur, which ruled out conjugation (Marrs, 1974). The genetic exchange vector, which was named as the gene transfer agent or G T A , behaved as discrete particles that sedimented in sucrose density gradients as a tight band at 70S, which is about the size of a ribosome and much smaller than any known d s D N A transducing phage (Solioz et al., 1975). A l l genetic markers examined could be transferred and ~4 x 10"4 is the maximum fraction of recipients that can acquire a given genetic marker, which is similar to other generalized transduction systems (Solioz et al, 1975; Solioz and Marrs, 1977). However, every G T A particle is thought to be capable of transduction, in contrast to generalized transduction systems, where only abnormal particles perform generalized transduction (Solioz et al., 1975). The properties described above, along with there being no plaques observed and no apparent cell lysis upon production of the particles, rules out G T A as being a typical phage-mediated generalized transduction system (Marrs, 1974). Yen et al. (Yen et al., 1979) isolated a G T A overproducing mutant strain, Y262, which aided in the further characterization of G T A because of the larger amounts of G T A particles. Isolation of D N A from the G T A particles revealed that this D N A is linear, double stranded, approximately 4.5 kbp in length and randomly packaged, as restriction digests of the D N A isolated from G T A showed similar patterns to restriction digests of chromosomal D N A from R. 1 capsulatus (Yen et al., 1979). A n electron micrograph of G T A particles was obtained, shown in Figure 2, which showed that G T A resembles a small, tailed bacteriophage, with a head diameter of approximately 30 nm, and a tail length of approximately 50 nm (Yen et al., 1979). This is in contrast to the d s D N A (48.5 kb) tailed phage X, which has a head diameter of 50 nm and a tail length of 150 nm (Hendrix, 1983). Although most of the physical characteristics were discovered for G T A at this time, little genetic information was known about G T A , and it was still not known how production of G T A was regulated. 1.4 Structural genes encoding GTA in R. capsulatus In 2000, through the use of a transposon-mutagenized library, Lang and Beatty (2000; 2001) found a gene cluster encoding the G T A structural genes within the R. capsulatus genome. Although the R. capsulatus genome includes a plasmid (http://www.ergo-light.com/ERGO/), the G T A genes are located in the single, 4.4 M b circular chromosome, and are flanked by typical bacterial genes (Figure 3). The G T A structural gene cluster is -15 kb in length, encodes 15 open reading frames (orfgJ to orfglS), and appears to be a polycistronic operon under the control of a single promoter (Lang and Beatty, 2000, 2001). Several of the open reading frames (ORFs) in the G T A gene cluster are homologous to known phage genes, such as the Streptomyces phage (j)C31 portal (orfg3) and capsid {orfg5) proteins, the E. coli T4 phage D N A packaging protein (orfgT), and the E. coli H K 9 7 phage prohead protease protein (prfg4) (Lang and Beatty, 2000). The G T A ORFs are similar only to phage structural genes and no homologues to phage replication or regulatory genes were found. A n interesting characteristic of the structural gene cluster for G T A is that the codon usage matches the codon usage of highly expressed R. capsulatus photosynthesis genes, implying that this cluster has been a part of the R. capsulatus genome longer than two putative prophages that are found in the R. capsulatus genome, whose 8 1— 1 lOOnm Figure 2 : Electron micrograph of G T A negatively stained with phosphotungstic acid. Taken from (Yen etal, 1979). 9 Figure 3: G T A structural gene cluster, cckA, ctrA and surrounding genes. Genes believed or known to be involved in G T A production are shown in blue, with G T A structural genes in dark blue and regulatory genes in light blue. Surrounding genes similar to motility (flagellar and chemotaxis) genes are shown in orange, genes similar to cellular genes of other known function are shown in yellow, genes similar to genes of unknown function are shown in red, and genes with no similarities are shown in purple. RRC03521 is annotated as a 3-oxoacyl- (acyl carrier protein) synthase while RRC03499 is a predicted serine acetyltransferase, Boxes drawn above the line represent genes oriented from left to right (5" to 3") and boxes drawn below the line represent genes oriented from right to left (3* to 5*). Modified from Lang and Beatty (2001). 10 codon usage is different (Lang and Beatty, 2000). Fifteen kb, however, is thought to be too short to encode a complete phage genome, as the complete genomes for H K 9 7 and cbC31 are approximately 40 kb. The head-to-tail structural regions of the H K 9 7 and 4>C31 genomes are 19 kb and 17 kb respectively (Smith et al, 1999), which are close in size to the G T A structural gene cluster. Since 15 genes is thought to be too few to encode a complete tailed d sDNA phage genome, it is possible that G T A uses regulatory genes from two prophages in the R. capsulatus genome (Haselkorn et al., 2001), or that there are other genes involved in G T A production that are yet to be found. 1.5 GTA regulatory genes in R. capsulatus In addition to the discovery of the G T A structural gene cluster, three genes have been found that encode proteins which regulate the expression of the G T A structural genes: Gtal , CtrA, and C c k A . Gtal is involved in quorum sensing, while CtrA and C c k A may be part of a sensor kinase (CckA)/response regulator (CtrA)-signalhng pathway, as close homologues of these two proteins have been characterized in Caulobacter crescentus (Jacobs et al., 2003; Lang and Beatty, 2000; Schaefer et al, 2002). The gtal gene encodes a long-chain acyl-homoserine lactone (acyl-HSL) synthase. A c y l -H S L s are associated with quorum sensing, where acyl-HSLs are secreted by cells into the medium and then used as signals for gene expression when taken up by other cells in the population. When gtal is mutated, G T A production decreases. Conversely, when exogenous acyl-HSL is added to the mutant culture, G T A production returns to normal levels. G T A levels are highest at stationary phase, when the cells are at the highest density, and so this is consistent with the discovery that quorum sensing regulates G T A activity (Schaefer et al, 2002). 11 The R. capsulatus ctrA gene product acts to positively regulate expression of the G T A structural gene cluster and is predicted to encode a response regulator protein (Lang and Beatty, 2000, 2002). Lang and Beatty (2000) inserted a K I X X cartridge (which contains the neo gene encoding neomycin/kanamycin phosphotransferase) into ctrA, which reduced G T A gene expression to an undetectable level (>103-fold). The C. crescentus and R. capsulatus ctrA genes have 71% identity and since the predicted D N A recognition helix-tum-helix of C. crescentus CtrA is identical to the corresponding region of the R. capsulatus C t rA sequence, Lang and Beatty assumed that the binding sites would be the same as well. By comparing the known C. crescentus CtrA-binding consensus sequence to the sequence upstream of the R. capsulatus ctrA gene, two putative binding sites for CtrA were found, suggesting that CtrA is self-regulated. However, as no predicted Ct rA binding sites were found upstream o f the G T A structural gene cluster, it was thought that there must be another factor that interacts with the G T A gene cluster promoter region or a different binding site for CtrA is used. That is, perhaps CtrA activates expression of another gene, or transfers a phosphate to another protein, which then binds to the G T A structural gene promoter region to activate transcription (Lang and Beatty, 2000). It is also possible that Ct rA binds to a non-canonical sequence in the G T A promoter, as appears to be the case for a minor fraction of CtrA-regulated genes in C. crescentus (Laub et al., 2002). The R. capsulatus cckA gene is also required for maximal expression of the G T A structural gene cluster, and is predicted to encode a sensor kinase protein which has 46 % identity (61 % similarity) to the C. crescentus C c k A protein (Lang and Beatty, 2000, 2002). In C. crescentus, the C c k A protein phosphorylates (activates for D N A binding) Ct rA (Jacobs et al., 2003). Therefore it was assumed that in R. capsulatus C c k A phosphorylates Ct rA as part of a sensor kinase/response regulator-signalling pathway to induce G T A gene expression (Lang and Beatty, 2000). A s explained in more detail below, when cckA is mutated, the level of G T A gene 12 expression and G T A activity decreases (50% decrease for expression of a plasmid-borne lacZ reporter driven by the G T A promoter, and 99% decrease for G T A transduction). But this decrease is not as low as in the ctrA mutant (85% decrease for expression of a plasmid-bome lacZ reporter driven by the G T A promoter and 100%) decrease for G T A transduction). This shows that C c k A is not as important as CtrA to G T A gene expression, and that induction of G T A gene expression is more complicated than a single system in which C c k A senses a signal to cause autophosphorylation and transfer of phosphate to CtrA (Lang and Beatty, 2001). It is also possible that C c k A acts in a different pathway than CtrA, and that there is more than one sensor kinase that activates CtrA. The 550 bp sequence located 5' of the second gene in the G T A structural gene cluster, orfg2, was identified as the promoter region, on the basis of a translationally in-frame fusion of orfg2 to the E. coli lacZ gene in plasmid p Y P (Lang and Beatty, 2000). It was assumed that the promoter is located somewhere in the 550 bp region 5' of orfgl. Since there are no canonical CtrA binding sites in the promoter region of the G T A gene cluster, it was thought that there must be another factor that is acted upon by CtrA. It was suggested that since a sequence similar to an R N A polymerase a-factor binding site (-10 promoter sequence) is found in the G T A structural gene promoter region, the activation of an alternative (stationary-phase) a-factor, or other transcription factor, is regulated by CtrA. This other factor would then directly enhance G T A structural gene expression (Lang and Beatty, 2001). 1.6 Overview of the thesis I hypothesized that there are G T A regulatory proteins other than Gtal and C c k A , since mutations in the genes for these proteins reduce G T A structural gene transcription and transduction activity, but do not completely abolish both of these activities. I hypothesized that 13 there are other proteins involved in the C c k A / C t r A pathway because: 1) a CtrA consensus binding site in the G T A promoter region has not been found; 2) the cckA knockout reduces G T A gene expression less severely than the ctrA knockout. I intended to identify these proteins that are in the same pathway as CtrA and C c k A , or that act independently to regulate G T A structural gene expression (such as a sigma factor or transcription factor), and that the predicted properties of these proteins would elucidate how G T A is regulated in R. capsulatus. M y initial approach was to use the plasmid p Y P (described in Materials and Methods and (Lang and Beatty, 2000) that drives lacZ expression from the G T A structural gene cluster promoter region to screen a R. capsulatus library of transposon mutants, and identify new genes needed to activate G T A gene expression. However, as my research progressed, I discovered multiple, interesting complexities that led me into new areas. I found and DNA-sequenced several mutations that reduced expression of the lacZ allele on p Y P , and I also discovered that the p Y P plasmid itself greatly reduced G T A production. In addition to the G T A structural gene promoter region, p Y P contains orfgl and a segment of orfgl fused translationally in-frame to the lacZ reporter gene (see Materials and Methods). Thus it was not clear whether it was the extra copies (~5 per cell) of the G T A structural gene promoter region on p Y P or the over-expression of orfgl, which reduced G T A expression in host cells. As the reasons for the effects of the transposon mutations on G T A gene expression were unclear, whereas the reduction in G T A production resulting from the presence of p Y P in cells appeared to be more amenable to experimental analysis, I changed the focus of my research. The new focus was to better localize the G T A structural gene cluster promoter, and evaluate the sequences on p Y P that reduce G T A production when that plasmid is present. This thesis summarizes my initial work on using the plasmid p Y P to identify transposon mutations of genes that appear to be needed for maximal expression of G T A structural genes. 14 Subsequent experiments were done on the G T A structural gene promoter region, to clarify whether the p Y P effect arises from the expression of G T A genes (orfgJ and orfg2::lacZ), in pYP, or the presence of activator binding sites in several copies relative to the chromosome. 1 also mapped the 5' ends of R N A s to two specific nucleotides in the promoter region, and propose -10 and -35 promoter sequences. Additionally, I used an immunoassay based on western blots of intact cells and culture supernatants probed with anti-GTA capsid protein antisera (Taylor, 2004), to evaluate the production of this protein (and by inference, production of G T A particles) in cultures of several strains of R. capsulatus. The data I obtained improve the understanding of G T A gene expression in terms of cellular responses to culture conditions, and identify likely -10 and -35 regions of the G T A promoter. 15 2 Materials and Methods 2.1 Bacterial Strains, plasmids and growth conditions The strains and plasmids used are described in Table 2. E. coli strains were grown in Luria-Bertani (LB) medium supplemented with 150 pg/ml ampicillin, 10 ug/ml tetracycline-HCl or 25 pg/ml kanamycin sulphate, as needed for selection for plasmids. R. capsulatus strains were grown aerobically in R C V minimal medium (Beatty and Gest, 1981) or photosynthetically in yeast extract/peptone/salts (YPS) medium (Wall et al., 1975) supplemented with either 0.5 ug/ml tetracycline-HCl or 10 ug/ml kanamycin sulphate, as needed for plasmid or mutant selection. BIO is a wild type strain of R. capsulatus that is rifampicin sensitive (Marrs, 1974) while SB 1003 is a rifampicin resistant strain of R. capsulatus created in a B10 derivative strain, B100 by G T A transduction with a spontaneous rifampicin resistant strain, BB101 (Yen and Marrs, 1976, 1977). Y262 is a G T A overproducer strain made by nitrosoguanidine mutagenesis of a spontaneous streptomycin-resistant strain, BB103 which is a derivative of B10 (Yen et al., 1979). The exact mutations that make Y262 an overproducer are not known, although it is assumed there are at least two separate mutations because two G T A transductions from Y262 were required to convert SB 1003 into a G T A overproducer (Yen et al, 1979). The strains Y K K R 2 (cck::KIXX) and Y C K F 2 (ctrA::KIXX), created in Y262(Lang, 2000; Lang and Beatty, 2000), were used as controls to compare to the G T A activity of the new mutants described later. The strain IKOI (gtaE), which was created in SB 1003, is a knockout mutant deficient in quorum sensing and G T A activity (Schaefer et al, 2002). 16 Table 2: Strains used in this study. Strain Genotype R. capsulatus BIO wild type Y262 Unknown mutations SB 1003 rif-10 Y C K F 2 ctrA::YAXX Y K K R 2 cckA::¥AXX IKOI gtalr.Q D W 5 puh A E. coli DH10B SI 7-1 Phenotype Source wild type G T A overproducer Rifampicin resistant, cured of phage Y262 background, no detectable G T A production Y262 background, reduced G T A production SB 1003 background, reduced G T A production, quorum sensing mutant Lacks R C H protein no photosynthetic growth. Cloning strain Cloning strain, capable of conjugation R. capsulatus. to (Marrs, 1974) (Yen etal., 1979) (Yen and Marrs, 1976) (Lang and Beatty, 2000) (Lang, 2000) (Schaefer etal., 2002) (Wong et al., 1996) Invitrogen (Simon etal, 1983) Table 3: Plasmids used for this study. Plasmid Description and Markers Source pXCA601 Promoter probe vector, T c R (Adams etal, 1989) p Y P Y262 G T A structural gene cluster promoter on a PstI to Sail fragment fused to lacZ in p X C A 6 0 1 , T c R (Lang, 2000) pYnP Y262 G T A structural gene cluster orfgl lacking the promoter region (EcoRI-Sal fragment) in p X C A 6 0 1 , T c R .(Lang, 2000) pSTU12 B10 structural gene cluster promoter on a PstI to H i n d l l l fragment fused to lacZ in p X C A 6 0 1 , T c R (Lang, 2000) pX/S5 Subclone for creation of p Y P (Lang, 2000) P 9 H S T U Subclone for creation of pSTU12 (Lang, 2000) pSMFOOl Y262 G T A structural gene cluster promoter deletion fused to lacZ in p X C A 6 0 1 , T c R This work pSMF002 Y262 G T A structural gene cluster promoter deletion fused to lacZ in p X C A 6 0 1 , T c R This work 17 The plasmid pXCA601 (Adams el al., 1989) was used to evaluate promoter activity of various GTA structural gene promoter region fusions to lacZ, using translationally in-frame fusions to the 8th codon of lacZ, using a BamHI site. The inserts fused to lacZm plasmids pYP and pYnP are shown in Figure 4. Plasmid pYP contains a translationally in-frame fusion of the GTA structural gene cluster orfg2' to 'lacZ under the control of the putative GTA promoter, which is presumed to be located in the 550 bp region 5' of the start codon for orfgl and 3' of the PstI site near the 3' end of the gene directly upstream of the GTA gene cluster (RRC03520) (Lang and Beatty, 2000). pYnP contains the same fusion to lacZ as pYP, although its 5' sequence extends only up to the EcoRI site that is located 3' of the start codon for orfgl, (Figure 4). Plasmid pYnP acts as a control to determine the basal level of lacZ expression from the pXCA601 plasmid, but as there is a transcriptional terminator inserted on the 5' side of the BamHI cloning sites in the lacZ gene, there is very little, if any, read through from other genes on the plasmid (Adams et al., 1989). Two GTA structural gene cluster promoter deletions were made to determine a smaller portion of the 1.1 kb predicted promoter region that contains the entire promoter. These are described in greater detail below in Section 2.6. 2.2 Creation and screening of a transposon mutagenized R. capsulatus Y262 library containing pYP A.S. Lang created a transposon-mutagenized library (Lang, 2000; Lang and Beatty, 2000) in Y262, which is a GTA overproducing strain of R. capsulatus, following the method of Simon et al. (1983). S17-l(pSUP2021) was used to transfer pSUP2021 into Y262 as this plasmid carries the Tn5 and cannot replicate in R. capsulatus. The resulting kanamycin resistant Y262 cells have Tn5 randomly inserted in their genome due to transposition events. The library contains approximately 2000 members and is stored in RCV medium (+20% glycerol) at -80°C. 18 Putative Rho-independent transcription terminator RRC03520 j orfgl orfg2' lacZ m Pst\ EcoR\ Sa/I B. orfg2' f f l l lacZ' | | | EcoR\ Sa/I Figure 4: Overview of capsulatus gene sequences present in plasmids p Y P (A) and pYnP (B). 19 Plasmid p Y P (described above) was conjugated into the Y262::Tn5 library so the library could be screened using "blue/white" screening, which in a red (carotenoid) pigmented bacteria, appear as purple/red colonies. The Y262::Tn5 library containing p Y P was grown in R C V medium and plated on R C V plates, containing X-gal (40 ug/ml), kanamycin sulphate (10 ug/ml) to select for Tn5 and tetracycline-HCl (0.5 ug/ml) to select for pYP , at approximately 300 colony forming units (CFUs) per plate. The plates were grown for two days under photosynthetic conditions and red colonies were picked for further analysis, as these wil l have low (3-galactosidase activity and hence low expression of the orfg2::lacZ fusion on pYP. As red to purple is a much more subjective colour difference than white to blue, it was necessary to have various other tests to ensure that the mutation did in fact reduce G T A gene transcription and biological activity. The first step was to make a stab plate of the colonies that appeared to have lower lacZ expression (red colonies) alongside control strains to compare levels of lacZ expression to ensure that it was not simply the size of the colony that made it appear lighter than those around it (Figure 5). The controls used were Y262(pYP) (normal expression; dark purple or blue colonies), Y K K R 2 ( p Y P ) (cckA mutant host with reduced expression; lighter purple colonies), Y C K F 2 ( p Y P ) (ctrA mutant host with low expression; reddish purple colonies), and Y262(pYnP) (no expression; red colonies). After this secondary screen, the potential mutants were then subjected to 3 more quantitative tests (described in detail below) to measure G T A gene expression: 1) (3-galactosidase specific activity (lacZ assays); 2) G T A activity ( G T A transduction bioassay); and 3) G T A capsid production ( G T A immunoassay). 20 1 » 2 *• 3 * 4 * 5 § 6 | # 7 8 ^ ' 9 « t 10 * Figure 5: Stab plate containing X-gal of mutants and control strains with either plasmid pYP or pYnP. Colonies 1-6 are mutants PS1-PS6, all containing plasmid p Y P ; 7 is Y262(pYnP) where pYnP is a lacZ fusion to orfgl that lacks the promoter region; 8 is Y C K F 2 ( p Y P ) which is ctrA'\ 9 is Y K K R 2 ( p Y P ) which is cckA~; and 10 is Y262(pYP) which is the parental strain of all the mutants. 21 2.3 Methods of measuring GTA 2.3.1 p-galactosidase assays measure transcription from the GTA promoter. The P-galactosidase specific activities of strains containing G T A O R F fusions to lacZ were quantified using the same method as Lang and Beatty (Lang and Beatty, 2000). The cultures were harvested at late stationary phase, as determined by monitoring culture turbidity. The culture turbidity was measured by light scattering using a Klett-Summerson photometer (filter #66, red) (Lang, 2000). P-galactosidase activity was determined by a colourometric assay of o-nitrophenol-P-D-galactoside cleavage and is expressed as Mil ler units (Miller, 1992), which are proportional to the increase in o-nitrophenol per minute, as measured by absorbance at 420 nm, per mg of protein. The amount of protein in samples was estimated by Lowry assay (Miller, 1992). Each P-galactosidase assay was performed in triplicate and the absorbance of the colourometric reaction averaged prior to converting to Mi l le r units. 2.3.2 Bioassays to determine frequency of gene transduction. The amount of active G T A produced by the mutant cultures was measured by G T A bioassays. Cultures of mutant strains of R. capsulatus were grown to stationary phase in 17 ml anaerobic screw cap tubes, photosynthetically under high light at 30-35° in Y P S medium with no antibiotics. A n overnight culture of D W 5 , which is a puhA' (reaction center H gene knockout) strain that can no longer grow photosynthetically (Wong et al., 1996), was grown aerobically in R C V at 30°C. The D W 5 cells were pelleted by centrifugation and resuspended in 3.5 ml I X G -buffer (10 m M Tr i s -HCl (pH 8.0), 1 m M M g C l 2 , 1 m M C a C l 2 , 1 m M N a C l , 500 pg/ml bovine serum albumin fraction V (Solioz et al., 1975). To separate G T A from bacterial cells, the 22 cultures were filtered through 0.22 or 0.45 um syringe filters (Millipore, Bedford, M A ) into plastic tubes and kept on ice. Plastic tubes must be used as G T A sticks to glass and decreases the efficiency of the assay. Culture filtrate (0.1 ml), DW5 strain of/?, capsulatus (0.1 ml), and I X G buffer (0.4 ml) were combined and incubated for 1 to 1.5 hours at 35°C with slow shaking. After 1.5 hours, R C V medium (0.9 ml) was added to each tube and incubated for 3-4 more hours at 35°C. The cultures were pelleted in 1.5 ml tubes, the supernatant decanted, and the bacterial cells were resuspended in 0.1 ml R C V . The cells were then plated on R C V or Y P S plates and incubated for 2-3 days in anaerobic Gas-Pak jars ( B B L , B D scientific, Franklin Lakes, NJ) under photosynthetic conditions. As D W 5 is puhA' and cannot grow photosynthetically, only D W 5 cells that receive puhA by G T A mediated transduction wi l l be able to grow photosynthetically. The numbers of puhA+ colonies were then counted on each plate. Control plates were also included that had only D W 5 + G buffer (to ensure that reversions were not occurring) and donor culture filtrate + G buffer (to ensure that all cells were removed by filtration). 2.3.3 GTA immunoassays of the major capsid protein {orfgS). The amount of capsid protein present either in the cell or in the supernatant of culture was measured by immunoblot. The antiserum used to visualize the capsid was obtained from rabbits inoculated with purified capsid protein (Taylor, 2004). The antiserum was crossreacted with sonicated M l 5 E. coli cells to decrease the non-specific binding observed with non-crossreacted antiserum (Taylor, 2004). Cell number was normalized based on the O D 6 6 o of the culture when it was harvested (equivalent of 100 pi of OD 66o=0.071), to ensure that a similar amount of total protein was present, and samples were boiled in 3 X loading dye ( N E B , ) for 5 minutes. 12% acrylamide S D S - P A G E gels with 5% stacking gels (acrylamide/bis 37.5:1) were used to separate the proteins and various cleavage products. Gels were run with the M i n i -23 P R O T E A N II systems (Bio-Rad) according to manufacturer's protocols. The proteins were then transferred to nitrocellulose membranes (Pall Corporation, Pensacola, Florida) by electroblotting at 100 volts for 1.5 hours. The membranes were blocked in 0.5% skim mi lk /TBS-T for 1 hour at room temperature. The membranes were incubated overnight at 4°C on a rocking platform with anti-capsid antiserum (1 in 10000 dilution) in 0.5% skim mi lk /TBS-T. The membranes were rinsed in T B S - T and subsequently washed once for 15 minutes and twice for 5 minutes at room temperature. Incubation in secondary antibody (donkey anti rabbit, H R P , Amersham) was done at room temperature in 0.5% skim mi lk /TBS-T at a 1 in 3000 dilution. Homemade E C L was then used to visualize the membranes (Diaz et al, 1998). The control bands on gels is M , the His-tagged major capsid protein isolated from E. coli, expression plasmid pREP (Taylor, 2004) that was used to make the anti capsid antibodies. A pre-stained ladder (NEB) was also used to estimate protein size. This assay is useful because it enables us to see when capsid is produced in the cell, and when it is released from the cell. Although this assay cannot differentiate whether the capsid protein is part of transductionally active G T A , it yields the maximal amount of G T A that is potentially made. 2.4 Identification of disrupted genes Genomic D N A was isolated from mutant cultures by multiple phenol/chloroform extractions, followed by ethanol precipitations (Sambrook et al., 1989) and 1 pg of D N A was digested with BamHl and iscoRI, or BamRl alone, following the suppliers' protocol (Invitrogen, Carlsbad, C A ) . The digested chromosomal D N A was run on a 1% agarose gel in 0.5X T B E buffer (Sambrook et al., 1989) at 40 volts for 16 hr. The D N A was transferred from the gel to a Biotrans nylon membrane ( ICN, Irvine, C A ) for 1 hr at 80 volts, in 0.5X T B E buffer. The membrane was then UV-crosslinked (BioRad, GS Gene linker) to bind the D N A , dried and 24 probed using the digoxigenin-dUTP (DIG) D N A labelling and detection kit (Roche Applied Science, Laval, Qc) to visualize the fragments that contain the Tn5 insertion. The hybridization and detection protocol supplied with the kit was used. The D N A used as a probe was isolated from p U C 4 K ! X X as a Sma\ fragment as described by Lang and Beatty (2000). This fragment contains the coding sequence of the neo gene which is present in the Tn5 transposon that was used to create the transposon mutant library. Once labelled, the probe was hybridized to the membrane for 16 hr at 68 °C and the hybridization pattern visualized by a colour-producing reaction that occurs on the DIG labelled probe. Figure 6 shows a Southern blot of 2 mutants, L M 4 and D5, as well as Y262 as a control (no Tn5 insertion). As the banding pattern for L M 4 and D5 is different, it is expected that these are two separate Tn5 insertions. There are two bands for each lane as the probe partially hybridizes to the inverted repeat on each side of the Tn5. BamHI cuts once in the middle of the Tn5 while EcoRI does not cut in the Tn5. If the banding pattern for both the BamHI alone and BamHI and EcoRI digested D N A had been the same, it would have meant that there was a BamHI site closer to the Tn5 than an EcoRI site, and pUC19 cut only with BamHI would have been used for cloning of the fragment. Plasmid pUC19 (NEB) was used as a cloning vector and was digested with BamHI and EcoRI, or BamHI alone, following the suppliers' protocol (Invitrogen, Carlsbad, C A ) . Genomic D N A restriction digested as above was then ligated into digested pUC19 using T4 D N A ligase (Invitrogen, Carlsbad, C A ) . These ligated vectors were electroporated into DH10B electrocompetent cells (Invitrogen, Carlsbad, C A ) , and colonies selected on L B agar containing kanamycin sulphate to select for clones that contain the neo gene. Several colonies were picked and the plasmids were isolated using a Qiagen Miniprep kit (Qiagen Sciences, Maryland, B A ) . The University of British Columbia's Nucleic A c i d and Protein synthesis unit (NAPS) 25 "2 •< r r — D o "obc ho O N ro ro M4- -w\ <b ladder 5-B -BamHI -EcoRI BamHl EcoRI <b ladder X CORI + -BamHI + + CO Ban CO am Ban 3 X X —1 ft Figure 6: Southern Blot of digested mutants to determine size and restriction ends of fragments for cloning. Two mutants are shown, L M 4 and D5, as well as Y262 as a negative control. There are no bands in either Y262 lane as there is no Tn5 insertion or neo gene found in that strain. As L M 4 and D5 show different banding patterns from each other, it is expected that these are separate Tn5 insertions. There are two bands in each digest lane as BamHI cuts the Tn5 once, and the neo gene overlaps the inverted repeats at one end of the Tn5, allowing it to bind to both sides of the Tn5 (although weaker on the non-/?eo gene side). Both the L M 4 and D5 fragments are BamHI to EcoRI fragments as opposed to BamHI only fragments, as the size of the bands decreases when cut with EcoRI (they would display the same banding pattern in both lanes i f they were BamHI only fragments). 26 sequenced the plasmids using -47M13, a p U C l 9-sequencing primer, and T N 5 E X T , a Tn5 specific primer (Table 4). The sequences were then sent to the U B i c B L A S T and aligned to the R. capsulatus genome using Generic genome browser version 1.58 (supported by the U . B . C . Bioinformatics Center, University of British Columbia; http: / / l u ri a. cmm t. ub c. ca/c gi -bin/gbrowse_imfi/rhodo). 2.5 Promoter deletions To assess which part of pYP was causing the observed decrease in G T A production, fusions of the RR03520 to orfgl region (Figure 4), varying in length and position, were made in p X C A 6 0 1 . For lacZ in pXCA601 to be translationally in-frame, a BamHI site must be inserted into the coding sequence of a gene such that there is a G A T codon in the G G A T C C BamHI site (Adams et al, 1989). Although there was a discrepancy between annotations as to which of the two potential translational start sites was correct, both are in the same reading frame and so either wi l l work for the fusion. Thus, to fuse orfgl to lacZ, primer G l B a m 2 (Table 4) was used. The first deletion (pSMFOOl) included sequences from the orfgl fusion to a position between the predicted sigma binding site and the predicted rho-independent termination hairpin, using G l P s t l (Table 4) as the second primer to insert a PstI site into the P C R product. The second deletion (pSMF002) was amplified using GlPs t2 (Table 4), which enabled the use of the native PstI site as in the original p Y P . It was thought comparison of the effects of pSMFOOl and pSMF002 to p Y P and pYnP would allow us to differentiate between an effect caused by orfgl and the 5' sequence: The fragments were P C R amplified from p Y P (pSMFOOl) or pX/S5 (pSMF002) and digested by BamHI and PstI for cloning. To facilitate cloning small fragments (220 bp and 520 bp) into a large (20 kb), low copy number vector, the fragments were first subcloned into pUC19 in E. coli. The pUC19 fusions were then digested, dephosphorylated 27 Table 4: Primer sequences and descriptions Name Sequence (5 '-3') Description -47M13 C G C C A G G G T T T T C C C A G T C A C G A C pUC19 primer (NEB) T N 5 E X T G A A C G T T A C C A T G T T A G G A G G T C Tn5 primer to IR ## bp from end. G l B a m 2 A C C C T G G A T C C C C A T G T C C A T G T C T Inserts BamHI site for fusion to lacZ. G l P s t l C G C C T G C A G C A A C C C T G A A T A T A G C Inserts PstI site 220bp upstream of orfgl translation start. GlPs t2 G C G A G C G T C T T T G T C A A C C Amplifies PstI site at the 3' end ofRRC03520. gtaPF G A C G C C C T A T A A C A C C T A T G T C A T sequencing primer used to sequence BIO and Y262 gtaPR A T C C G C A C C C A T T C C G C C sequencing primer used to sequence BIO and Y262 GSP1 C T T G T C T G C G A A G T T T T T C A R . A . C . E . primer for c D N A creation GSP3 G C A A T T T C C C G A T A A A G C T C C T C R . A . C . E . primer for amplification of c D N A 28 using Antarctic phosphatase (NEB) and ligated into digested pXCA601 without any isolation step to separate the fragments from p U C l 9. The ligations were then electroporated into D H 1 OB electrocompetent cells, and screened by restriction digest to ensure the correct fragment was present. Plasmids, pSMFOOl and pSMF002 were conjugated into Y262 using the helper strain HB101(pRK2013) (Ditta et al, 1985). The fusions were then tested by P-galactosidase assay and G T A immunoassay (as described above) to determine the effect they have on G T A gene transcription and G T A production. 2.6 5' R.A.C.E. analysis of mRNA 5' Rapid amplification of c D N A ends (R.A.C.E . ) (Invitrogen, Carlsbad, C A ) was used to determine the transcriptional start site for the G T A orfgJ, and presumably the G T A gene cluster. The protocol supplied with the kit was used (Invitrogen, Carlsbad, C A ) . The R N A used for the procedure was isolated from both late log (280 K U , dense, but still growing exponentially) and early stationary (375 K U , dense, but growth is slowed considerably from log phase) phase cultures using RNeasy mini prep kit following the suppliers protocol (Qiagen, Maryland, B A ? ) . Approximately 5 x 1 0 cells were used for the preps, which yielded -25 ug of R N A . 3-4 ug of this was then used for R . A . C . E . . The R N A was converted to c D N A using superscript II (supplied with R . A . C . E . kit) and primer GSP1 (Table 4); whose position relative to the predicted translational start site for g l is shown in Figure 16. GSP3 (Table 4, Figure 16) was used to amplify the c D N A with the kit supplied reverse primers. 29 3 Results 3.1 The search for GTA regulatory mutants 3.1.1 Screening of the transposon mutagenized library Screening of the Y262::Tn5(pYP) library (described in Materials and Methods) yielded 60 potential mutants that appeared to have reduced (3-galactosidase production driven by the G T A structural gene promoter, which were selected for further screening. Although there were a large number of suspected mutants, many of these were false positives due to the difficulty in distinguishing between "blue and white" in pigmented R. capsulatus. Figure 5 (Materials and Methods) demonstrates this difficulty by comparing several mutants and control strains on a stab plate which contained X-gal . Further investigation of these 60 mutants was conducted through the use of more quantitative assays designed to test both transcription off the G T A promoter using the orfglv.lacZ fusion on p Y P (Figure 4), and G T A transduction using a bioassay. The combined data from these two approaches led me to focus on 12 mutants that appeared to have the greatest reductions in G T A gene expression and transduction. The (3-galactosidase assays were done first to determine mutants with low G T A gene expression from the G T A promoter driving expression of the pYP orfgl:: lacZ gene (data not shown). Tn5 insertions in genes whose products regulate G T A gene expression, either directly or indirectly, would show reduced (3-galactosidase specific activity compared to Y262(pYP) , the G T A overproducer parental strain. Mutants that had ( 3 -galactosidase activity of no more than 50% the (3-galactosidase specific activity of Y262pYP were chosen to be further screened by the G T A bioassay. Figure 7 shows (3-galactosidase expression for 6 mutants, as well as 4 control strains. 30 (pYP) (pYP) (pYP) (pYP) (pYP) (pYP) (PYP) (pYP) ( p Y p ) (pYnP) (ctrA-) (cckA-) Figure 7: P-galactosidase assays performed in triplicate on 6 mutants (A 13, A24, A27, D3, L M 4 , and P6) all containing the lacZ fusion plasmid p Y P as well as 4 control strains: Y262(pYP), Y C K F 2 ( p Y P ) , Y K K R 2 ( p Y P ) , and Y262(pYnP). Y262 is the parental strain for all my Tn5 mutants, while Y C K F 2 is the ctrA' strain and Y K K R 2 is the cckA' strain. The error bars indicate standard deviation over 2 separate cultures and 2 or 3 dilutions of sonicated cells normalized to protein concentration. 31 Y262(pYP) had the highest level of lacZ expression, which was expected as Y262 is the G T A overproducer strain, and has been shown to contain the highest level of G T A m R N A (Lang, 2000). Y262(pYnP), which contains the expression plasmid with the orfgl r.lacZ fusion minus the promoter region (Figure 4), had undetectable levels of lacZ expression; this was also expected as there is little read through on this reporter vector (Adams et al., 1989). The cckA mutant, Y K K R 2 ( p Y P ) , showed only a 50% decrease compared to Y262(pYP) , while the ctrA mutant, Y C K F 2 ( p Y P ) , and the 6 new mutants had similar levels of lacZ expression, which were approximately 10-15 % that of Y262(pYP). The G T A bioassay measures G T A transduction activity based on the transfer of an essential allele {puhA) for growth under photosynthetic conditions to the DW5 puhA mutant (Wong et al., 1996). Figure 8 shows G T A bioassay results for 37 mutants selected from the (3-galactosidase assay secondary screen. The 16 mutants that had less than 10% of the G T A transduction of Y262 were chosen for further analysis. Figure 9 shows G T A bioassay results done in triplicate on the same 6 mutants as in Figure 7, and the variability in results. The most surprising result is that Y262(pYnP) produces more than 200%> more G T A transductants than Y262(pYP), as it was expected that these two strains would produce similar amounts, as discussed further below (Section 3.2.2). The results for the Tn5 mutants show the variability with this transduction assay as the error is sometimes as large as the actual number (i.e. LM4(pYP) ) . The (3-galactosidase data correspond loosely with the data received from the transduction assay, although differences do appear. For example, P6(pYP) had reduced (3-galactosidase specific activity similar to the ctrA mutant Y C K F 2 ( p Y P ) , although P6(pYP) had almost the same amount of G T A transduction activity as Y262(pYP) . 32 E 0 10 20 30 40 50 60 % of Y262 GTA Figure 8: Overview chart of G T A transduction activity for 37 mutants. The values shown for each mutant are the % of Y262 G T A transduction (typically ~10 3 colonies). 33 800 700 1 600 > CD J 500 | 400 </> c A 1 3 A 2 4 A 2 7 D3 L M 4 P 6 Y C K F 2 Y K K R 2 Y262 Y262 (pYP) (pYP) (pYP) (pYP) (pYP) (pYP) (pYP) (pYP) (pYP) (pYnP) (ctrA-) (cckA-) Figure 9: G T A assays performed in triplicate (described in Materials and Methods) on 6 mutants and 4 control strains. The # o f transduction events is equal to the number of colonies o f the photosynthesis incompetent mutant D W 5 that arose on plates incubated under condition requiring photosynthetic growth, indicating that D W 5 received the mutant allele from the G T A donor by G T A transduction. Error bars indicate standard deviation. 34 3.1.2 Studies on 16 mutants found to have reduced GTA expression The Tn5 insertion point in each of 16 mutants was mapped using Southern blotting, followed by D N A sequencing and B L A S T analysis of clearly unique mutants to determine which gene was disrupted, and therefore causing the observed effect. Figure 6 shows how Southern blots of restriction digested chromosomal D N A of the mutants determined i f each insertion was unique, which reduced the number of mutants from 16 to 12, as 4 had similar banding patterns to others indicating that the transposon was inserted in the same spot. The D N A fragments containing the neo gene and flanking R. capsulatus sequences were then cloned into pUC19 and sequenced using a standard pUC19 primer and a primer to the inverted repeat segment of the Tn5 (Tn5EXT, Table 4). The genes which were disrupted in these twelve mutants are listed in Table 5. While several of these transposon insertions were in hypothetical genes, some were in interesting putative genes. However, because of the great variety of Tn5-disrupted genes, none of which are obviously a sigma factor or a transcription regulator, it is difficult to interpret these results. It seems that many genes of uncertain function significantly affect G T A structural gene transcription, and transduction. Furthermore, some of these Tn5 insertions appear to be in a gene located 5' o f other co-transcribed genes which could result in a phenotype due to a polar effect on an operon, and not solely due to the mutated gene. Therefore additional experiments would have to be done to unambiguously correlate specific genes to the observed phenotype, and to determine the genuine biological function of specific genes. 35 Table 5: 12 mutants and their corresponding disrupted genes and annotated gene function. mutant Disrupted O R F 1 Annotated or predicted gene function1 A l 3513 Phage Prohead Protease - G T A gene cluster A 1 3 * 2344 Sensory transduction protein/histidine kinase A24* 337 Hypothetical protein W (LytR/AlgR family response regulator) A25 2660 hypothetical protein (RecB family exonucleases) A27* 1308 Hypothetical cytosolic protein A28 4035 Membrane-bound Lytic Murein Transglycosylase B D3* 4708 ** (similarity to 2-component hybrid sensor and regulators) L M 4 * 1241 Omega-amino acid-pyruvate aminotransferase PI 4540 4-aminobutyrate aminotransferase P4 2693 Type III Restriction-Modification System Methylation Subunit P6* 2590 hypothetical protein in phage cluster PS11 1930 Type I restriction-modification system restriction subunit 1. O R F number and annotation from http://www.ergo-light.com/ERGO/ * indicates G T A transduction and gene expression data for these 6 mutants shown in Figures 7 and 8. ** indicates no annotation for this gene. 36 3.2 Western blot analyses of GTA capsid protein levels A n anti-capsid (Orfg5) antiserum was used to probe western blots of intact cells and cell-free culture supernatants to evaluate the relative amounts of this capsid protein as an index of G T A gene expression and release of G T A particles from cells (Taylor, 2004). The antiserum was found to bind to a number of proteins (bands in blots of S D S - P A G E ) in intact cells, but the use of the orfg5 Tn5 knockout A l (see Table 5) as a negative control simplified the analysis. Figure 10A shows a western blot that compares A l ( p Y P ) and Y262, while Figure 10B gives the same western blot, but with the bands present in A l subtracted from Y262, using Adobe Photoshop (© 1990-2002, Adobe Systems Incorporated). The His-tagged recombinant capsid protein expressed from the pREP expression plasmid in E. coli M l 5 cells, is included as an additional control. As with homologous capsid proteins of d s D N A tailed phage (Duda et al, 1995), this G T A capsid protein is made as a pro-protein of 42.2 kDa that is cleaved on the C-terminal side of a lysine residue to produce the 26 kDa mature capsid protein found in G T A particles (Lang and Beatty, 2000). This cleavage is thought to be done by the protease encoded by orfg4 o f the G T A structural gene cluster (Figure 3). In contrast, the recombinant His-tagged protein produced in E. coli is made as a 43.1 kDa pro-protein, but it is thought to be cleaved by a chemotrypsin-like protease to yield a 36.3 kDa species, which would explain the lower band in the immunoblots (Taylor, 2004). Mass spectrometry was done on both of these bands to establish their identity, which is in fact the product of orfg5 (Taylor, 2004). Thus the recombinant protein yields two prominent capsid bands at 43.1 and 36.3 kDa, whereas intact cells of R. capsulatus yield the 42.2 kDa pro-protein and 26 kDa mature protein bands (FigurelO), and cell-free culture supernatants contain the 26 kDa mature protein. 37 A. 19 hrs 68 hrs A l ( p Y P ) M 42 kDa 26 kDa B. 42 kDa 26 kDa Figure 10: G T A immunoassays: resolving the extra bands. A , original immunoblots of Y262(pYP) whole cell samples at 19 and 68 hours o f growth, A l ( p Y P ) which is the Tn5 capsid mutant, and M (recombinant, His-tagged Orfg5 purified from E. coli). B, Photoshop altered version of immunoblots from A . B y copying the A l ( p Y P ) lane, inversing the colour, and overlaying it onto the other lanes, it is possible to cancel out bands that appear in both lanes. Bands assigned to the 42 kDa proprotein and 26 kDa mature Orfg5 capsid protein are labelled. 38 3.2.1 The pYP effect While testing strains in the G T A anti-capsid (orfg5) immunoassay and the G T A bioassay, it was discovered that Y262(pYP) makes reduced amounts of G T A , although Y262(pYnP) makes amounts equal to Y262. Y262(pYnP) was cured of its plasmid by repeated subculture in the absence of tetracycline and tested to ensure that Y262 had not reverted, and plasmid p Y P was re-introduced. However, the same results were obtained from this new Y262(pYP) strain. Figure 11 shows western blots of Y262(pYnP), the Y262 that was cured of pYnP and the reconstructed Y262(pYP), probed with the anti-capsid antiserum, which proves that it is p Y P that causes the observed decrease in G T A production. There are three general possibilities that could cause this decrease in the presence of plasmid p Y P . The first possibility is that regulation of the G T A gene cluster is tightly regulated and additional copies of an activator-binding site on pYP could be binding an activator protein, resulting in less of this protein being available to bind to the chromosomal promoter. This hypothesis seems plausible as there is readily detectable lacZ expression in Y262(pYP) (Figure 7), which is regulated by the culture growth phase (Lang and Beatty, 2000); however, as there is reduced production of G T A in Y262(pYP), this would indicate reduced expression of the chromosomal G T A structural genes. Another possibility is that the expression of orfgl on p Y P , and/or the expression of the orfg2 segment fused to lacZ, inhibits transcription of the G T A gene cluster. Lastly, overproduction of Orfgl and/or the Orfg2::LacZ fusion protein from p Y P may interfere with a posttranscriptional step in G T A production, such as assembly of the mature G T A particles. This is possible as there is some capsid pro-protein seen in the Y262(pYP) lane, although very little seems to be cleaved into mature capsid, and none is seen outside of the cell (Figure 11). 39 Figure 11: Immunoassay of Y262, Y262(pYP) and Y262(pYnP). probed with anti-capsid (orfgS) antisera. A . shows whole cells; B . shows cell free culture supernatant. The first lane (M) is the His-tagged capsid protein that was purified from E. coli M15 cells and used to create the a-capsid antiserum, with proteins running at 43.1 kDa and 36.3 kDa. These bands were attributed to the full-length, His-tagged Orfg5, and a cleavage product due to an unidentified E. coli protease (Taylor, 2004). For Y262(pYnP), Y262(pYP) and Y262, the prominent band in both the supernatant and the cell is 26 kDa, The 42.2 kDa Orfg5 protein was reported to be cleaved by an R. capsulatus protease, most likely the orjg4 product, to yield the 26 kDa protein in the process of G T A particle maturation (Lang and Beatty, 2000). 40 3.2.2 Time course analysis of capsid production in Y262 and B10 A bioassay comparison of G T A transduction between Y262 ( G T A over-producer) (Yen et al., 1979) and BIO (wild type) (Marrs, 1974) indicated a difference of 3 orders of magnitude. However, nothing was known about the relative kinetics of the intracellular and extracellular production of G T A in these two strains. Therefore I used the capsid immunoassay to investigate these questions, over a growth curve in which Y262 and BIO grew identically (Figure 12A). B y comparing capsid production and release over the growth curve for these two strains, the difference is very noticeable as seen in Figure 12. In BIO, both the capsid precursor and cleaved product appeared to be present in the cell in very low amounts starting at 21 hours after inoculation of the culture, and cell-free G T A was undetectable at all time points. However, in Y262, capsid production was observed in cells starting in late-log phase (19 hours post inoculation), with capsid release in later-log/ early stationary phase (21 hours). It is interesting to note that there appears to be less G T A present in the supernatant at 72 hours post inoculation, but that could be due to incorrect loading of the sample or a bubble between the gel and the membrane not allowing proper transfer instead of a decrease in the amount of cell-free G T A particles. These results confirm that the increase in G T A transduction by Y262 over BIO is due to an increase in the number of G T A particles produced in Y262. The relative intracellular amounts of the precursor and cleaved products in Y262 compared to BIO may be due to one or more mutations in Y262 that causes the over expression of a positive regulator of the G T A gene cluster, therefore allowing more G T A to be produced by each cell. Thus the difference in G T A transduction frequency between BIO and Y262 is due to an increase in G T A structural gene transcription/translation in Y262, and not due to increased release of G T A from cells. 41 A . 1 0 B. 15 17 19 21 23.5 25 36 48 72 hrs M C. 15 17 19 21 23.5 25 36 48 72 hrs M c e l l , 26 k D a HI — sup. m 26 kDa mm •» — -43 kDa Figure 12: Time course of Y262 capsid production compared to BK) . A , the growth curve and time points when samples were taken for Y262 and BIO. B , immunoblots of Y262 cells (top) and cell free culture supernatant (bottom). C, immunoblots of BIO cells (top) and cell free culture supernatants (bottom). Anti capsid antiserum was used to visualize capsid, and therefore G T A in samples. Cel l numbers loaded per sample were normalized as described in Materials and Methods. 42 3.2.3 Time course analysis of capsid production in ctrA' and cckA' strains Recall that the cckA mutant, Y K K . R 2 , had reduced G T A transduction and (3-galactosidase expression from p Y P , although not as reduced as the ctrA mutant, Y C K F 2 (see Figures 7 and 9). B y comparing these two regulatory mutant strains to the overproducing strain in western blots, it was possible to see differences between capsid production and release in these strains over a growth curve (Figure 13). For Y C K F 2 {ctrA"), there appears to be very little, i f any capsid produced in the cell (Figure 13 B)or released out of the cell (data not shown). However, for Y K K R 2 , there appears to be a lot of both the capsid proprotein as well as the mature capsid protein within cells (Figure 13 C), however this mature capsid protein was never seen in the supernatant of the cultures (data not shown). When Y K K R 2 is compared side by side with Y262, and the same, amounts of cells are loaded for each strain, there is more of the proprotein present in the cell, as well as mature capsid protein (Figure 13 D). This is consistent with the results obtained earlier that show that Y K K R 2 ( p Y P ) has only 50% of the (3-galactosidase specific activity of Y262(pYP) and very little G T A transduction is detected for Y K K R 2 (Figures 7 and 9). From this is would appear that C c k A is somehow involved in the maturation of the G T A or release of the G T A particles from the cell. It would be interesting to determine i f the mature capsid protein seen in the cell is part of mature G T A particles, or i f the reason that Y K K R 2 rarely shows G T A transduction is due to a deficiency in a regulatory element that promotes G T A assembly. 43 0.0 10.0 20.0 30.0 40.0 50.0 60.0 Time (hrs) YCKF2 - x - YKKR2 B . M 13.5 15 17.5 19 21 23 26.5 49.5 68 hrs C.I3.5 15 17.5 19 21 23 26.5 49.5 68hrs M 42 kDa 26 kDa mm ••<* 4m ffll <M> _ _ ^ H M b 4 m «u» ^ » MW Y262 YCKF2 YKKR2 M D. 40 kDa _ . , ^ i m k t 43/42 kDa 25 kDa =— « B P ' 26 kDa Figure 13: Time course o f capsid production in ctrA' and cckA' mutant whole cell samples. A , growth curve and time points when samples were taken. B, immunoblots of Y C K F 2 (ctrA') and C, Y K K R 2 (cckA). D, a comparison of cell samples for Y262, Y C K F 2 , and Y K K R 2 at the 68 hour time point. Only cell samples are shown as there is no capsid detected in the supernatant for either Y C K F 2 or Y K X R 2 . Anti-capsid antiserum was used for the immunoblots to visualize capsid, and therefore G T A in the samples. Cel l numbers loaded per sample were normalized as described in Materials and Methods. 44 3.2.4 Immunoblots of B10, SB1003 and IKOI BIO is a wild type strain of R. capsulatus that produces low levels of G T A compared to the overproducer Y262 (see above). SB1003 is the strain of R. capsulatus used for genome sequencing (http://www.ergo-light.com/ERGO/). IKOI is a derivative of SB1003 which has the gtal gene knocked out, which reduces G T A production compared to the parental strain (Schaefer et al., 2002). B y comparing G T A immunoassay results for these three strains in Figure 14, it appears that SB1003 and IKOI produce and release more capsid than BIO. The amounts of both the precursor and the mature capsid protein appear to be similar in BIO and SB1003 cells (Figure 14A), however the amounts of capsid found in the cell-free supernatant of the culture is very much higher in the SB1003 sample than the BIO sample. IKOI also has greater levels of capsid than BIO in the cell samples as well as the cell-free supernatant samples (Figure 14B). It appears that IKOI has more capsid precursor protein as well as more mature capsid protein than BIO in the cell, and it also has more mature capsid in the cell-free supernatant as well (Figure 14B). The capsid levels seen in IKOI samples are reminiscent of the levels seen in Y K K R 2 (cckA') where there seems to be a buildup of both capsid precursor and mature capsid protein within cells, but low levels of G T A transduction activity (Schaefer et al., 2002). The reason for these observed differences is unknown, as the only known difference between BIO and SB1003 is that SB1003 is rifampicin resistant (Yen and Marrs, 1976). SB1003 was created from a poorly characterized "phage-free" BIO derivative called B100, with rifampicin resistance being transferred by GTA-mediated transduction to B100 from another BIO derived strain (Solioz, 1975; Yen and Marrs, 1976, 1977). It has been observed that SB 1003 produces greater amounts of photosynthetic complexes than BIO (J.T. Beatty, personal 45 Figure 14: BIO capsid production compared to SB 1003 and IKOI. A . shows immunoblots of BIO and SB 1003 both cell (upper blot) and cell free supernatant (lower blot) samples. B . shows immunoblots of BIO and IKOI (gtal) both cell (upper blot) and cell free supernatant (lower blot) samples. Anti-capsid antiserum was used for the immunoblots to visualize capsid, and therefore G T A in the samples. Cell numbers loaded per sample were normalized as described in Materials and Methods. 46 communication), but it is not clear whether this is due to the rifampicin-resistant allele or to another, unknown, genetic difference. Further experiments, such as transduction bioassays, would need to be performed to determine i f the amounts of this capsid protein seen in the SB 1003 immunoassay correspond to the numbers of functional G T A particles. 3.3 GTA promoter region analysis 3.3.1 DNA sequencing Fragments of the G T A structural gene cluster promoter region were sequenced from two different strains (Y262 and BIO) to determine whether the difference in G T A expression between those two strains might be due to a sequence change in the promoter region. These sequences were then compared to the genome-sequenced strain, SB 1003 (http://www.ergo-light.com/ERGO/). Strain SB 1003, like strain BIO, was thought to not overexpress G T A genes, although my western blot analyses indicated that SB 1003 produces more capsid protein both in the cell and in the cell free supernatant of cultures than B10 (see Section 3.2.3.3). Plasmid pX/S5 (Lang, 2000) was used for sequencing the Y262 G T A structural gene cluster promoter region, as it contains 1.8 kb of sequence from an X h o l site (in RRC03520) to a Sail site (in orfg2, used to create plasmid p Y P fusion to lacZ), which includes the G T A structural gene cluster promoter (Figure 15). Four primers were used to provide overlapping sequences, with 2 (gtaPR and gtaPF: sequences of primers in Table 4) priming in the promoter region, and the other two priming off the plasmid vector (M13 primers F and R). Plasmid p9HSTU (Lang, 2000) was used for sequencing the B10 G T A structural gene cluster promoter region, in the same way as pX/S5 , as p 9 H S T U contains 2.3 kb of sequence from a K p n l site (upstream of RRC03520) to a H i n d l l l site (200 bp downstream of the Sail site 47 P r e d i c t e d R h o -i n d e p e n d e n t t ranscr ip t ion t e rmina tor g t a P R R R C 0 3 5 2 0 \ ID Kpn\ Xho\ Pst\ orfgl orfg2' g t a P F EcoR\ Sa/I Hind\\\ Figure 15: The G T A structural gene cluster promoter: overview. The position of sequencing primers gtaPR and gtaPF are shown with arrows. Further sequencing was done with M l 3 primers as these fragments (Y262 and BIO) are in M l 3 derivative plasmids. For the Y262 promoter, X h o l to Sail was sequenced, and for BIO, K p n l to H i n d l l l was sequenced. For comparison to p Y P and pYnP, see Figure 4. 48 used to create pYP) , which includes the G T A promoter (see Figure 15). Neither the BIO nor the Y262 strain had differences in their G T A gene cluster promoter region from the SB1003 sequence, nor from each other. A total of 1.8 overlapping kb, including part of org!, all of orfgl and part of RRC03520, was sequenced. Thus it appears that the reason for the overexpression of G T A in Y262 is due to one or more mutations in ^raws-active regulatory proteins, as opposed to a czs-active sequence in the G T A structural gene cluster promoter region. This also corresponds with the finding that when plasmid p Y P is used for (3-galactosidase assays in BIO, the levels are not as high as in Y262(pYP) (Lang, 2000). These data also indicate that the increased levels of the G T A capsid protein detected in SB 1003, relative to B10, are due to a trans-active factor. 3.3.2 Softberry's BPROM analysis B P R O M by Softberry is a computer program that was designed to search for bacterial promoters in D N A sequences based on sigma70 promoters, the major E.coli promoter class (http://www.softberry.com/benT.phtml?topic=bprom&group=programs&subgroup=gfindb). The predicted -10 and -35 sites 5' of the G T A orfgl are shown in Figure 16. The -10 site has similarity to sites bound by sigma factor rpoD15 (Gilman et al, 1981). B P R O M also predicts -10 and -35 sites near the start codon for orfgl (Figure 15B), although the -10 sequence does not have a special similarity to any known sigma factor binding site. The problem with B P R O M is that it is biased towards prokaryotes with low % G C , while R. capsulatus has an overall G C content of 65.5%. However the G C content of the predicted G T A structural gene promoter region is 54%, and the G C content of the orfgl-orfgl5 region is 69% (http://www.infobiogen.fr/services/analyseq/cgi-bin/freqsq_in.pl). 49 512309 CGGCTGCAGA CCGATCCGGC GGTGATTTAC GGCGTGACCA AGGGGCAGGG CGTGCTGGGG CGCGGTTTGC 512239 GGCAATCGGA GCTGCGGCGC GAGAGGGGGT A T A A G AO'OTA TGTGATCGAC GGGCTGCCGC CGGGGCCGAT 512169 CTGCAACCCC GGGACGCAGG CGATCCGCGC GGCGCTGAAC CCTGATTCGA CGAAGTTCCT GTATTTCGTG 512099 GCGGACGGCA CCGGCGGGCA TGCTTTTGCC GAGACGATCA CCGAGCATAA r; p :'• f>. O C ' CCGGAACGTC AAAACCCCGG GCGCGCTGGC 512029 GGGAGATCGA GAAGACCCAA AAGC^GGGCG CAAGCGACGG i'.iX'v, O O ~J L A A A C T G A A G G CTTCGTCCGG 511959 GGTTTTTTCT TTTCAGCGGG TGCAACCCTG AATATAGCAC r T G A G T T T G C GAACGCTTSA »AGG:^ AGAGAT 511889 AAGGCATGCT AGGAGAGGTG GGCAAGCGCC GCGGGTGACC 1 (A) TGGACATGGG GTGTGCGCTT T T T T C A T T T C GCTCGTGCGG 511819 ACAGGCATGA GAGGCGGGTC ACGCAAGACA GTTCAAGGGT GGGGACGCTC CTCCGGTGGA 511749 TTTGCTGGAG GSP3 GAGACGGAGG AGCTTTATCG GGAAATTGCC GGGGAACTGG CCCTGGCGAT GAAAGGGGTT 511679 511609 CGCCAGGGCG TGGTGATGGA T ? i V ^ AGGCGAAGGA AGAAAGGGTG GGCCAAGGCC GCCGCGCAGG GSP1 CGTGTTGAAA AACTTCGCAG CGGTCAAGGA ACAAGTTGCC CCTTCGCGCG GGTGTCGGAG GCGTTCCAGA CCGGAAGCGA 511539 GCTTGACCTG GACGCCGCCC GGGCTGAGAT CGGGCGCCGC CTGGCTTGCC TGCGCGACGC CGCAGGAGGT 511469 -OACGCCTTT CTGGGGGGGC TTGGGAACAA 'TGCGCTTTTG GCGCTGCCCT GGATTTTCGA 7"' ^ ATTCTGGGCG 511399 CTGCCGCATC AGCTGCCCCC GGTGGGGGCG TGGAAAAGCT GGGTGATCAT H j GGGCGGGCGC GGCGCGGGCA 511329 AGACCCGGGC CGGGGGGGAA TGGGTGGGGA TGCAGGTCGA GGGGGCGGGT CCGGCCGATG CCGGGCCCGC 511259 GCATCGGGTG GCGCTGGTGG GCGAGACCTT TGATCAGGTG CGCGACGTGA TGATTTTCGG CGAGAGCGGG 511189 ATTTTGGCCT GTTCTCCGCC GGACCGGCGC CCGGAGTGGG AGGCGACGAA GCGGCGGCTG GTCTGGGCGA 511119 ATGGCGCGAC GGCGCAGGCC TATTCGGCGC AGGAGCCCGA GGCGCTGCGC GGGCCGCAAT TCGACGCCGC 511049 CTGGGTCGAC GAGCTGGCGA AATGGAGACG GGCCGAGGAG ACCTGGGACA TGCTGCAATT CGCGCTGCGG 510979 CTGGGCAAGC ATCCGCAGCA GGTGATCACC ACGACGCCGC GCAATGTGGG GGTGCTGAAG GCGATCCTCA H i n d i I I AAAGCTTCCT 510909 ACAACCCCTC GACGGTGGTG ACGCATGCGC CGACCGAGGC GAACCGGGCC TATCTGGCGG Figure 16: The G T A structural gene cluster promoter: sequence view. Restriction site for noted enzymes are in purple. Start codon for noted genes (A=Andrew Lang annotated, E=Ergo annotated) are in green. Stop codon for noted genes are in red. Putative Rho-independent transcription terminator is shown in brown. Sequencing primers, name noted above starting with gta (sequences in Table 4) are in orange. R . A . C . E . primers, name noted above starting with GSP (sequences in Table 4) are in blue. 5' m R N A ends determined by R . A . C . E . are l^f t3^3f f fTOI B P R O M predicted -10 and -35 sites are highlighted in yellow. Proposed -10 and -35 sites based on R . A . C . E . data are highlighted in ljght blue.; The underlined bases G G T T C A were changed to G G A T C C (BamHI site) and the underlined bases G G T G C A were changed to T G C G C A (PstI site). 50 3.3.3 5' end mapping of GTA mRNA (R.A.C.E.) Figure 16 shows the two R N A 5' ends that were determined for the m R N A transcript of orfgl using R . A . C . E . (see Materials and Methods). The first is located 23 bp 5'(upstream) of the stop codon for RRC03520, which encodes a conserved hypothetical protein. The second is 111 bp upstream of the predicted start codon for orfgl , and overlapping the B P R O M predicted -10 sequence. In terms of localization of a promoter, these results may be interpreted in three general ways: 1 ) Both 5' ends arise from transcription initiation (i.e., there are two separate promoters); 2) The 5'-most end arises from transcription initiation, and the other end is the result of posttranscriptional cleavage of this transcript, or of premature termination of the reverse transcriptase reaction; 3) Both 5' ends result from m R N A cleavage, or premature termination of the reverse transcriptase, and the promoter is located closer to the PstI site in RRC03520 (Figure 16). I attempted to distinguish between these possibilities by fusing different lengths of the promoter region to lacZ on plasmid p X C A 6 0 1 , and measuring the relative amounts of P-galactosidase specific activities in Y262 cells that contain these plasmids (see below). 3.3.4 Promoter deletions To determine the minimal sequence required to encode the entire promoter, two plasmids were made (pSMFOOl and pSMF002, described in Material and Methods), which both differ from p Y P by having the translationally in-frame fusion to lacZ at the 5th codon of orfgl. The original p Y P plasmid contained 1.1 kb of sequence, with the fusion to lacZ located 400 bp into the orfg2 coding region, including orfgl and extending in the 5' direction to a PstI site located 320 bp into the upstream putative gene, RRC03520. The short orfgl fusion (160 bp, pSMFOOl) included the predicted sigma binding site but not the predicted rho-independent 51 termination hairpin (Figure 16). This deletion was made to evaluate the minimal sequence required for promoter activity. The longer orfgl fusion (530 bp, pSMF002) used the same PstI site as in p Y P . These two orfgl fusions of varying length in pSMFOOl and pSMF002 were used in attempts to localize the promoter to a smaller region. In parallel, pSMFOOl and pSMF002 were used to investigate sequences that could cause the p Y P effect (reduction of G T A expression; see Section 3.2.2). These two plasmids were introduced into Y262, and the P-galactosidase specific activities of cells grown photosynthetically to late stationary phase were measured. Unfortunately neither of these fusions showed promoter activity that was detectable in the p-galactosidase assay (data not shown). There are three main reasons why this could be: 1) the fusions are not actually in-frame with the lacZ gene due to an error in the cloning procedure; 2) the annotated start codon for orfgl is not the actual start codon and therefore the fusions are not in frame with the correct start codon; 3) the promoter driving expression of the orfg2::lacZ fusion in p Y P is located 3' of the 5 t h codon of orfgl. The D N A sequences of the orfgl::lacZ fusions in pSMFOOl and pSMF002 were determined, and it was found that plasmid pSMF002 contained an insert of non-/?. capsulatus D N A , whereas pSMFOOl contained the expected sequence. 3.3.5 Summary of data and proposed promoter for GTA structural genes. The R . A . C . E . data yield the most reliable data for localization of the G T A structural gene promoter, because the promoter deletions were not functional. Using the 5' most postion of the two m R N A 5' ends detected, it appears that the promoter for the G T A gene cluster is further upstream that what was previously thought, and is located within the upstream conserved hypothetical protein gene (RRC03520; see Figures 3 and 16). The R . A . C . E . data also show that 52 the Softberry B P R O M software is not very accurate when predicting -10 and -35 sites in R. capsulatus, most likely due to a bias towards more AT-r ich sites in E. coli. 3.3.6 Immunoassays of Y262(pSMF001)and Y262(pSMF002) Even though the promoter deletions did not produce any detectable P-galactosidase specific activity, they were tested by G T A immunoassay to see i f the sequences contained in them cause the p Y P effect. Although pSMF002 was not the desired sequence, pSMFOOl showed no apparent p Y P effect (Figure 17). This result does not distinguish between an effect due to the promoter region, or one due to the Orfg2::LacZ fusion protein. However, the blot of Y262(pSTU12) gave more capsid in the cell than Y262(pYP) and these two plasmids differ only in the position of the Orfg2::LacZ fusion (Figure 16 and Table 3). Therefore it appears that it is whether or not the Orfg2::LacZ fusion protein is made (pYP vs. pYnP), or the exact nature of the fusion protein (pYP vs. pSTU12), that affects the level of G T A production (the p Y P effect). 53 M p Y P p Y n P p S M F O O l pSMF002 pSTU12 capsid" M W Figure 17: G T A immunoassay of pSMFOOl and pSMF002 compared to controls. Cell samples are shown in (A), while supernatant samples are shown in (B). A l l plasmids are in Y262, with the capsid" strain being A l ( p Y P ) (Table 5), which has a transposon insertion in orfg5 of the G T A structural gene cluster. Anti-capsid antiserum was used for the immunoblots to visualize capsid, and therefore G T A in the samples. Cell numbers loaded per sample were normalized as described in Materials and Methods. 54 4 Discussion As regulation of GTA in R. capsulatus was so poorly understood, I undertook this research to attempt to elucidate the regulatory pathway or network that determines when GTA is made and released from the cell. Although I faced many problems, and was unsuccessful in determining a single "master element" that regulates GTA, I found a number of genes that appear to be needed for maximal expression of the GTA structural genes. I also studied the promoter region of the GTA structural gene cluster to locate the position of a plausible promoter sequence, and discovered interesting differences in GTA production between several R. capsulatus strains. These results are discussed below in more detail. 4.1 The surprising variety of genes needed for maximal production of GTA. In theory, a promoter fusion to lacZ is a powerful method for finding regulatory mutants that no longer positively or negatively regulate a gene of interest. I found 12 genes that are listed in Table 5 that appear to regulate GTA in R. capsulatus, by using a plasmid construct with the promoter for the GTA structural gene cluster fused to lacZ to screen a library of transposon mutagenized cells. The Tn5 insertion in mutant A l mapped to orfg5 of the GTA structural gene cluster. A l was later used as a capsid" control in immunoblots to resolve the 'extra' bands seen with the anti-capsid antiserum. It was also a nice check to ensure that this system can find mutants that genuinely affect GTA production, although there appears to be some sort of feedback regulation as it would not be expected that the transcription of the GTA gene cluster (as measured by the (3-55 galactosidase activity of pYP) would be affected by a mutation in one of the chromosomally-located structural genes. Four of the Tn5 mutations that were sequenced mapped to genes simply annotated as hypothetical proteins. One large problem I faced while deciphering which genes were involved in G T A regulation was a poorly annotated genome with a large number of genes annotated as hypothetical or conserved. However, upon closer inspection of one of the hypothetical genes (RRC02590, disrupted in mutant P6; Table 5), I found that RRC02590 appears to be in the middle of an apparent phage gene cluster. There are in fact 6 such putative phage gene clusters in the R. capsulatus genome, one of which is the G T A structural gene cluster. Two are annotated as prophages R c M 1 and RcP 1 (named based on the contigs they were originally found on (Haselkom et al., 2001)), and the other 3 appear to be sequences acquired by duplication of the other phage clusters, or the remnants of past prophages. The Tn5 insertion in mutant P6 (Table 5) was mapped to a putative phage gene cluster separate from the G T A gene cluster and the 2 prophages. This is extremely interesting since the G T A structural gene cluster does not appear to contain enough genes to encode a complete d s D N A phage, therefore G T A could be using prophage genes (or remnants) to provide essential functions. Another of the hypothetical proteins (RRC02660, disrupted in mutant A25 ; Table 5) had similarity to a RecB family of exonucleases (e value of 3e-65). In E. coli, the RecB nuclease domain of the R e c B C D nuclease-helicase generates single-strand regions at the ends of D N A duplexes and is though to be involved in horizontal gene transfer (Kowalczykowski et al., 1994). Perhaps RRC02660 is a gene in R. capsulatus that encodes a protein needed for processing of the D N A during maturation of the G T A transducing particles. Several of the Tn5 insertions mapped to genes annotated as sensory transduction or response regulator proteins, which was quite exciting. One of the hypothetical proteins 56 (RRC00337, disrupted in mutant A24; Table 5) had sequence similarity to a response regulator of the L y t R / A l g R family (e value of 3e-08), which could directly or indirectly (as for CtrA) regulate the expression of the G T A structural genes. The Tn5 insertion in mutant A13 (RRC02344; Table 5) is in a putative sensory transduction/histidine kinase gene, and while the gene which contains the Tn5 insertion in mutant D3 is not annotated, I found that this gene has similarity to a 2-component hybrid sensor regulator protein (e value of 5.88e-06). It is possible that these proteins interact with CtrA, C c k A or Gtal as part of a complicated regulatory network that leads to G T A structural gene transcription and/or particle assembly. The Tn5 insertion in mutant P1 did not appear at first to be of interest as it was in an O R F (RRC04540) annotated to encode a 4-aminobutyrate aminotransferase; but upon further inspection I found a putative homoserine kinase gene located directly downstream of this gene, perhaps in an operon. As homoserine lactone has been determined to be part of the regulation of quorum sensing and, directly or indirectly, G T A expression (Schaefer et al, 2002), it is conceivable that this is a kinase needed for phosphorylation of this homoserine lactone as part of G T A regulation. Two of the Tn5 insertions mapped to homologues of restriction modification system subunits, one to a Type 1 system (RRC01930 in mutant PS11; Table 5) and the other to a Type III system (RRC02693 in mutant P4; Table 5). Although G T A particles may be filled with D N A using a headful mechanism whereby the D N A is inserted into the premade capsid head until the head is full, and then the D N A gets cut, it is possible that the D N A is cut using a semi-random restriction modification system that cuts the D N A prior to packaging it into the G T A particle head. Although the annotated putative functions for the 12 genes listed in Table 5 sometimes do not correlate to what we believe should be regulating G T A activity, the similar phenotyp'es of 57 the diverse mutants indicate an extraordinary complexity of this system in R. capsulatus, consistent with the idea that this bacteriophage-like particle is regulated by the cell. This could demonstrate that G T A has been co-evolving with R. capsulatus for a long time to become deeply rooted in cellular processes, as opposed to being a recently acquired prophage that has become defective. M y finding of multiple genes that appear to regulate different aspects of G T A activity also disputes our past assumption that G T A is regulated by a simple two-component system and a quorum sensing system, with just three key regulatory proteins (i.e. CtrA, C c k A and Gtal). It is also interesting to note that some of the genes that I discovered seem to be involved with the regulation of G T A post-transcriptionally, and so it is possible that there is a feedback mechanism that prevents more G T A m R N A from being transcribed i f there is a build-up of unmatured G T A particle components in the cell (as in the cckA' and gtal mutants). 4.2 The western blot approach The G T A immunoassay developed by Taylor (2004) is a great tool for determining what is happening with G T A production. Although my experiments do not determine whether the mature (26 kDa) capsid protein (Orfg5) is part of a complete phage head, it can be assumed that i f this capsid protein has been cleaved, which is the form seen outside of the cell, that it is indeed able to be incorporated into a complete phage head. In the cckA' mutant, however, the mature (26 kDa) capsid protein appears to be made to high amounts in the cell (Figure 13), but it does not appear to be released from the cell, and so indicates that this mutation prevents assembly of the mature G T A particles, or release of G T A from the cell. 58 4.2.1 The pYP effect While testing control strains for G T A activity with and without plasmid p Y P , it was discovered that the presence of pYP reduced G T A transduction activity (measured by the G T A bioassay) and capsid production (measured by G T A immunoassay) in all strains. However, pYnP, a similar construct to p Y P except for the absence of the promoter region for the G T A gene cluster, did not have the same effect on G T A transduction levels (Figure 9), or on capsid levels as measured by the G T A immunoassay (Figure 1 1). Therefore it must be the sequence present in p Y P and absent from the pYnP construct that reduces G T A structural gene expression. The ongoing D N A sequence determination of pSMFOOl and pSMF002 may clarify this question. 4.2.2 Time course analysis of capsid production in Y262 and B10 The production of G T A in R. capsulatus was previously determined to be at the highest amounts in stationary phase, using the bioassay which measures G T A transduction (Yen et al., 1979). The transcription of the G T A gene cluster, measured by monitoring (3-galactosidase specific activity using plasmid p Y P (described in Material and Methods), was shown to increase throughout the growth curve and reach a maximum in late stationary phase (Lang, 2000). The immunoblots of capsid protein in the cell and in cell-free culture supernatant in Figure 12 support the bioassay data, and give us a little more insight into the production and release of G T A in Y262 as well as in BIO. B y comparing the immunoblots of Y262 in Figure 12B to the growth curve shown in Figure 12A, it is apparent that G T A is made in late-log phase, and released very shortly (approximately 2 hours) after it accumulates in the cell. This improves our understanding of when G T A is released from the cell, and adds that the mature capsid protein, and presumably G T A , accumulates-in the cell before being released, as opposed to being released as fast as it is 59 made. As we have no understanding of how G T A is released from the cell, this accumulation of G T A in the cell prior to release could mean three things: 1) that the accumulation of G T A is needed for it to be released, i.e. the release of G T A is regulated by the amount of G T A precursors in the cell; 2) that G T A is released by lysis of a small number of cells in the population and that this accumulation of G T A causes a few cells to lyse and release the G T A into the culture supernatant (recall that no plaques were observed with G T A , meaning that either no cell lysis occurs, or only a small subset of the cells lyse); 3) the assembly of the G T A particles takes a few hours to complete, and that is why we see mature capsid protein 2 hours prior to release of G T A into the culture supernatant. Although it is not possible to determine which of these options is correct, the immunoblot of Y262 over a time-course gives us a better view of when capsid is made and when G T A is released from the cell over the growth curve. B y comparing the amount of capsid seen in Y262 and BIO, we are able to clearly see differences in capsid production, and therefore G T A production in these two strains. Previous work showed that BIO had less G T A transduction activity (Yen et al, 1979), less of G T A orfg4 transcript and lower (3-galactosidase specific activity measured from the lacZ gene fusion on pYP than Y262(Lang, 2000). The immunoblot shown in Figure 12C supports these data, as there is very little capsid protein seen in the BIO cell samples, and no detectable capsid protein in the BIO cell-free culture supernatant samples. Therefore it can be concluded that the reason that Y262 has more G T A transduction activity than BIO is due to an increase in G T A structural gene transcription/translation in Y262, and not due to increased release of G T A from cells. 60 4.2.3 Time course analysis of capsid production in ctrA' and cckA' mutants Two mutant strains, Y C K F 2 (ctrA') and Y K K R 2 (cckA'), known to be defective in G T A transduction, were investigated over a time-course using the G T A immunoassay to attempt to elucidate why these strains have low/undetectable G T A transduction activity. From the immunoblots in Figure 13, it is possible to deduce possible mechanisms that are defective in these two mutants. It was previously thought that CtrA and C c k A were involved in a two component signalling pathway similar to one seen in C. crescentus, but my results indicate that the pathway is more complex than a simple two component system, and it seems as though CtrA and C c k A do not necessarily operate in the same pathway as previously thought. From the immunoblots of Y C K F 2 in Figure 13B, it is clear that very little, i f any capsid is made at any point over the growth curve for this mutant. This is consistent with the (3-galactosidase data from Y C K F 2 ( p Y P ) shown in Figure 7, in which the levels of (3-galactosidase were very low compared to Y262(pYP). This also confirms our previous assumption that CtrA is very important to the regulation of transcription of the G T A structural gene cluster, and that in the Y C K F 2 mutant lacking this protein, there is little, i f any transcription or expression of proteins from the G T A gene cluster. From the immunoblots of Y K K R 2 (cckA mutant) in Figure 13C, it is seen that large amounts of capsid, both the pro-protein and the mature, cleaved protein are produced. However, there is little mature capsid seen outside of the cell, which would explain why Y K K R 2 ' s transduction frequency is almost non-existent. This also correlates with the P-galactosidase data in Figure 7 that shows that the transcription from the G T A promoter is only decreased by approximately 50% in Y K K R 2 ( p Y P ) . These data indicate that C c k A plays a role in post-transcriptional, and even post-translational processing of the G T A structural gene cluster, or in 61 the assembly or release of the mature G T A particles from the cell. 1 no longer assume that C c k A and CtrA are inextricable parts of a two-component system, as two things would need to be occurring for that to work with the data I have shown: 1) C c k A would also have to regulate some other part of G T A assembly or post-translationally processing as described above, as well as activate CtrA for there to be as much capsid seen in the cell, compared to the ctrA mutant; 2) there must be some other sensor kinase protein that can activate CtrA to promote transcription of the G T A structural gene cluster for there to be such a high level of both transcription of the G T A gene cluster (as shown by the [3-galactosidase activity in Figure 7), and expression of the capsid protein (as shown by the immunoblot in Figure 13C) in the cckA' strain. 4.2.4 Immunoblots of B10, SB1003 and IKOI Anther interesting complexity between strains is the paradox of BIO compared to SB1003 and IKOI (the gtal mutant derived from SB1003). It was previously thought that BIO and SB1003 would have similar G T A levels, and that IKOI would have lower levels than both, as SB 1003 is a BIO derivative, and IKOI (quorum sensing mutant) has been shown to be defective in G T A transduction. However, the levels of capsid seen in these 3 strains vary considerably, as shown in Figure 14. Figure 14A compares SB 1003 to B10, in both cell and cell-free culture supernatant samples. The levels of capsid seen in the cell appear similar, but the levels differ considerably in the cell-free culture supernatant samples. In B10, no capsid protein is seen, however in SB 1003, there is quite a bit of the mature capsid seen. SB 1003 is a rifampicin resistant derivative of B10, that was created by G T A transduction from a rifampicin resistant strain and a poorly characterized strain (B100) that was deemed to be cured of lytic phages (Solioz, 1975). It has been observed that SB 1003 produces greater amounts of photosynthetic complexes than B10 62 (J.T. Beatty, personal communication), and so it is possible that the mutation that makes SB 1003 rifampicin resistant (thought to be an amino acid change in the R N A polymerase-subunit that prevents rifampicin from binding to it) could also increase the affinity of the R N A polymerase for certain promoters, such as the photosynthesis gene puf operon and the G T A structural gene cluster. Figure 14B compares BIO to IKOI, which is a quorum sensing mutant (gtaf) that was created in SB 1003 (Schaefer et al, 2002). Again, the levels of capsid compared to B10 are increased, however in this case it is the capsid levels in the cell that are increased the most dramatically. There is some mature capsid protein seen in the cell-free culture supernatant sample, although more than B10, and less than SB1003, which is consistent with the work of Schaefer et al. (2002), who reported that IKOI had reduced G T A transduction activity compared to SB1003. The intracellular levels of capsid are elevated in the IKOI strain, however, and are similar to those seen in Figure 13C for Y K K R 2 (cckA). There appears to be a buildup of both the capsid proprotein and the mature capsid protein, and it is conceivable that the extracellular mature capsid protein seen is due to lysis of cells in the culture rather than active release of the G T A . This is an interesting finding as it seems to support the theory that G T A release is due to lysis of a subset of cells in a population, perhaps due to a quorum sensing signal telling the cells that the culture is overcrowded. It has been observed that photosynthetically grown cultures of IKOI do not enter death phase as readily as Y262 cultures, (i.e. culture densities, as measured by OD660, stay constant for longer than 70 hours post inoculation when Y262 and B10 cultures have started to decline in density; M . Leung, personal communication). Although these immunoblots do not give conclusive results as to what is happening in regards to G T A production and release, they give a better picture of what is occurring in regard 63 to capsid production and processing, especially in the mutant strains Y C K F 2 (ctrA'), Y K K R 2 (cckA') and IKOI (gtaT), compared to control strains. 4.3 GTA structural gene cluster promoter analysis Prior investigation of the promoter for the G T A structural gene cluster was limited to sequencing a short region (470 bp) and looking for a CtrA consensus binding site, although nothing of obvious significance was found in either (Lang, 2000). The promoter for the G T A gene cluster was thought to be located between the start codon for orfgl and the stop codon for the upstream gene (RRC03520), which is not thought to be involved in the G T A gene cluster, although it is transcribed in the same direction (Lang, 2000; Lang and Beatty, 2002). I undertook this promoter analysis research because I was interested in discovering which part of the putative G T A structural gene cluster was responsible for the observed pYP effect that I discovered. D N A sequencing of the putative promoter region was done to discover i f there were any differences between the various strains of R. capsulatus that we knew had different G T A transduction levels. Softberry's B P R O M software was used to predict -10 and -35 sites, rapid amplification of c D N A ends (R.A.C.E . ) was used to determine the 5' ends of the G T A orfgl, and presumably the entire G T A gene cluster. Promoter region deletions were made to attempt to discover the region of the p Y P plasmid fusion that causes the p Y P effect, and the minimal region needed for full promoter activity. 64 4.3.1 Sequence differences between the GTA promoter in Y262, B10 and SB1003 Lang (2000) hypothesized that a m-acting regulatory sequence in the promoter region of the G T A gene cluster caused the observed G T A activity differences between Y262 and BIO. Two promoter fusions, p Y P and pSTU12, were made using D N A directly cloned from two strains that had differing G T A activities, Y262 and BIO respectively, and were tested by Lang for promoter activity in Y262 (Lang, 2000). It was found that the fusion plasmid containing the G T A promoter region from B10, p S T U l 2 , produced unmeasurable levels of P-galactosidase, while the Y262 fusion plasmid, p Y P , produced readily detectable P-galactosidase levels (Lang, 2000). It was previously thought that the reason for the difference in P-galactosidase specific activity between the B10 orfgl'r.'lacZ fusion and the Y262 orfgl':: 'lacZ fusion was due to a exac t ing regulatory sequence that was mutated in Y262 that either prevented a negative regulatory protein from binding or allowed a positive regulatory protein to bind more specifically, which in both cases would cause the up-regulation seen in Y262 compared to B10. However my D N A sequencing data show that there is no difference between strains Y262, B10 or SB1003 over a substantial region encompassing the predicted G T A structural gene cluster promoter. Therefore there must be one or more trans-acting factor(s) mutated in Y262 and SB 1003 to cause the observed increase in production of G T A . To explain the reduced P-galactosidase specific activity seen from the B10 orfgl'r.'lacZ fusion compared to the Y262 orfgl':: 'lacZ fusion (Lang, 2000), it must be the difference between fusion sites that were used to create the two plasmids, p Y P and pSTU12 that cause this difference. That is, although both plasmids contain exactly the same G T A structural gene promoter region, these plasmids differ in the sequence fused translationally in frame to lacZ. Thus the two hybrid P-galactosidase proteins 65 produced contain different N-terminal sequences, both in length and amino acid sequence composition, which probably accounts for the difference in P-galactosidase specific activity. When Y262(pSTU12) was compared to Y262(pYP) using the immunoassay, there was reduced amounts of capsid seen in the supernatant and the cell samples of Y262(pSTU12), although they were not as reduced as in Y262(pYP) (Figure 17). A s the sequences contained within the promoter region and orfgl differ only in the fusion joint of orfg2 to lacZ, it must be the nature of the Orfg2::LacZ fusion that explains the difference in both p-galactosidase specific activity and G T A immunoassay capsid levels. Another interesting finding was that there are equal levels of ctrA m R N A in Y262 and BIO, whereas there is almost no detectable org4 (from the G T A gene cluster) m R N A seen in BIO and a greater amount in Y262 (Lang, 2000). It was hypothesized that this could be explained by there being a mutation in the promoter that would allow a regulatory protein (i.e., CtrA) to bind with higher affinity to the G T A gene cluster promoter in Y262. However as there is no sequence difference in the G T A promoter region between these two strains, there must be a change in the expression or activation of CtrA that causes the difference in G T A transcript levels between the two strains, or a protein other than Ct rA induces the transcription of the G T A structural gene cluster. 4.3.2 Predicted promoter region for GTA: R.A.C.E. and Softberry analysis Softberry's B P R O M software predicted a -10 and -35 site 100 bp upstream of the start codon for orfgl. Although B P R O M is biased towards AT-r ich organisms, which R. capsulatus is not, the putative promoter region for the G T A gene cluster is actually quite A T rich (46%) compared to the average over the genome (32%). The 5' end mapping of orfgl m R N A gave two 5'ends. This could be due to the reverse transcriptase reaction terminating prematurely, two 66 messages with the shorter being cleaved from the longer, or two different promoters initiating transcription. The shorter 5' end overlaps with the B P R O M predicted -10 site. The longer 5' end overlaps gene RRC03520 by 23 bp, and so it is possible that this is a misannotated gene, or that the promoter for G T A is located within the coding sequence of this gene. Multiple promoters within gene coding regions have been documented in R. capsulatus (Wellington and Beatty, 1991; Wellington et al, 1991). From my results, it is not possible to conclusively determine exactly where the promoter for G T A is located, although I suggest that the promoter is in fact further upstream than previously thought, because one of the R N A 5' ends was located 20 bp 5' of the stop codon of the upstream gene, RRC03520. Although more experiments could be preformed to provide conclusive evidence of the exact location of the promoter, I suggest -35 and -10 sites in Figure 16. 4.3.3 Promoter deletions Neither the pSMFOOl nor the pSMF002 lacZ fusion yielded P-galactosidase activity, although it was later discovered that the pSMF002 fusion was non-7?, capsulatus D N A , and therefore would not be expected to have P-galactosidase activity. Since pSMFOOl {orfgl fusion position shown in Figure 16) had no detectable p-galactosidase, it is possible that orfgl is not translated, although the R . A . C . E . data show that orfgl is transcribed. There could be something about the 5 Orfgl amino acids fused to the N-terminus of P-galactosidase in pSMFOOl that inhibits P-galactosidase activity in the fusion protein, but this is unlikely because of the extremely robust nature of such fusions (Silhavy and Beckwith, 1985). It is conceivable that the G T A structural gene promoter is located 3' of the orfglr.lacZ fusion joint in pSMFOOl . However, this seems unlikely because of the strongly conserved Orfgl-homologous sequences 67 (using the same reading frame as in the pSMFOOl fusion of orfgl to lacZ) located 5' of Orfg2 homologues in several other bacteria that contain G T A - l i k e gene clusters (Lang et al, 2002). The most probable explanation is that pSMFOOl does not contain the entire G T A structural gene cluster promoter, and therefore, does not transcribe the orfglr.lacZ fusion. To ensure that orfgl is transcribed/translated, and that the fusion point is indeed correct, pSMF002 wil l have to be made and tested. If pSMF002 has (3-galactosidase activity, then it can be assumed that pSMFOOl does not contain the entire G T A structural gene cluster promoter, however i f pSMF002 does not have (3-galactosidase activity, then either the fusion point is not to the correct start codon for orfgl or orfgl is not translated. However, the absence of (3-galactosidase expression in pSMFOOl is consistent with the promoter sequence suggested in Figure 16, which is absent from pSMFOOl . 4.3.4 GTA immunoassays of Y262(pSMF001) and Y262(pSMF002) Although pSMF002 was determined to contain non-/?. capsulatus D N A , pSMFOOl was the expected fusion. When Y262(pSMF001) was compared to Y262(pYP), Y262(pYnP) and Y262(pSTUl2) , it was apparent that pSMFOOl did not exhibit the p Y P effect seen in both p Y P and pSTU12 (Figure 17). However, pSTU12 is seen to have less of an effect on capsid production than p Y P does. A s Y262(pSTU12) has no detectable (3-galactosidase specific activity, it can be assumed that this fusion makes a non-functional p-galactosidase enzyme, which leads us to believe that it is the fusion protein itself that causes the observed effect on capsid production. B y comparing this to pSMFOOl , either pSMFOOl does not contain the entire G T A structural gene cluster promoter, or the Orfgl ::LacZ fusion protein is non-functional. Further deletions would have to be made to determine which of these predictions is correct. 68 4.4 Future Research There are several paths that this research could take. For the twelve mutants discovered to have an effect on G T A production (Table 5), each gene would have to be knocked out with a translationally inframe (non-polar) deletion and complemented in trans with only the gene knocked out, transcribed using a promoter like the puf promoter as done for the ctrA and cckA knock outs (Lang and Beatty, 2000, 2001). Even so, it would be difficult to ascribe a biochemical function to these gene products, especially the hypothetical proteins. Further studies of G T A production in SB 1003, compared to B10 and Y262, could help elucidate key differences between these strains that are not known at this time. G T A transduction assays (bioassay) of SB 1003 compared to Y262 would show i f the capsid seen in the cell-free supernatant samples of SB1003 are transductionally active. Comparative genomics and transcriptomics between these strains would also be interesting to do, once a microarray for R. capsulatus is available. Such work may give a clue into the mutations that make Y262 a G T A over producing strain compared to B10, and the difference that 1 found between SB 1003 and B10. Introducing plasmid p Y P into SB 1003 to measure transcription levels from the G T A structural gene cluster promoter would also be an interesting follow up to these immunoassays. If the levels of transcription are similar to the transcription levels in Y262, it could be that one of the mutations that makes Y262 a G T A overproducer is the same that makes SB 1003 rifampicin resistant. Further D N A sequence analysis and comparisons of known key G T A regulatory factor sequences between Y262, B10 and SB 1003 could be used to design directed mutagenesis experiments to explore how the different phenotypes shown for G T A production and transduction activity relate to potential gene sequence differences. 69 Additional promoter deletions could be made to p Y P , using various combinations of 5 : sequences in fusions of both orfgl and orfgl to lacZ. A translationally in-frame (nonpolar) deletion of orfgl could also be created. Such experiments would determine i f orfgl is a real gene, and whether orfgl is in fact the first real gene in this cluster. R . A . C . 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