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Understanding the type I secretion of the S-layer protein RsaA in Caulobacter crescentus Toporowski, Michael Cameron 2004

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Understanding the type I secretion of the S-layer protein RsaA in Caulobacter crescentus by Michael Cameron Toporowski B.Sc, The University of British Columbia, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming \ o the required standard THE UNIVERSITY OF BRITISH C O L U M B I A April 2004 ©Michael Cameron Toporowski, 2004 Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Name of Author (please print) Date (dd/mm/yyyy) Title of Thesis: Lj ^ c'U r s-fxnV7i e, iy p<_J^ S c c - ^ f - y o ^ ? LZLZ Th fAy - / W / - f / ^ /£ s« A Degree: iPQ. S<~ , Year: Departmentof W i c ^ b r c h ^ M4 J ^ M , ^ . The University of British Columbia ~~ ^ Vancouver, BC Canada ABSTRACT The transport of RsaA, the S-layer subunit protein of Caulobacter crescentus, is mediated by A B C transporter (type I) secretion. The A B C transporter and membrane fusion protein (MFP) components were previously reported as downstream of rsaA, however the two outer membrane proteins (OMP) were not described. This study aims to elucidate the transcriptional regulation of the rsaADE genes as well as the role of two putative OMP genes, rsaFa and rsaFb, in the secretion of RsaA. The rsaADE genes were previously thought to be transcribed as an operon in a similar fashion to that of the Escherichia coli alpha-hemolysin (HlyA) system. Here I show that contrary to previous hypotheses, the rsaD and rsaE genes appear to be transcribed together using a promoter found between rsaA and rsaD suggesting that they are transcribed irrespective of rsaA. The outer membrane proteins of this system had been suggested, but not characterized. Two candidates for the OMP, rsaFa and rsaFb, were identified by similarity to the E. coli HlyA secretion OMP TolC, using the available C. crescentus genome sequence and were modeled using the solved TolC structure. rsaFa was found several Kb downstream of the other transporter genes, while rsaFb is in an apparently random location. The rsaF genes were disrupted to determine i f they were involved in RsaA secretion. Knockout of rsaFa reduced secretion to -54% of wild type levels while the rsaFb knockout reduced secretion levels to -76%. When expression of both proteins was eliminated there was no RsaA secretion, but a residual level of -9% remained intact inside the cell, suggesting posttransiational down regulation. Complementation with ii either of the individual rsaF genes using a multi-copy vector (and demonstration of overexpression) did not restore RsaA secretion to wild type levels indicating both rsaFa and rsaFb are required for normal levels of S-layer secretion. However, overexpression of rsaFa (with normal rsaFb levels) in concert with multi-copy expression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already high level type I secretion system. This is the only known example of type I secretion requiring two outer membrane proteins to assemble a fully functional system. It appears that production of RsaA is self-regulated, such that a build up of any amount of RsaA inside the cell due to blockage of transport limits RsaA production and severely impedes cell growth showing no signs of RsaA degradation. Secretion of RsaA appears to be a function of the number and type of outer membrane proteins as well as the amount of RsaA produced. iii T A B L E O F C O N T E N T S A B S T R A C T 11 T A B L E OF CONTENTS iv LIST OF T A B L E S vi LIST OF FIGURES V l l LIST OF ABBREVIATIONS ix A C K N O W L E D G E M E N T S x 1. INTRODUCTION 1 1.01- Caulobacter crescentus 1.02- Surface layer proteins 1.03- Transport of S-layers 1.04- S-layer of C. crescentus 1.05- Biotechnology applications of the C. crescentus S-layer 1.06- Transport systems 1.07- Type I secretion 1.08- The TolC protein 1.09- Type I organization 1.10- Transcriptional regulation 1.11- The RsaA secretion system 1.12- Summary of the study 2.01- Bacterial strains, plasmids, and growth conditions 2.02- D N A extraction, purification and separation 2.03- Plasmid and D N A manipulations (rsaADE studies) 2.04- Plasmid and D N A manipulations (rsaFa and rsaFb studies) 2.05- Internal deletions in the rsaF genes 2.06- Knockout construction 2.07- RsaADE gene transcription studies 2.08- Antibody production 2.09- Protein techniques 2.10- SDS-PAGE and western blotting 2. E X P E R I M E N T A L PROCEDURES 14 2.11- Electron microscopy 30 2.12- Bioinformatic analysis and protein threading 31 3. RESULTS- Transcriptional regulation of the rsaADE genes 32 3.01- Identification of the rsaADE genes 32 3.02- Identification of a potential rsaD promoter 32 3.03- Absence of rsaE promoter determined 34 3.04- Identification of the rsaDE promoter 35 4. RESULTS-Identification and characterization of the outer membrane proteins of the RsaA secretion system 40 4.01- Identification of the two rsaF genes 40 4.02- Internal deletions of the rsaF genes 46 4.03- Disruption of the rsaFa and rsaFb genes 47 4.04- Effect of disruption of the rsaF genes on S-layer secretion 49 4.05- Complementation of the secretion deficient JS1009 strain 52 4.06- Production of RsaA appears to be regulated when secretion is impeded 56 4.07- Coordinate Overexpression of RsaA and RsaF 62 5. DISCUSSION A N D CONCLUSION 71 REFERENCES 76 v LIST OF T A B L E S Table 1. Bacterial strains and Plasmids 15 Table 2. Comparison of RsaA levels as determined by whole-culture preparations or low pH extraction 51 Table 3. Generation times of the C. crescentus mutant strains 60 Table 4. Levels of RsaA determined by whole-culture preparations 66 vi L I S T O F F I G U R E S Figure 1-1. C . crescentus cell cycle 2 Figure 1-2.3-D hexagonal array of the S-layer 4 Figure 1-3. Cartoon depictions of the type I through type V transport systems 8 Figure 1-4. Ribbon structure of the E . coli TolC protein 10 Figure 3-1. In-silico predicted rsaD promoter orientation 33 Figure 3-2. Predicted rsaD promoter sites 34 Figure 3.3 a-b. Characterization of CB15A B15 35 Figure 3-4 a-c. Characterization of the CB15A-rsaA strain 37 Figure 3-5 a-b. Characterization of the CB15A B15: R A T l f i C m strain 39 Figure 4-1. Relative location of the rsaA secretion apparatus 40 Figure 4-2. ClustalW alignment of the RsaFa, RsaFb and TolC proteins created using the MacVector 6.0 program 42 Figure 4-3. Predicted 3D-ribbon and space-fill models of RsaFa and RsaFb as well as the TolC monomer 43 Figure 4-4. Cartoon depiction of charged regions that may block RsaA transport 45 Figure 4-5. P C R confirmation of rsaFa knockout 47 Figure 4-6. P C R confirmation of rsaFb knockout 48 Figure 4-7. RsaFa and RsaFb levels in wild type and rsaF knockout strains 49 Figure 4-8. a- b. Effect of disruption of the rsaF genes on S-layer secretion 50 Figure 4-9. Determination of internal levels of RsaA in the rsaF double knockout 52 Figure 4-10. Complementation of the rsaF genes in trans recovers S-layer secretion 53 Figure 4-11. Expression of the RsaF proteins in the complemented JS1009 strain 54 Figure 4-12. Cartoon depiction of heterotrimer formation 55 Figure 4-13. RsaA production and RsaA secretion levels are comparable suggesting that little residual S-layer is left inside of the cell 56 Figure 4-14. Impeded RsaA transport in rsaF (JS1009) mutant and RsaA (Hpsl2furin) mutant.... 58 Figure 4-15 a. Exponential growth curve of knockout and modified RsaA strain 59 Figure 4-15 b. Logarithmic growth curve of knockout and modified RsaA strain 59 Figure 4-16. Cartoon depiction of hypothesized autoregulation of the rsaA gene 61 Figure 4-17. a-c. Levels of aggregate production in the JS1001 as compared to strains with additional copies of the transporter components 64 Figure 4-18. Effect of RsaF overexpression in the JS1001 strain 65 Figure 4-19. Levels of RsaFa and RsaFb in JS1001 strain 65 Figure 4-20. Colloidal gold labeling of surface displayed RsaF 67 Figure 4-21. Effect of RsaFa and rsaA overexpression in the JS1001 strain 69 Figure 4-22. Cartoon of RsaA secretion and transport system in the overexpressing strains 70 viii LIST OF A B B R E V I A T I O N S A B C ATP-Binding Cassette Ap ampicillin B L A S T Basic Local Alignment Search Tool bp base pair C-terminus carboxy terminus Cm Chloramphenicol D N A deoxyribonucleic acid DNAse deoxyribonuclease g grams Glu Glatamic acid/ Glutamate hr hour kb kilobases kDa kilodaltons K m kanamycin L B Luria-Bertani broth LPS lipopolysaccharide min minute MFP Membrane Fusion Protein mg milligram ml millilitre pl microlitre p.g microgram NaCl sodium chloride N-terminus amino terminus OD600 optical density at absorbance of 600nm OMP Outer Membrane Protein P A G E polyacrylamide gel electrophoresis PBS phosphate buffer salts PCR polymerase chain reaction P Y E peptone yeast extract RNAse Ribonuclease S-layer surface layer S-LPS smooth lipopolysaccharide SDS sodium dodecyl sulphate Sm streptomycin TBS Tris-buffered saline (lOmM tris-HCL (pH 7.5), 0.9%NaCL) TIGR The Institute for Genomic Research tris Tris (hydroxymethyl) methylamine A C K N O W L E D G E M E N T S I would like to thank my supervisor, Dr. John Smit for his advice and guidance throughout the course of this project. I would also like to thank past and previous members of the Smit lab, especially Dr. John Nomellini for help and assistance as well as countless discussions about numerous aspects throughout this project. I owe him countless beers. I would like to thank Andrea Pusic and Natalie Drouillard for their technical assistance at various stages of this work. I thank Dr. Peter Awram for starting the work on the RsaA secretion apparatus. I also thank Assaf Levi for the construction of the CB15 A-rsaA clone. Last but not least, I would like to thank my parents, my brother, and my girlfriend who put up with me during my masters thesis; without their love and support I couldn't have done this. 1. INTRODUCTION The S-layer of Caulobacter crescentus is a secreted by a highly proficient type I secretion system. The type I secretion system was initially discovered and partially characterized in the Smit lab by Dr. Peter Awram. The A B C transporter and Membrane Fusion Protein (MFP) were characterized, but the Outer Membrane Protein (OMP) was only identified. This thesis focuses on tying up some loose ends, and in turn unraveling a few more, in the secretion of the S-layer of C crescentus. Characterization of the OMP units and a better understanding of the transcriptional control of the other type I secretion sub-units has led to a greater understanding of type I secretion systems as well as proven useful for biotechnology applications of the C. crescentus S-layer. Results suggest that the type I component genes are transcribed separately from the transported RsaA. Identification of a separate promoter in between rsaA and rsaD shows that the rsaADE gene set is not co-transcribed like previous studies hypothesized (4, 30). Also presented is the characterization of the two OMPs (RsaFa and RsaFb) which are associated with the RsaA secretion system. The data suggests that both rsaF genes are required for wild type S-layer secretion levels. Regulation of RsaA production and secretion appears dependant on proper secretion, suggesting possible autoregulation. Furthermore, levels of S-layer secretion can be increased when both rsaFa and rsaA copies are increased. L01- Caulobacter crescentus 1 C. crescentus is a non-pathogenic Gram-negative bacterium common in fresh water and soils. Of the Caulobacter sp., C. crescentus, which derives its name from its crescent shape, has been well studied in numerous areas of research. This bacterium has a dimorphic developmental lifestyle (18, 28, 50) switching between a motile swarmer phase and a stalked phase often being affixed to a surface (Fig. 1-1.). Swarmer cells have single flagellum, pili and holdfast (an adhesin) at one pole (52). Cells wil l then lose the flagellum and form a stalk from the cell envelope. These stalked cells will divide to produce a new swarmer cell with the flagellum being created at the pole furthest from the stalked cell. Often C. crescentus cells will form rosettes with multiple cells binding at one central point by the distal end of their stalks (50). Older and poorly growing cells become elongated and are often oddly shaped becoming twisted and bent. For detailed review on cell cycle see Quardokus and Brun, 2003. Figure 1-1. C. crescentus cell cycle Cartoon representation of the dimorphic lifestyle of C. crescentus. (Figure from Quardokis and Brun, 2003). 7.02- Surface layer proteins Surface layers (S-layers) are common in many genera of microorganisms, including Gram-negative bacteria, Gram-positive bacteria and Archeabacteria. S-layers are two-dimensional arrays that cover the outside of the cell. S-layers may function as protective barriers and molecular sieves, promote cell adhesion and surface recognition Non-motile 2 and maintain cell shape and envelope rigidity (34). Thousands of copies of nearly always a single protein or glycoprotein self-assemble into a crystalline-like lattice (58). S-protein represents approximately 10-15% of the total cellular protein of the bacterial cell(16). For reviews on S-layers see Beveridge et. al., 1997; Boot and Pouwells, 1996; Sleytr and Messner, 1983. 1.03- Transport of S-layers The majority of the S-layer transport systems that have been discovered are type II systems in which an N-terminal signal system directs export across the inner membrane using the general secretion pathway (GSP) and secretion from the bacterium then occurs via a protein specific mechanism. An example of this is the S-proteins of Aeromonas that are transported across the cytoplasmic membrane via the GSP, but require substrate specific terminal branches if the GSP to transport across the outer membrane. The S-layer proteins of C. crescentus (4) and those of Campylobacter fetus (68) and Serratia marcescens(35), however, are secreted by a type I mechanism likely allowing for high levels of S-layer secretion. 1.04- S-layer of C. crescentus The Gram-negative bacterium Caulobacter crescentus is covered by an S-layer which minimally acts as a physical barrier to Bdelvibrio-like parasites and lytic enzymes (37). This crystalline surface layer is composed of a hexagonal array of the 98-kDa protein RsaA (63). The six RsaA subunits form a ring-like, circular structure that interconnects with other rings to form a two-dimensional hexagonal array (62) (Fig. 1-3 2.)- The S-layer is anchored to C. crescentus via an interaction with an outer membrane smooth lipopolysaccharide (S-LPS)(5, 71). Ca is required for the proper crystallization of RsaA into the S-layer and its removal using EGTA disrupts S-layer structure (46, 71). Production of the C. crescentus S-layer has been estimated to be 10-12% of total cell protein with approximately 40,000 RsaA subunits attached to the surface of the cell(6, 9, 62). The level of secretion observed in C. crescentus appears to be one of the highest levels of S-layer secretion with only C. fetus having similar amounts. RsaA synthesis occurs without need for induction and the protein is produced continuously throughout the cell cycle (24, 60). Figure 1-2. 3-D hexagonal array of the S-layer. (figure from Smit et al, 1992) 1.05- Biotechnology applications of the C. crescentus S-layer The S-layer of C. crescentus can be used for multiple biotechnology applications. Currently the S-layer protein has been used for heterologous protein production as well as protein and epitope display applications. The S-layer covers the entire surface of the bacterium. An uncleaved C-terminal secretion signal directs the secretion of RsaA (10, 12-14). When the native RsaA protein is secreted, the S-layer is attached to the outer membrane via S-LPS(5, 71). If the S-LPS is absent or disrupted, the S-layer forms aggregates which are up to 90% pure RsaA and can be easily collected. It is desirable to 4 produce large amounts of recombinant protein that can be easily purified. The C-terminal 2_j  secretion signal and Ca binding domain, responsible for aggregation, can be fused to a desired protein allowing recombinant proteins to be secreted by the RsaA transport system. Heterologous proteins aggregate in the culture medium and are easily purified using mesh filter. This process has been shown to be viable and recombinant proteins have been expressed and purified from C. crescentus{\\). Up until 2003, the C. crescentus S-layer system was marketed as the PurePro™ expression system (Invitrogen The S-layer has additional applications in the field of protein and epitope display. Initial studies used to define functional regions of the S-layer protein revealed the presence of sites that could be used for the display of small peptides. Efforts have since shown that these sites can accommodate upwards of 200 amino acid inserts (47). Since the S-layer covers the entire bacteria, this leads to thousands of copies of epitopes or proteins displayed per cell. Therefore numerous potential applications, from gene fragment display to whole cell vaccines are possible. To increase the potential applications of this system, it is vital to understand the S-layer transport apparatus. Understanding potential zones of hindrance to RsaA transport is key to allowing secretion of all types of heterologous proteins, including those with positive charges. As well, identifying any potential bottlenecks in RsaA transport is important so that increased levels of S-layer secretion can be achieved. To address some of these questions this thesis examines the production of the RsaA secretion apparatus as well as characterization and overexpression of the OMPs. 5 1.06- Transport systems There are five classes of secretion systems in Gram-negative bacteria that are well described (Fig. 1-3.). These systems have all been named type I through type V . The type I system requires three proteins to form a pore which spans through the inner and outer membranes allowing the protein to be secreted. This is the method by which RsaA is secreted and thus is discussed in depth below. The type II secretion system is likely the most commonly used transport system in Gram-negative bacteria. The type II secretion system is associated with the general secretion pathway (GSP) with acts as a common protein transporter. Type II systems employ the GSP for export across the inner membrane and then use a complex of 12-16 proteins for secretion to the outside of the bacterium (67). The secreted proteins utilize a Sec-dependant N-terminal signal sequence to direct transport across the inner membrane by the Sec pathway (51). Proteins are transported across the inner membrane in an unfolded state and then fold in the periplasm. Folded proteins are often needed for the outer membrane component to recognize it for secretion. The type III system is a Sec-independent secretion system used to translocate proteins into the extracellular environment, or directly into eukaryotic cells. These secretion systems are usually associated with bacterial pathogenesis, but are also involved in flagellum production. The systems are assembled from over 20 different structural proteins, including 10 that have counterparts in the flagellar export pathway(49). Type III systems are able to transport proteins across three membranes using a complex secretion apparatus. 6 Type IV secretion systems are involved in Sec dependant transport. The type IV system functions to transport D N A from bacteria to bacteria, as well as proteins from bacteria to eukaryotic cells. It has been found to facilitate the transport of multi-subunit proteins across bacterial membranes (19). It shares some similarities with the type II system utilizing the GSP system for translocation across the inner membrane. The type IV system is still not well understood, but the VirB system of Agrobacterium tumefaciens has been found to utilize 10 proteins for the transport mechanism. 7 T y p e I M Q V t t C S U y>\ Type QI accretion Type V Msretioci OM I M B T » * II D Type IV K O K I O M rM O M ff. f*n*u4j HnM ATP ^ ATF A O M V I M O M IM Figure 1-3. Cartoon depictions of the type I through type V transport systems. Depicts the organization of systems in the bacterial membranes and periplasmic space, (figure from Sharff etal, 2001) The type V system, or autotransport system consists of both the transporter protein and the transported protein. Type V secretion is a terminal branch of the GSP that exports proteins with diverse functionalities, including proteases, toxins, adhesins, and invasins(31). A typical autotransporter contains three domains: an amino-terminal signal sequence for secretion across the inner membrane by the Sec system, an internal 8 passenger or functional domain, and a carboxy-terminal p-domain (31). The (3-domain forms a pore in the outer membrane and then the passenger domain is transported to the outside of the cell. 1.07- Type I secretion Type I secretion is a sec-independent pathway which secretes the protein from the cytoplasm across the outer membrane without any interaction with the periplasm. Type I proteins utilize a C-terminal secretion signal that is usually in the last 60 amino acids of the protein (7). Proteins secreted by this pathway include Escherichia coli a-hemolysin and other bacterial R T X toxins and proteases from Erwinia chyrsanthemi, S. marcescens and Pseudomonas aeruginosa^'A, 73). The R T X motifs contain a variable number of glycine and aspartic rich repeats in the C-terminal half of the protein. This R T X repeat is likely not the primary secretion signal, with the amino acid sequence and secondary structure of the C-terminus being more important (12, 22, 73). The type I secretion apparatus is composed of a three-component ATP-binding cassette (ABC) based exporter. The A B C transporter, which most likely forms a homodimer in the inner membrane, engages the C-terminal sequence of the substrate protein and hydrolyzes ATP during the transport process. The membrane fusion protein (MFP) is anchored in the inner membrane by a single transmembrane domain, as well as bound to the A B C transporter, and appears to span the periplasm(67). The MFP is thought to interact with the A B C transporter protein and the last component, the outer membrane protein (OMP), forming a channel that extends from the cytoplasm through the two membranes to the outside of the cell. 9 1.08- The TolC protein The E. coli HlyA system is the best characterized type I secretion system (15, 26, 43). In particular, the OMP TolC has been extensively characterized and is the prototype of a large family of OMPs from various Gram-negative bacteria. The structure of TolC has been solved to 2.1 A (36) (Fig. 1-4.), and multiple studies have been carried out on its multifunctional nature. TolC appears to be very promiscuous, interacting with different inner membrane translocases involved in protein secretion, drug efflux and cations including the HlyA, AcrA, and CvaA systems (25, 32, 74). It has been suggested that the reason TolC is a multifunctional protein may be that its gene does not belong to any export operon (2, 3). Figure 1-4. Ribbon structure of the E. coli TolC protein (Figure from Koronakis et al. 2000) 1.09- Type I organization Genome organization of most type I systems typically have the ABC transporter and MFP genes adjacent to the gene for the secreted protein on the 3' side, whereas the OMP gene location can vary. In some cases, the genes for all three transport components are immediately adjacent to the substrate gene(s)(20, 38, 68). In other type I systems, only the ABC-transporter and MFP genes are located next to the substrate gene (39, 40). The OMP commonly lies far from the other components. TolC from the E. coli a-10 hemolysin (HlyA) system, and HasF from S. marcescens are examples of this and are not located hear any export secretion systems (8, 74). 1.10- Transcriptional regulation Transcriptional regulation of the type I components is not well studied. The majority of type I genes are organized together and are assumed to be transcribed together. Co-transcription of the type I transporter genes is based on previous experiments with the HlyA system of E. coli. Transcript analysis using rifampicin resistance suggested that hlyABCD were co-transcribed (75). To date, only two of the well characterized type I systems appear to have separate promoters for the transported protein as well as the transporter components. Both the S. marcesens (Lip) and C. fetus (SapA) systems have separate promoters for the S-layer gene and the transporter genes. The sapDEF genes are transcribed inversely from sapA using a separate promoter. The lipBCD genes are located immediately downstream of slaA but a separate promoter exists for lipBCD gene set. The type I apparatus is still not well understood and transcription of its components still appears relatively unknown. 1.11- The RsaA secretion system The RsaA secretion system has all the usual characteristics of a type I secretion system. The secretion signal is located within the C-terminal 82 amino acids of RsaA (13). The A B C transporter (RsaD) and MFP (RsaE) have been characterized (4). The rsaD and rsaE genes are located downstream of rsaA and transcription of the RsaA secretion apparatus had been assumed to occur in a fashion similar to the HlyA system 11 with rsaADE being co-transcribed using the rsaA promoter. When initial studies were done, an OMP candidate did not immediately follow rsaE, indicating that that system resembles the Hly and Has systems. In that study they randomly mutagenized the genome of C. crescentus using the Tn5 transposon and identified several mutants that were deficient in RsaA secretion. Numerous Tn5 insertions were identified in rsaA, rsaD and rsaE, but none were found in an OMP component suggesting either that interfering with the OMP was lethal to the cell or that there were multiple genes involved in the formation of the outer membrane component of the secretion system. The two putative rsaF genes were later identified using the C. crescentus CB15 genome sequence, but characterization was not carried out. LI 2- Summary of the study I was able to elucidate the transcriptional regulation of rsaADE. Molecular based approaches to using an rsaA deficient strain as well as an rsaD knockout strain led to the discovery that rsaD and rsaE are transcribed using a separate promoter predicted to be just downstream of the rsaA. I have also identified two genes, rsaFa and rsaFb that code for OMPs involved in S-layer transport. Eliminating the function of either of these genes results in decreased RsaA secretion indicating that either protein can function as the OMP component of the secretion system. However, knocking out both genes completely eliminates RsaA secretion. Contrary to what previous studies suggested (30, 53), I demonstrate that RsaF (a and b) are involved in RsaA secretion and that both are required for maximal S-layer secretion. Data also suggests there is possible auto-regulation of RsaA expression. 12 Secretion is apparently a cooperative process as it was not possible to restore wild type level of RsaA secretion by overexpression of individual OMPs. Interestingly, i f both OMPs were present, cooperative expression of additional RsaFa and RsaA led to levels of RsaA secretion significantly above wild type levels. 13 2. EXPERIMENTAL PROCEDURES 2.01- Bacterial strains, plasmids, and growth conditions A l l of the strains and plasmids used in this study are listed in (Table 1.). E. coli DH5ct was used for all E. coli cloning manipulations, except for the use of Rb404 (17) for the use of non-methylated Clal site digestion. As well, E. coli JM109 (76) was used for induced production of GST tagged RsaE. E. coli was grown at 37°C in Luria broth (1% tryptone, 0.5% NaCl, 0.5% yeast extract) with 1.3% agar for plates. C. crescentus strains were grown at 30°C in P Y E medium (0.2% peptone, 0.1% yeast extract, 0.01% CaCb, 0.02% MgS04) with 1.2% agar for plates. Ampicillin was used at 50 ug/ml, kanamycin was used at 50ug/ml, chloramphenicol was used at 20 jig/ml, and streptomycin was used at 50 |ag/ml in E.coli. Kanamycin was used at 25 (J.g/ml, chloramphenicol was used at 2 u.g/ml, and streptomycin was used at lOug/ml in C. crescentus when needed. JS1001 strains utilized for aggregated protein experiments were grown in 1.25 L M l 1 HIGG media (0.15% glucose, 0.15% glutamate, 2 mM phosphate, 0.5 mM calcium, 5 m M imidazole, 1% ammonium chloride). Cultures were grown for 4 days at 30°C at a shake speed of 60 R P M in Fernbach flasks, to maximize aeration. Aggregates were then recovered by coarse filtration using using nylon mesh as previously described (13) and cell densities (OD6oo) were taken for all cultures. Aggregates were washed with dH 2 0 until no C. crescentus cells were seen attached under compound microscope. Aggregated proteins were kept at -80 °C and consequently lyophilized removing all traces of water. 14 Strain or plasmid Relevant character ist ics Reference or source Bacterial strains C. crescentus NA1000 Ap r syn-1000; variant of wild-type strain CB15 that synchronizes well A T C C 19089 JS1001 S - L P S mutant of NA1000, sheds S-layer into medium Edwards and Smit, 1991 JS1003 NA1000 with rsaA interupted by K S A C Km' cassette Edwards and Smit, 1991 JS1007 Sm', NA1000 rsaFa- strain This study JS1008 Cm', NA1000 rsaFb- strain This study JS1009 Cm' , Sm' , NA1000 rsaFa-/rsaFb- strain This study B15 Km', Sm r , NA1000 Tn5 insertion in the rsaD gene Awram and Smit, 1998 A-rsaA CB15, rsaA gene knocked out deleting rsaA promoter and portion of rsaA gene This study E. co/ / DH5D recA1, endA1, gyrA96, thi, hsdR17, SupE44, relA1, LacZYA-arfF Invitrogen JM109 recA1, endA1, gyrA96, thi, hsdR17, SupE44, relA1, Laclq, LacZM15 Yanisch-Perron etal . , 1985 R M 0 4 F-dam-3, dam-6, metB1, galK2, galT22lacY1, thi-1, tonA31, tsx-78, mtl-1, supE44 Brent and Ptashne, 1980 Plasmids pHP45 Ap r , Sm r , plasmid from which omega Sm cassette removed Fel layetal. ,1998 ; PUC4KISS Km', omega Km cassette removed for pBBR4 Taylor and Rose, 1988 pBSKI+ Ap', LacZ, Cloning vector Stratagene pBSKII Ap r , LacZ, Cloning vector Stratagene pBSKIIEEH Ap', modified pBSKSII cloning vector with EcoRI, EcoRV, H/ndlll modified M C S This study pBSKIIESH Apr, modified pBSKSII cloning vector with EcoRI, Stul, Hind\\\ modified M C S This study PTZ18UCHE Cm' , cloning vector This study pTZ18UCHE:reaF/*N0C Cm' , cloning vector with rsaFb fragment missing N terminus and C terminus This study pBSKIIEEH: rsaFaOSm Ap', Sm', rsaFa gene fragment with Sm cassette inserted at Psfl site This study pBBR4 Km', broad host range plasmid derived from pBBR1 This study pBBR4: rsaFa rsaFa +, Km', rsaFa gene inserted EcoRI/ SamHI in pBBR4 This study pBBR4: rsaFb rsaFb +, Km', rsaFb gene inserted EcoRI/ SamHI in pBBR4 This study pWB9A19 Cm', Sm', rsaA gene and rsaA promoter strain This study pWB9Hps12 Cm', Sm', rsaA containing with SamHI site at a.a. 723 Bingle etal . , 1997 pWB9:Hps12furin Cm' , Sm', rsaA containing with furin cleavage site (RKKR) in SamHI site at a.a. 723 This study pGEX4T3 Ap', G S T tagged expression vector Amersham pGEX4T3: rsaFa Ap', GST tagged expression vector with in frame BamHI-EcoRI rsaFa gene This study pK18mobsacB Km', Suc s , E.coli based suicide vector Schafer et al., 1994 pK18mobsacB: rsaFaDSm Km', Suc s , E.coli based suicide vector with r s a F a D S m fragment This study pRAT1 Ap r , rsaA+, rsaD+, rsaE* Awram and Smit, 1998 pRAT9 Cm', rsaD+, rsaE+ pBBR1 based plasmid Awram and Smit, 1998 puC19 Ap', LacZ, ColE1 cloning vector Vieria and Messing, 1982 puC19:RAT1Cm Ap', Cm', containing the RATI gene set with a Cm cassette interupting the rsaA gene This study pBBR4:RAT1Cm Km', Cm' , broad host range with RATI gene set, Cm cassette interupting the rsaA gene This study pGEX4T3: rsaD Ap', G S T tagged expression vector with inframe rsaD gene This study pGEX4T3: rsaE Ap', G S T tagged expression vector with inframe rsaE gene This study pAL1 Sm', Sue 3 , E.coli based p N P T S ! 38 suicide vector with A-rsaA fragment This study i 15 2.02- DNA extraction, purification and separation Chromosomal extraction was done by phenol chloroform extraction. 3 ml volumes of logarithmic phase culture were centrifuged at 16000 X g for 2 min. Cell pellets were resuspended in 0.5 ml of 10 mM tris-HCL. Lysozyme was added to final concentration of 300 ug/ml and incubated at 37°C for 10 min. SDS was added to 0.3% and incubated at 60 °C for 20 min. Proteinase K and RNAseA were added to 300|_ig/ml and incubated 4 hr. D N A was then purified by 2 phenol/chlorform extractions (1:1:1), one phenol extraction followed by phenol/ chloroform extraction. Isolation of plasmid D N A was done utilizing the Qiaprep spin mini prep (Qiagen) system. Isolated plasmid D N A was eluted in TE buffer or dt^O. Restriction enzyme digestions were done with Invitrogen or New England Biolabs Inc. enzymes and buffers as specified by manufacturers. D N A fragments were analyzed by agarose gel electrophoresis using a horizontal apparatus with a TBE buffer system. 0.9% agarose gels were used for D N A separation with 0.5 ug/ml EtBr added to the molten gel solution prior to casting. Separation of D N A fragments was conducted using a voltage range of 80-120V. D N A bands were isolated and excised from gels using a scalpel. D N A was purified using the Qiaex II gel extraction kit (Qiagen) using the manufacturers' protocols. 2.03- Plasmid and DNA manipulations (rsaADE studies) Standard methods of D N A manipulation and isolation were used (55). Electroporation of C. crescentus was performed as previously described (27). A l l PCR products were generated using Platinum Pfx D N A polymerase (Invitrogen) following the 16 manufacturers suggested protocols, except for the p A L l construction which utilized Taq D N A polymerase. NA1000 chromosomal D N A was used as the template for all PCR products, except the p A L l fragments in which CB15 chromosomal D N A was used. A fragment containing the rsaD gene was amplified by PCR using the primers 5'-C C G A A T T C C A T G T T C A A G C G C A G C - 3 ' and 5'-G C G G C C G C T C T G G A C G C G C T G C A A - 3 ' incorporating EcoRI and Noil restriction sites. This gene fragment was inserted into the EcoRV site of the pBSKI + plasmid. The pBSKI + : rsaD plasmid was cut with .EcoRI and Notl releasing the rsaD fragment. This fragment was inserted into EcoRl-Notl cut pGEX4T3 plasmid. The pGEX4T3: rsaD construct is an in-frame insertion of the rsaD gene so that it has an attached GST tag. Another fragment containing the rsaE gene was amplified by PCR using the primers 5 ' - C C G A A T T C C A T G A A G C C C C C C A A G - 3 ' and 5'-GCGGCCGCTCTCCTCGCGCATCGT-3 ' incorporating EcoRI and Notl restriction sites. This fragment was inserted into the pBSKI + plasmid at the EcoRV site. The pBSKI + : rsaE plasmid was digested using EcoRI and Notl releasing the rsaE fragment. The fragment was then ligated into EcoRl-Notl cut pGEX4T3 plasmid. The pGEX4T3: rsaE construct is an in-frame insertion of the rsaE gene so that it has an attached GST tag. Construction of the p A L l plasmid was carried out by Assaf Levi. Plasmid p A L l was constructed in order to create an in-frame deletion of the complete rsaA coding region. A PCR product encoding a 1.0 kb region upstream of the rsaA gene was amplified using the primers 5 ' -GGATCCGGCGTTCGAGCTGCTGCTGA-3 ' and 5'-G A A T T C T C A C C T G G C G G G T G A G T G A G - 3 ' introducing BamHl and£coRI sites. 17 Another PCR product was created using the primers 5'-G A A A T T C C G C T C G C C T A A G C G A A C G T C - 3 ' and 5'-A C T A G T G G C C G A G A T C T T G C C G T C G A - 3 ' amplifying a 1.0 kb region containing the end of the rsaA gene and incorporating EcoRI and Spel sites. Fragments were ligated into the pGEM-5ZT(+) vector at the EcoRV site using the pGEM-T® easy kit (Promega). The resulting fragments were digested with EcoRI, BamRI and Spel, and ligated into BamHl and Spel cut pNPTS138 plasmid(33). This resulted in creation of plasmid pALlwhich was transformed, by electroporation, into the E. coli DH10B strain (Invitrogen) and selected by blue-white screening. The pUC19: R A T I plasmid was created using the pUC19 plasmid (70) and the p R A T l plasmid (4). The RATI fragment was removed from the p R A T l plasmid as an EcoRI- Sstl fragment and ligated into an EcoRI- Sstl cut pUC19 plasmid. Interruption of the rsaA gene in the pUC19: R A T I construct was done by antibiotic insertion. The pUC19: RATI QCm was created using the pUC19: R A T I plasmid and the QCm cassette from the pHP45QCm plasmid (21). The QCm cassette was removed from the pHP45QCm plasmid as a BamEI fragment and blunted using T4 polymerase. The pUC19: RATI plasmid was electroporated into the Rb404 E. coli strain to stop D A M methylation. The isolated pUC19: R A T I construct was digested using Clal and the ends were blunted using T4 D N A polymerase. The blunted QCm cassette was then ligated into the blunted Clal site of the pUC19: RATI plasmid resulting in the pUC19: R A T I QCm plasmid. The pBBR4 and pUC19: p R A T l Q C m vectors were then used to create a broad host range construct containing the R A T I QCm fragment. The EcoRI-Sstl cut RATI QCm 18 fragment from the pUC19: R A T l Q C m was ligated into the EcoRl-Sstl cut pBBR4 plasmid creating the pBBR4: R A T l Q C m plasmid. 2.04- Plasmid and DNA manipulations (rsaFa and rsaFb studies) Standard methods of D N A manipulation, isolation, PCR product generation and cloning procedures were carried out as above. Plasmids used for internal deletions were made with the rsaF genes (a or b) and flanking regions to encourage homologous recombination. A PCR product containing the rsaFa gene and flanking regions of 1008 bp 5' and 139 bp 3' was generated using the primers 5'- G C C A C G C C C G G C G T C C A G T C C G A - 3 ' and 5'-G A G C T C C C T A G A G C G T T C T C C G A T C C G T G C G - 3 ' . This fragment was blunt end ligated into the pBSKI + plasmid at the EcoKV site and called pBSKI: rsaFa EX. A PCR product containing rsaFb and flanking regions of 795 bp 5' and 858 bp 3' was generated using the primers 5 ' - C G C C G G C T T C G C A G C G A T G A G C C C -3' and 5'-C C C G G A G G C C T C C C A G G C G G C G T A - 3 ' . This fragment was blunt end ligated into the Stul site of the pBSKIIESH plasmid and called pBSKIIESH: rsaFbEX. A PCR product containing rsaFa was generated using primers 5'-C G C G G A T C C A T G C G A G T G C T G T C G A A A G T T C T G T C -3' and 5'-C C G G G A A T T C T A G T T G C G G G G C G C G G T C T G G A C -3'. Another PCR product containing rsaFb was created using primers 5'-C G C G G A T C C A T G T T G A T G T C G A A C C G T C G A C G G G -3' and 5'-C C G G G A A T T C T A T T T C G A G C C G C T C G G G G G C T T -3'. PCR products were blunt 19 end ligated into the EcoRV site of the pBSKIIEEH vector and called pBSKIIEEH: rsaFa and pBSKIIEEH: rsaFb respectively. The pBSKIIEEH vector was constructed by Dr. lohn Nomellini from the plasmid pBSKII (Stratagene). The BssHl fragment containing the multiple cloning site was removed and replaced with annealed oligonucleotides 5'-C G C G C T G A A T T C G G A T A T C T T A A G C T T G G - 3 ' and 5'-C G C G C C A A G C T T A A G A T A T C C G A A T T C A G - 3' forming EcoRI, EcoRV and Hindlll sites. Similarly, the pBSKIIESH plasmid was created from the plasmid pBSKII (Stratagene). The BssRl fragment containing the multiple cloning site was removed and replaced with annealed oligonucleotides 5'-C G C G C T G A A T T C G A G G C C T T T A A G C T T G G -3' and 5'-C G C G C C A A G C T T A A A G G G C T C G A A T T C A G -3' forming EcoRI, Stul and Hindlll sites. These both result in smaller simpler plasmids that can be digested easily for blunt end cloning. Dr. lohn Nomellini constructed the plasmid pBBR4 from plasmids p B B R l M C S and pUC4 KISS (65). The Q-Km fragment from pUC4 KISS was removed using Pstl and the ends were blunted using T4 polymerase. A 0.3-kbp portion of the Cm r - encoding gene was removed from p B B R l M C S by cutting with Dral and replaced with the blunted Q-K m fragment, producing a K m r broad-host-range vector that replicates in C. crescentus. Plasmids pBBR4: rsaFa and pBBR4: rsaFb were made by removing the BamHl- EcoRI fragment of the pBBR4 plasmid and replacing it with the BamHI-EcoRI fragment from pBSKIIEEH: rsaFa and pBSKIIEEH: rsaFb plasmids respectively. 20 The plasmid pGEX4T3: rsaFa was constructed from plasmids pBSKIIEEH: rsaFa and pGEX4T3 (Amersham). Using incorporated restriction sites generated from the initial PCR primers, the BamHl- EcoRI fragment containing rsaFa was cloned in-frame into BamRl-EcoRl cut pGEX4T3 plasmid. The pBSKIIEEH: rsaFaQSm plasmid was created using the plasmids pBSKIIEEH rsaFa and pHP45Q(21). The QSm cassette was removed from the pHP45Q plasmid as a Smal fragment. This fragment was then blunt-end ligated into the pBSKIIEEH: rsaFa plasmid at a T4 polymerase blunted Pstl site inside the rsaFa gene. The resulting plasmid pBSKIIEEH: rsaFaQSm was then used to make the pK18mobsacB: rsaFaQSm plasmid. The EcoRl-Hindlll fragment containing the rsaFaQSm fragment was cloned into EcoRl-Hindlll cut pK18mobsacB plasmid. A PCR product containing a truncated form of rsaFb with the N-and C-terminus missing was generated using the primers 5 ' -GAAGCCGACGTGCTGTCT- 3' and 5'-TGTAGGAGGTTTTCGGGTCA-3 ' . This PCR product was blunt ligated into the Stul site of the pBSKIIESH vector using T4 D N A ligase creating pBSKIIESH: rsaFb ANAC plasmid. The pTZ18U CHE plasmid was constructed by Dr. Peter Awram using inverse PCR with the primers 5 ' - G A G G C C T A G T A C T C T G T C A G A C C A A G T T T A C T C A T A - 3 ' and 5 ' - G A G G C C T A C T C T T C C T T T T T C A A T A T T A T T G A A - 3 'to create the backbone of the pTZ18U plasmid without the Ap r cassette. The CHE (chloramphenicol) fragment was created as a PCR product using the pMMB206 plasmid (44) and the primers 5'-G G A A G A T C T G T T A A C T T T T C A G G A G C T A A G G A A G C T - 3 ' and 5'-G G A A G A T C T G T T A A C A C A A T A A C T G C C T T A A A A A A A T T A - 3 ' . The pTZ18U backbone product was cut with Stul and blunt-ligated with Hpal cut CHE fragment 21 creating the pTZ18UCHE plasmid. The pTZ18UCHE: rsaFb&NAC plasmid was then made by Dr. John Nomellini using the pTZ18UCHE plasmid and the pBSKIIESH: rsaFbANAC plasmids. The EcoRI- Hindlll fragment from the pTZ18UCHE plasmid was removed and replaced with the EcoRI- Hindlll fragment containing the rsaFbANAC fragment from the pBSKIIESH: rsaFb&NAC plasmid. The pWB9: rsaAAP was created as previously described (12). The pWB9 Hpsl2furin construct was made by Dr. John Nomellini using the BamHl site at a.a. 723 (Hpsl2) (11). Two oligonucleotides oligonucleotides 5'-T C G A G A C C C G A T G C G C A A G A A A C G G G -3' and 5'-CCCGTTTCTTGCGCATCGGGTC -3' were annealed together and then ligated into the pUC9CXS plasmid Xhol - Stul in a similar manner as the pillin epitope (11). The resulting plasmid was then digested using BamHl releasing the R K K R furin containing fragment. This fragment was then inserted into the BamHl site at a.a. 723 in rsaAAP and forward orientation of the fragment was confirmed by Cm resistance. The Cm r cassette was removed by excising with BgUl and then ligated back the two complementary ends. The rsaAAP Hpsl2furin fragment was then removed as an EcoRI-Sstl fragment and ligated into EcoRI-Sstl cut pWB9KSAC plasmid. 2.05- Internal deletions in the rsaF genes Internal deletion of the rsaF genes were done using the pBSKI: rsaFaEX and pBSKI: rsaFbEXplasmids. Internal deletions were done using unique restriction enzyme sites in either rsaF gene. Three separate internal deletions were done in the rsaFa gene. The first is a deletion utilizing the EcoNl restriction enzyme sites. There are 3 EcoNl 22 sites in the rsaFaEX fragment, one 185 bp 5' of the rsaFa gene, one 721 bp into the gene and another 1099 bp into the gene. Digestion of the pBSKI: rsaFaEX zt the three sites and consequent re-ligation to remove the internal fragments leads to a deletion of-1.3 Kb and the resulting plasmid called pBSKI: rsaFaAN. The second internal deletion was made in the pBSKI: rsaFaEX plasmid using the EcoRV and PstI restriction enzyme sites. The EcoRV site is 135 bp 5' of the start site and the PstI site is 503 bp 3' from the start of the gene. The pBSKI: rsaFaEX was digested with PstI and EcoRV, blunt ended using T4 D N A polymerase and then ligated together removing a fragment of 638 bp. The resulting plasmid was called pBSKI: rsaFaAVP. The last internal deletion used the PstI and Kpnl restriction sites. The pBSKI: rsaFaEX plasmid was digested with PstI and Kpnl and blunt ended using T4 D N A polymerase and ligated. The resulting plasmid pBSKI: xsaYaAKP has an 852 bp deletion. A l l rsaFa internal deletion fragments were removed as EcoRI-Hindlll fragments and ligated into .EcoRI- Hindlll cut pK18mobsacB plasmid. The one rsaFb gene internal deletion was made in the pBSKIIESH: rsaFbEX plasmid. The .EcoRV and Ncol sites in the rsaFbEX fragment. The .EcoRV site is 227 bp 3' of the start site and the Ncol site is 174 5' of the end of the gene. After digestion of the pBSKIIESH plasmid with £coRV and Ncol and then ends were blunted using T4 D N A polymerase and ligated back together. The resulting plasmid pBSKIIESH: rsaFbAVNhas an internal deletion of 1051 bp. The pBSKIIESH: rsaFb AVNwas digested with £coRI and Hindlll releasing the rsaFb AVN fragment. This fragment was ligated into £coRI-Hindlll cut pK18mobsacB plasmid. 23 2.06- Knockout construction Knockout of rsaFa in the CB2A strain was done using the pK18mobsacB: rsaFa AKP plasmid. Primary recombination of the plasmid was selected for using Km resistance. Five concurrent sub-culturing events were used to encourage a second recombination event. Secondary selection on 5% sucrose P Y E plates and subsequent replica plating on P Y E and P Y E K m plates was used to confirm a second recombination event. Colonies were then screened using the primers 5'-C G C C G G C T T C G C A G C G A T G A G C C C -3' and 5'-C C C G G A G G C C T C C C A G G C G G C G T A - 3 ' to confirm that appropriate gene replacement occurred. A strain confirmed to possess only the internal deletion form of rsaFa was designated CB2A: rsaFaAKP. Since internal deletion knockouts were hard to obtain, alternate methods were undertaken. Knockouts of the two rsaF genes were done in wild type (S-layer positive) NA1000 C. crescentus. rsaFa was destroyed through gene replacement of an rsaFa fragment containing an internal QSm cassette. The pK18mobsacB plasmid was used as a suicide vector to incorporate the antibiotic ablated rsaFa. The pK18mobsacB: rsaFaQSm plasmid was electroporated into NA1000 cells. Primary selection on P Y E Sm/ K m plates was used to determine if recombination had occurred. Five concurrent sub-culturing events were used to encourage a second recombination event. Secondary selection on 5% sucrose P Y E plates and subsequent replica plating on P Y E Sm and P Y E K m plates was used to confirm a second recombination event. Colonies were then screened by PCR using primers 5 ' - C G C G G A T C C A T G C G A G T G C T G T C G A A A G T T C T G T C -3' and 5'-C C G G G A A T T C T A G T T G C G G G G C G C G G T C T G G A C - 3 ' to determine i f the 24 recombination event resulted in restoration of wild type rsaFa or incorporation of the rsaFaQSm gene fragment. Destruction of rsaFb was done via insertional inactivation using an N - and C-terminally deleted rsaFb fragment. The non-replicatable pTZ18U CHE: rsaFb ANAC plasmid was electroporated into NA1000 competent cells and using Cm' cassette on the plasmid selection for insertional inactivation. Recombination of the pTZ18UCHE: rsaFb&NAC plasmid resulted in loss of the full rsaFb gene leaving independent N -terminal deleted and C-terminal deleted rsaFb gene fragments. Colonies were screened by PCR using the primers 5' - G A G G C C T A C T C T T C C T T T T T C A A T A T T A T T G A A - 3' and 5 ' - G G A C G A C G C T G A C C A G C A C C C C C T G C T -3'. The double rsaF knockout was created by gene replacement using the pK18mobsacB: rsaFaQSm plasmid and JS1008 (rsaFb) competent cells. Screening for homologous recombination of the pK18mobsacB: rsaFaQSm was done in the same manner as the single rsaFaQSm knockout. The only change to the protocol was that Cm was used in all media to maintain the rsaFb knockout. PCR confirmations of both rsaF knockouts were done using the primers and conditions as stated above. 2.07- RsaADE gene transcription studies The CB15 A-rsaA strain was created using the p A L l plasmid. Delivery of the p A L l plasmid into the CB15 strain was done by conjugation with the E. coli LS980 (match maker) and MT607 helper strain (D. Alley). The helper strain utilizes vector pRK600 a derivative of pRK2013, Cm r , containing a Tn9 insertion, Co lE l ori, and tra 25 functions from pRK2013 (57). Gene replacement was confirmed by PCR analysis (not shown) and the resulting strain was called CB15 A-rsaA. The CB15A Tn5 mutant B15 was made and confirmed as previously described (4). The pBBR4: R A T l Q C m plasmid was electroporated into the B15 strain and cells were selected for resistance to Km, Cm, and Sm. 2.08- Antibody production Antibodies used to detect RsaA were prepared by Dr. John Nomellini using a form of RsaA containing N and C-terminal portions of the protein, referred to as anti 188/784. This internal deletion form of RsaA was previously described in linker mutagenesis studies (12). Essentially, the N-terminal 1-188 a.a. fragment was removed as an EcoKl-BamHl fragment and the C-terminal 784-1025 a.a. fragment was removed as a BamHl-Hindlll fragment. The two fragments were ligated together in-frame at the BamHl site and then ligated into EcoRl-Hindlll cut pUC8 plasmid. The Hindlll cut pUC8: 188-784 was ligated to Hindlll cut pKT215 vector and transformed into C. crescentus forming aggregated protein which was used to make antibodies against both termini. Aggregated 188/784 protein was collected and washed with d H 2 0 to remove any attached C. crescentus cells. Aggregates were solubilized with 4 M Urea and dialyzed (30,000MW dialysis tubing) in dF^O to remove all traces of urea. Samples were then injected into New Zealand white rabbits and rabbit serum was collected and processed using standard protocols (55). Polyclonal antibodies were produced against RsaFa using a GST tagged protein. The pGEX4T3: rsaFa plasmid was expressed in E. coli (DH5a), but unfortunately the protein was only produced in the form of inclusion bodies. Thus protein was extracted by 26 inclusion body preparations. Inclusion body preparations were done by growing cells at 30°C to 1.0 OD600 and centrifuging cells and resuspending in 1XPBS buffer. Resuspended cells were incubated with lysozyme (100p.g/ml) for lhr at 25°C and then RNAseA (50p.g/ml) and DNAsel (lug/ml) were added and incubated for an additional hr at 25°C. After incubation, 10%SDS was added as well as SDS sample buffer at a 1:1:1 ratio. Samples were boiled for 5 min and then put on ice for 15 min. The inclusion body preparation was then centrifuged at 16,000g for 10 min. The pellet was then recovered and solublized using 4 M urea. Protein was dialyzed (30,000MW dialysis tubing) in dt^O for two days to remove all traces of urea. New Zealand white rabbits were injected with prepared protein samples containing a 1:1 ratio (volume) of protein to incomplete freunds adjuvant. Rabbit serum was collected and processed using standard protocols (55). The RsaD antibodies were made in a similar fashion to the anti-RsaFa antibodies. The pGEX4T3: rsaD plasmid was expressed in E. coli DH5a and similarly, the protein was only produced in the form of inclusion bodies. Inclusion bodies were prepared same as above. Dialyzed protein was then injected into New Zealand white rabbits and the rabbit serum was collected and processed using standard protocols (55). The RsaE antibodies were made using a GST tagged protein. The pGEX4T3: rsaE plasmid was expressed in the E. coli JM109 strain and soluble protein was produced and purified. JM109 cells with the pGEX4T3: rsaE plasmid were grown to OD6oo ~0.8 and the incubated with 0.1 m M IPTG at 30°C for 3 hrs. Cells were then pelleted and resuspended in cold buffer (PBS/ 0.5 % Tween-20/ I M NaCl/lOmM DTT/ ImM PMSF). Resuspended cells were sonicated and then centrifuged and supernatant saved. Supernatant was added to Glutathione Sepharose beads (Sigma) and rocked for 1 hr. 27 After rocking, beads were centrifuged and supernatant was aspirated. Beads were washed 3 times using 1XPBS. GST-tagged RsaE was eluted using elution buffer (50mM tris-HCl pH8/10 m M reduced Glutathione(Sigma)). Eluted protein fractions were then pooled and dialyzed against dEf^O. Dialyzed protein was then injected into New Zealand white rabbits and the rabbit serum was collected and processed using standard protocols (55). 2.09- Protein techniques Surface protein from C. crescentus cells was extracted by low pH extraction as previously described (72). Cell pellets were washed twice using 10 mM HEPES (pH 7.2) buffer and then release of the S-layer was facilitated using lOOmM HEPES at pH 2.0. To compare the amounts of surface layer protein extracted from different mutants, normalized levels of cells (determined by OD600) growing at log phase were used and equal amounts of extracted protein samples were loaded onto protein gels. Whole-cell-protein preparations were done with normalized levels of cells (determined by OD600) growing at log phase. Cultures were centrifuged and cell pellets were washed twice with lOmM tris-HCl pH 8. Cells were resuspended in lOmM tris-HCl pH8 and lysozme (lOOug/ml) was added and incubated at 25°C for 15 minutes, then RNAseA (50ug/ml) and DNAsel (lug/ml) were added and incubated at 37°C for 30 minutes. Equal amounts of whole-cell-protein preparations were loaded onto protein gels. Note that whole cell preparations done to determine internal levels of S-layer were altered for certain strains. The wild type S-layer positive NA1000 has RsaA attached to its surface and thus the strain was subjected to low pH extraction before whole cell preparation was done to remove attached RsaA. As well, the S-layer shedding JS1001 28 strain was poured through fine mesh removing aggregates so that they did not affect RsaA levels present in the whole-cell preparation. Whole culture preparations were done with normalized levels of cells (determined by OD600) growing (in liquid PYE) at log phase. Whole cultures were normalized so that lml of 0.6 OD600 cells were used for all cultures, thus making sure that any aggregated proteins were very small and well dispersed. Cultures were incubated with Lysozme (100 Lig/ml) and incubated at 25°C for 15 minutes, and then RNAseA (50 Lig/ml) and DNAsel (1 p.g/ml) were added and incubated at 37°C for 30 minutes. Powdered urea was added to make the final concentration of culture equal to 2M urea. Addition of urea is needed to solubilize any micro-aggregates produced by S-layer shedding strains. Equal amounts of whole culture protein preparations were loaded onto protein gels. Aggregated proteins were collected as previously described (13). Cultures were grown for 4 days at 30°C in 2.8L Fernbach flasks shaken at 60 rpm and then aggregated protein was collected using a fine mesh. Collected aggregates were washed with dFLO and then centrifuged at 6000 X g for a few seconds. Washing and centrifugation was repeated until few to no C. crescentus cells were seen attached to the aggregates under the compound microscope. Wet weights were taken and then aggregates were kept at -80°C. Frozen aggregates were then lyophilized until completely dry and weight taken. 2.10- SDS-PAGE and western blotting Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was done using 4% stacking, and 7.5% or 12% (as indicated) separating gels. Coomassie stained SDS-PAGE gels and western immunoblotting were done as previously described 29 (55). After transfer of proteins to 0.2 urn BioTrace NT nitrocellulose membrane (PALL), blots were blocked using Blotto (3% skim milk , 0.9% NaCl, and 20mM Tris). Western blots were probed with primary rabbit polyclonal antibodies, and antibody binding was visualized by either colorimetric or chemiluminescence developing methods. Colorimetric blotting was done using goat anti-rabbit serum coupled to horseradish peroxidase and color forming reagents as previously described (61). Chemiluminescent blotting was done using the Amersham Biosciences E C L western blotting kit in accordance with the manufacturer's protocol. Anti-188/784 antibodies were incubated at 1/15000 dilutions for colorimetric and 1/30000 dilutions for chemiluminescent western blotting. Incubation of primary anti-RsaFa was done at a 1/1000 dilution for colorimetric and 1/5000 dilution for chemiluminescent western blotting. Anti-RsaD antibodies were incubated at a 1/1000 dilution for colorimetric and 1/2000 dilution for chemiluminescent westerns. Anti-RsaE antibodies were used at 1/5000 dilution for colorimetric and 1/10000 dilution for chemiluminescent western blotting. Incubation of secondary anti-rabbit HRP was used at 1/5000 dilution for colorimetric and 1/15000 dilution for chemiluminescence western blotting. Kodak X - O M A T LS film was used for visualization of chemiluminescent blots while spot densitometry was done using a Bio-Rad VersaDoc 5000 system and the Quantity One (V4.3.0) program. 2.11- Electron microscopy A l l electron microscopy studies were carried out by Dr. J. Smit. Protein A -colloidal gold immunolabelling of C. crescentus strains was performed as described 30 previously (60) using the anti-RsaFa antibody and 5 nm protein A-colloidal gold label prepared as previously described (59). The antibody was preabsorbed with JS1009 cells. Cells (1.0 OD600 ) were centrefuged at 16000 X g for 5 min, and then the cell pellet was resuspended in 10 m M Tris-HCl pH 8 and sonicated for 5 sec using a microprobe. Sonicated cells were then centrifuged at 16000 X g for 2 min and the supernatant was removed. Anti-RsaFa rabbit serum was added to resuspend the pellet, and the suspension was then put on ice for 1 h. The serum suspension was centrifuged at 16000 X g for 3 min and then the adsorbed serum was collected. Cells were imaged unstained by whole mount transmission electron microscopy. 2.12- Bioinformatic analysis and protein threading A l l sequences were obtained from The Institute for Genomic Research (TIGR) C. crescentus genome database. The Biology Workbench website http://workbench.sdsc.edu/ was utilized for protein sequence alignments using BLASTP sequence alignment tools (1). ClustalW alignments of RsaFa, RsaFb and TolC were done using Mac Vector V 6.0. Protein threading was done using the Swiss Model program(56) using the TIGR RsaFa (CC1015) and RsaFb ( C C D 18) protein sequences and the TolC Pdb file (IEK9). Cartoon models of the predicted threaded structures were generated using the Deep View/ Swiss-Pdb Viewer V3.7(29, 48) and the Swiss Model generated RsaFa and RsaFb Pdb files. 3D rendering was used to generate the space fill and ribbon models. Ribbon models were altered with colored sections to define a-helical and [3-sheet segments. Electrostatic surfaces were predicted using electrostatic potentials of-3.00 to 3.00. 31 3. R E S U L T S - Transcriptional regulation of the rsaADE genes 3.01- Identification of the rsaADE genes The rsaA gene and its respective secretion apparatus were previously identified in our lab. The genes encoding the A B C transporter (rsaD) and the MFP (rsaE) were characterized by Dr. Peter Awram using Tn5 insertions. rsaA and the first two transporter components were found next to each other, but the two OMPs were located elsewhere. The promoter for rsaA was identified when the S-layer protein was initially characterized (61). rsaA contains a strong promoter and is often used in gene expression studies as a housekeeping gene(42) since rsaA is transcribed throughout the cell cycle with little to no change(24). A r/zo-independent terminator is found 40 bp 3' of the rsaA translational stop, which is located 162 bp 5' from the rsaD start codon. Based on studies defining the transcriptional regulation of the E. coli HlyA system(75), it was assumed that the promoter from rsaA read through the r/zo-independent terminator on occasion allowing for the transcription of rsaD and rsaE. Since no promoters were identified at the time, this hypothesis was assumed to be true. 3.02- Identification of a potential rsaD promoter When I suspected that a promoter upstream of rsaD existed, I decided to use in-silico methods to elucidate possible promoter sites. The Softberry B P R O M program was used to predict i f there were any possible promoter sequences. The nucleotide sequence from the stop codon of rsaA to the start codon of rsaD was used as a possible region of interest. When the 242 bp region was inserted into the B P R O M program it predicted a promoter -140 bp 5' of rsaD (Fig. 3-1.). The putative -35 site (rsaD -35(1)) is located 32 from 147 to 141 bp upstream from the rsaD start codon. The putative -10 site is located 123 to 115 bp 5'of the rsaD start site. The ribosomal binding site was predicted to be in the region 11 to 7 bp 5' of the start codon. A n alternate -35 site (rsaD -35(2)) was found closer to the -10 site after analysis of the intergenic space using a previously predicted consensus C. crescentus promoter sequence (41). The rsaD -35(1) site lies 19 bp away from the -10 site, whereas the rsaD -35(2) site is only 10 bp from the -10 site. Of the two predicted -35 sites, the latter -35 site makes the spacing of the predicted (-35, -10) binding sites similar to those of identified promoters in C. crescentus. As well, the rsaD -35(2) site has a better fit with the consensus -35 site as the TTG bases are highly conserved with the second T being conserved for all identified promoter sites (Fig. 3-2.). Although the B P R O M program boasts 80 % accuracy in the identification of promoters, these are based on the E. coli olO and may not relate directly to C. crescentus. I therefore suggest that the architecture of the promoter region consists of the rsaD -35(2) site and the predicted -10 site. These results are significant as they predict a potential promoter site to support the later molecular based findings, described below. ^ ^ ^ ^ ^ ^ • • ^ • £ ' : " J > T A J J G C G A A C G T C T G A T C C T C G C C T A G G C G A G G A T C G C T A G A C T A A G A G A C C C C G T C T T C C - A A A 1 G G A G G C G G G G T C T T T C T J J A T G G G C G C T A C G C G C T G G C C G G C C T O g C C J A G T T C C G G T G ^ ^ T G A T T T A G C G G G A C T G G G G G G C T T G C T C A C T T T C C G C C A C A A T T T C G T G G T C G A G A C G G C G C C T T A G T T G T T A C T G T A C A T G G C C G C G T C G G T T C G C G C G G C G T C C T G A A G G C T C A C A A T G T T C A A G C G C A G C G G C G C G A A G C C G A C G A T C T T C G A C C A G G C C G T G C T G G T C G C C C G C C C G G C G G T G A T C A C C G C C A T G G T C T T C A G C T T C T T C A T C A A C A T T C T G G C C C T G G T C A G C C C G C T G T A C A T G ,—, CZI - rho indep terminator - rsaA - predicted promoter 1 = 1 ~ r s a D • - predicted RBS Figure 3-1. In-silico predicted rsaD promoter orientation. Predicted rsaD promoter found using the Softberry B P R O M program. Identified -10 and -35 sites are highlighted ~115 bp upstream of the rsaD gene. Two potential -35 sites are shown as both show similarity to consensus C .crescentus promoter sequences. The promoter site is located far enough away from the rho-independent terminator to allow for polymerase binding. Consensus -35 - T T G ACQ Consensus -10 - G C T A N A ( A A T ) C rsaD -35(1) - T G G C C G rsaD-10 - G C T A T G A T rsaD -35(2) - T T G C C T Figure 3-2. Predicted rsaD promoter sites. Predicted rsaD promoters as compared to predicted consensus promoter sequences. Both -35 sites appear similar to the consensus site, but the rsaD -35 (2) site appears a better fit due to the consensus T T G bases being identical. In addition, the 135 bp region separating rsaD and rsaE was also analyzed to determine if there was a potential promoter. The B P R O M program predicted that there were no promoters in the region, and no sites show significant similarity to predicted C. crescentus promoter sequences. Experiments detailed below further suggest that there is no promoter between the rsaD and rsaE genes. 3.03- Absence of rsaE promoter determined As stated, rsaE is located directly after rsaD with only 135 bp separating the two genes. When studies were carried out by Dr. Peter Awram, there were no convenient tools to identify a possible promoter and thus the system was assumed to be analogous to HlyA. Creation of polyclonal antibodies to RsaD and RsaE has allowed for determination of any possible rsaE promoter. Using the previously created Tn5 rsaD knockout strain (B15) I was able to show that there was no promoter between the two genes. Whole-cell protein samples of the B15 (rsaD) and wild type NA1000 strains were run on SDS-PAGE gels, and western blots were probed with anti-RsaD antibodies to determine presence or absence of RsaD (Fig. 3-3a.). As expected, the NA1000 strain contained RsaD whereas no RsaD was present in the BI5 strain. Note, that since the RsaD antibodies were generated using protein from inclusion bodies, they resulted in high background levels. Therefore, further results show only anti-RsaE westerns, despite both westerns being carried out. Western blots using anti-RsaE antibodies were conducted, and the NA1000 strain showed the presence of RsaE while the B15 strain did not ( F i g . 3-3b.). If there was a rsaE promoter located in the intergenic space between rsaD and rsaE, then presumably the promoter would still be active despite the Tn5 insertion. Thus since no rsaE product could be detected in the rsaD Tn5 mutant, the rsaD and rsaE genes must have been transcribed using the same promoter. B15 NA1000 B15 NA1000 -60kDa -40kDa Figure 3.3 a-b. Characterization of CB15A B15. a. Absence o f RsaD in the C B 1 5 A B15 strain (rsaD). Chemiluminescent western blot using anti-RsaD shows no RsaD (62.0 kDa) in the B15 strain but RsaD is present in N A 1 0 0 0 . b. Absence o f RsaE in B15 strain is shown by chemiluminescent western blot. Us ing anti-RsaE no RsaE (48.4 kDa) is seen in the B 1 5 strain (rsaD) where as RsaE is present in the N A 1 0 0 0 strain. 3.04-Identification of the rsaDE promoter To elucidate the transcriptional control of rsaD and rsaE, two separate molecular methods were utilized. The first involved the CB15 A-rsaA strain, created by Assaf Levi, which contained a deleted rsaA and rsaA promoter region. A region starting 242 bp 5' of the translational start site of rsaA and continuing approximately 1 kb further upstream was amplified, therefore excluding the rsaA promoter. Another fragment beginning a few 35 bp 5' of the rsaA translational stop codon and continuing ~1 kb 3' of rsaA was amplified. These two products were ligated together making a fragment that did not contain rsaA and its promoter (see materials and methods). The ligated product was then put into the pNPTS138 suicide vector and used for gene replacement. Loss of rsaA was confirmed by PCR screening for a deletion form product of rsaA (not shown) and the resulting strain was called CB15 A-rsaA. Western blotting using the anti 188/784 RsaA antibodies shows that no RsaA is produced by the CB15 A-rsaA strain (Fig. 3-4 a.). The CB15 A-rsaA was then used to examine transcription of rsaADE. If transcription of rsaD and rsaE is dependent on the rsaA promoter, then transcription of rsaD and rsaE in the CB15 A-rsaA strain cannot occur using the rsaA promoter as it is deleted. On the other hand, i f transcription of rsaD and rsaE is independent of the rsaA promoter, then another promoter must be present between rsaA and rsaD. Western blots were done to determine i f RsaD and RsaE were present. Both the wild type NA1000 and the CB15A-rsaA strain contain RsaD (not shown) as well as RsaE (Fig. 3-4 b.). In order to confirm that the transporter proteins are active, a plasmid borne copy of rsaA was complemented into the CB15 A-rsaA strain. If rsaD and rsaE are transcribed and the proteins are active, then addition of the rsaA gene should lead to S-layer secretion. Secretion of S-layer is evident in the complemented strain (Fig. 3-4 c ) . Levels of S-layer secretion appear to be similar to wild-type levels, suggesting that production of RsaD and RsaE is not affected by the loss of the chromosomal copy of rsaA and its promoter. 36 N A 1 nnO A-rsnA R1S \-rsnA N A I nnn A-rsaA A-rsaA:vWB9 NA1000 C Figure 3-4 a-c. Characterization of the CB15A-rsaA strain. a. Anti-188/784 RsaA chemiluminescent western of whole culture preparation reveals that there is no RsaA in the CB15A-rsaA strain (rsaA'), unlike wild type NA1000. RsaA marked by arrow. b. Chemiluminescent western blot using Anti-RsaE shows presence of RsaE in NA1000 and CB15 A-rsaA strain {rsaA') while the CB15A B15 strain (rsaD) shows no RsaE. RsaE is marked with arrow. c. Anti- 188/784 RsaA colorimetric western of complemented CBl5A-rsaA strain shows presence of RsaA. No RsaA is present in the CB15A-rsa,4 strain (rsaA')but wild type NA1000 RsaA levels are restored in the CB\5A-rsaA: pWB9: rsaA strain. RsaA marked by arrow. 37 To confirm that the putative rsaDE promoter was actually driving transcription of rsaD and rsaE, a second molecular method was undertaken. The B15 Tn5 clone shows no secretion of RsaA since it lacks a functional copy of rsaD (4). Confirmation that it lacks both RsaD and RsaE was shown above (F ig. 3-3.). A strategy using trans-complementation of a plasmid borne copy of rsaADE was adopted to determine transcriptional control. The R A T I fragment(4), which includes rsaADE as well as flanking regions (total length ~ 1 lkb), was used. The RATI fragment was modified so that rsaA was knocked out, but rsaD and rsaE were not. An QCm cassette was inserted into rsaA destroying the gene, and the possibility of read-through transcription using the rsaA promoter. Therefore, i f complementation led to RsaA secretion then rsaD and rsaE must be transcribed using a separate promoter in the intergenic space between rsaA and rsaD. The modified R A T I QCm fragment was inserted into a broad host range plasmid and electroporated into the B15 strain. Western blots of low pH extracted protein showed that addition of the modified R A T I QCm fragment led to RsaA secretion (Fig.3-5 a.). This suggested that the plasmid borne rsaD and rsaE were transcribed and that the S-layer was secreted. To confirm that the plasmid borne rsaD and rsaE were transcribed, western blots were done revealing that both RsaD (not shown) and RsaE were present in the complemented strain (Fig.3-5 b.). 38 wt B15 B15:comp B15:comp B15 wt Figure 3-5 a-b. Characterization of the CB15A BIS: RATI QCm strain. a. Anti-188/784 RsaA chemiluminescent western blot of the complemented B15: RATI QCm (B15:comp) strain shows restoration of RsaA secretion (denoted by arrow). NAI 000 (wt) shows RsaA while the CB15A B15 strain (rsaD) does not. Complementation of the B15 strain with pBBR4: RATI QCm leads to restoration of RsaA secretion suggesting rsaD and rsaE are transcribed. b. Chemiluminescent western blot using anti- RsaE confirms presence of the rsaE gene product. B15: RATI QCm (B15:comp) shows presence of RsaE (shown with arrow) while the B15 strain (rsaD) does not. Levels of RsaE in the complemented strain appear higher than wild type NAI 000 (wt) levels. The combined results suggest that the rsaA promoter was not used for the transcription of the downstream transporter components rsaD and rsaE. Transcription must have occurred at a site just downstream of rsaA after the rAo-independent terminator. Both rsaD and rsaE must be co-transcribed, as no separate promoter exists for rsaE. 39 4. RESULTS-Identification and characterization of the outer membrane proteins of the RsaA secretion system 4.01- Identification of the two rsaF genes Two possible candidates for the OMP were identified in the C. crescentus genome (45) with similarity to the E. coli TolC protein sequence. These have been named rsaFa and rsaFb. RsaFa has 23% identity and 45% similarity to E. coli TolC, and RsaFb has 25% identity and 47% similarity to TolC as determined by local sequence alignment (see materials and methods). rsaFa (1581 bp) is located downstream of rsaADE after a gap of 5025 bp coding for five S-LPS-related genes(5). rsaFb (1452 bp) however, is located 322 Kb (303 genes) away from rsaFa and is flanked by genes of unknown function (Fig. 4-1.). The two rsaF genes share 39% identity and 60% similarity which suggests that RsaFa and RsaFb may have arisen by gene duplication. Unknown function Figure 4-1. Relative location of the rsaA secretion apparatus. The rsaD and rsaE genes are located 3' of the rsaA gene. The rsaFa gene is downstream of 5-S-LPS genes. The rsaFb gene is located 322 kb away flanked by 2 genes of unknown function. The genomic organization of these genes is different from that seen for other OMP components. In many systems all three components of the transporter system are found in sequential order and appear to be part of a single operon. Here are two separate 40 genes, both of which appear to be separately transcribed from the rest of the secretion components unlike that found with other OMP type I secretion components, except the E. coli TolC and S. marcescens HasF proteins (8). The RsaF proteins were aligned with the TolC sequence using Mac Vector 6.0 ClustalW, and predicted a-helix and P-sheet segments were identified (Fig. 4-2.). The RsaFs have a similar predicted secondary structure to TolC and regions of similarity are clearly seen. To predict the tertiary protein structure, the two RsaF proteins were modeled using the solved x-ray structure of the E. coli TolC. Electrostatic surface models show differences between RsaFa and RsaFb as well as significant differences compared to the TolC monomer (Fig. 4-3.). TolC has a predicted negative charge throughout the ct-helical and p- barrel segments. The RsaF models have neutral charges throughout their inner surface, with zones of positive and negative charge exposed at various regions. Both RsaFa and RsaFb exhibit a negative charge in the entrance to the a-helical periplasmic spanning portion, and a gradual progression to positive charges where the monomer shifts from a-helical to p- barrel segments. RsaFa appears to have more positively charged areas than RsaFb, but it does have zones of negative charge, facing the internal chamber of the a-helical segment, caused by clusters of Glu residues (e.g. amino acids 338-340). Both RsaF proteins have regions of positive charge at the entrance and spanning the P-barrel region of the protein. 41 ClusfelW Formalted Align rmnls 170 ISO ISO « "plvJR Q l - O Q ,T-7 I q[ffTl n[V"G]lJV A I .% D V Q N A H A' Q [*]v k Q B D i r t S NJ a A F L i V G I I T Q ' T D V Q O S O A R [A|V>[ t- Q K o LpT7T |T |T ii|k Y| *\V[T]Q y r t T P y Q Q A K A R TrtC flwF* • l V | - |Mtp u w n a n\, K P l FA G B L [Tl T S Q V R p L F rRBRTt N A R V R flirafa yf E Q Qi E Rl ( i _____ *'° Ljv D y L|_HJA] L R T T I D 4a? I V L N A Q A E L|q|N A Q I In 5 ' 9 430 M'.W O J N I a-helical regions (3-sheet regions Figure 4-2. ClustalW alignment of the RsaFa, RsaFb and TolC proteins created using the MacVector 6.0 program. ClustalW alignment shows similarity between both RsaFa and RsaFb as well as TolC. Inserted arrow and rectangles show regions predicted to form a-helical ribbons and (3-sheets. Shown are predicted consensus regions of the three proteins and are not specific to a single protein. Areas of divergence are seen mostly in areas where loops would occur. The RsaF proteins likely form a similar 3D structure as TolC since the predicted secondary structure of the proteins appear similar. The alignment denotes identical and similar amino acids with boxes. Identical amino acids are shaded and similar amino acids are in bold type. 42 Figure 4-3. Predicted 3D-ribbon and space-fill models of RsaFa and RsaFb as well as the TolC monomer. Threaded 3D-ribbon structures of RsaFa (a) and RsaFb (b) as well as the TolC (c) monomer as shown by Deep View/Swiss-Pdb-Viewer v3.7. Ribbon structures were predicted by the Swiss Model program and then modified with colored sections to emphasize separation of the a-helical and f3-barrel regions. Electrostatic surfaces are shown on space-fill models of the monomers. Electrostatic potentials of-3.00 to 3.00 were used to show areas of negative (red) and positive (blue) charge. RsaFa (1) and RsaFb (2) show more neutral and positively charged zones than the TolC (3) monomer. Negatively charged zones appear at the periplasmic entrance of all three proteins. Regions of positive charge appear in both RsaF proteins at the entrance to the p-barrel. As well a cluster of Glu residues (338-340) forms a zone of negative charge in the a-helical channel of RsaFa. Identification of possible OMPs was done by searching for homologs of TolC in C. crescentus using the BLASTp tool on TIGR. RsaFa and RsaFb show significant similarity to the a-Hemolysin TolC protein, whereas the other protein hits appear more related to other non-type I OMPs. Initial B L A S T analysis of the CB15 genome showed 4 hits. Two (RsaFa and RsaFb) had significant scores and low E values (1.1 e"22 (RsaFb) and 1.3 e"16 (RsaFa)) while the other hits (CC0806 and CC1785) had moderate scores and E values. CC0806 and CC1785 are likely not type I OMPs as a search against a non-redundant database indicated that CC0806 and CC1785 share higher percent identity with lipoproteins (approx. 40-48%) and TIGRFAMs and Pfams group them in the lipoprotein family. ClustalW alignment of the RsaF proteins and TolC revealed significant similarity between the three proteins. The high similarity of RsaFa and RsaFb suggests that they may be orthologues formed through gene duplication. When predicted secondary regions of the RsaF proteins are compared with TolC, the predicted ct-helical regions appear in stretches of similar amino acids, whereas regions where the sequences show less similarity are in predicted loop and p-sheet regions (Fig. 4-2.). If RsaFa and RsaFb were actually crystallized, their 3-D structure may not be identical to the predicted Swiss Model files. However, the predicted structures do give greater insight into the OMPs possible form and function. Predicted charged regions are of interest as heterologous proteins expressed using the C. crescentus secretion system may be blocked from transport due to charge (47). It is likely that transported proteins carrying a charge would interact with either the negatively charged regions predicted to face the internal cavity of the a-helical region, or with positively charged regions in the 44 P-barrel region (Fig. 4-4.). Regions of charge may function as a 'filtration device' blocking the transport of improperly formed or folded RsaA monomers before they reach the bacterial surface. C. crescentus has been shown to possess a protease (sapA) (69) that scans the S-layer cleaving improperly made RsaA. Due to high levels of C-terminally secreted protein being produced the possible filtration function of RsaF may be an additional level of control in order to inhibit the passage of malformed RsaA. Figure 4-4. Cartoon depiction of charged regions that may block RsaA transport Negatively charged regions in the a-helical region such as the stretches of Glu residues may block transport. Alternatively, positively charged regions located throughout the |3-barrel region may also hinder RsaA secretion. Charged zones may act as a filter for improperly made or folded RsaA monomers. When improperly formed RsaA monomers interact with charged regions in the outer membrane protein, secretion is blocked and the RsaA monomer is pushed back into the cytoplasm. AS 4.02- Internal deletions of the rsaF genes Initially I intended to knockout rsaFa and rsaFb by gene replacement using internally deleted forms of the rsaFs. A 2.7 kb fragment, containing rsaFa and flanking regions of-1000 bp 5' and -200 bp 3' was amplified by PCR. Internal deletions of-1.3 kb, 852 bp, and 638 bp were created in rsaFa (outlined in materials and methods). Attempts to insert an internal deletion form by gene replacement using a suicide vector (pK18mobsacB) consistently resulted in reversion to the wild type rsaFa. Gene replacements of altered forms of rsaFa were attempted in both the NA1000 (S-layer+) and CB2A (S-layer) strains. From the many hundreds of clones screened by PCR, only the rsaFaAKP (852 bp) internal deletion resulted in a correct gene replacement event in the CB2A strain. This gene replacement was confirmed by PCR (data not shown) and the strain was called CB2A rsaFaAKP. rsaFb was also targeted for gene swapping using internal deletions. An internal deletion in rsaFb of 1051 bp was made. A l l attempts to get an internal deletion form to recombine properly failed. Since antibiotic insertion and N - and C-terminal methods of destroying the rsaF genes had worked, efforts to create internal deletions were stopped. 46 4.03- Disruption of the rsaFa and rsaFb genes Since the internal deletion strategy was unsuccessful, knockouts were constructed by homologous recombination of inactivated rsaF genes in the wild type S-layer positive C. crescentus strain NA1000 using alternate methods. Disruption of the rsaFa gene was , ? ^ 4 performed by gene replacement of an rsaFa gene construct containing an antibiotic-resistance cassette. The rsaFaQSm gene fragment was inserted into the chromosome using a suicide vector and a mutant resulting from the gene replacement event was identified by PCR (Fig. 4-5.) and has been designated JS1007. Figure 4-5. PCR confirmation of rsaFa knockout. Antibiotic insertion form of rsaFa shown in at 3.5Kb in the JS1007 strain. Lanel- NA1000, Lane 2-JS1007, Lane 3- pBSKIIEEH: rsaFanSm, Lane 4- l-Hindlll marker (sizes from top to bottom 22kb, 12kb, 6.6kb, 4.4kb, 2.4kb, 2.2kb). The rsaFb gene was disrupted via insertional inactivation, using an N - and C-terminally deleted rsaFb gene fragment (rsaFb AN AC). The rsaFb ANAC gene fragment was inserted into the chromosome via homologous recombination of a non-replicating plasmid, resulting in tandem non-functional copies of the rsaFb gene. Confirmation by PCR showed that there were only disrupted forms of rsaFb in the chromosome (Fig. 4-6.). Using one primer from the plasmid and one primer from the gene I was able to confirm insertion, after which a mutant was selected and called JS1008. In order to create the double-knockout strain, the rsaFa gene was disrupted in the JS1008 strain in the same 47 manner as in the JS1007 strain. This double rsaF knockout strain was confirmed by PCR (not shown) for disruption of both rsaF genes and designated JS1009. To determine that RsaFa and RsaFb were not produced in the knockout strains, western blot analysis was done using polyclonal anti-RsaFa antibodies. Rabbit polyclonal antibodies were generated against a GST tagged rsaFa gene product. The proteins can be differentiated by size; RsaFa is 57.5 kDa and RsaFb is 1 2 3 4 50.2 kDa. Cross-reactivity between the two proteins was evident probably due to the similarity of the RsaFs. For this reason, anti-RsaFb antibodies were not produced. Curiously, the anti-RsaFa antibodies reacted better with the RsaFb protein. Whether this is due to increased binding efficiency, or relative levels of the RsaF proteins, is unknown. Figure 4-6. PCR confirmation of rsaFb knockout. N-term C-term deletion form of rsaFb shown in at -600 bp in the JS1009 and JS1008 strain. Lanel- lOObp ladder, Lane 2- JS1009, Lane 3- JS1008, Lane 4- X-Hindlll marker (sizes from top to bottom 22kb, 12kb, 6.6kb, 4.4kb, 2.4kb, 2.2kb). The cross reactivity of the polyclonal antibodies allowed us to see the loss of the RsaF proteins in the knockout strains (Fig. 4-7.). Progressive loss of RsaFa and RsaFb was evident in western blots of the rsaF knockouts. Densitometry analysis of the western blots showed that levels of the remaining RsaF in the single rsaF knockouts are the same as those observed in the wild-type strains. This suggests that both rsaFa and rsaFb are similar to the E. coli tolC and S. marcescens hasF, and are transcribed separately from an export/ secretion system. 48 wt AFb AFa AFa/Fb Figure 4-7. RsaFa and RsaFb levels in wild type and rsaF kDa-RsaFa knockout strains. Loss of RsaF proteins was kDa-RsaFb determined using whole-culture-protein samples of the wild type and knockout strains. Developed film of chemiluminescent anti-RsaFa western blot showed loss of the RsaF proteins. The amount of protein loaded per lane corresponded to 20(4.1 of whole-culture preparation and samples were run on 12% SDS-PAGE gel and transferred to nitrocellulose membrane. Wild type NA1000, independent rsaFa and rsaFb knockouts and double rsaF knockouts were compared. Both RsaFa (~57kDa) and RsaFb (~50kDa) can be seen in the wild type NA1000 (wt). Loss of RsaFb was evident in the JS1008 strain (rsaFb') (AFb) as only RsaFa was seen. Similarly, only RsaFb was seen in the JS1007 (rsaFa') strain (AFa). The RsaF double knockout JS1009 (rsaFa/rsaFb') (AFa/Fb) showed neither RsaFa nor RsaFb. 4.04- Effect of disruption of the rsaF genes on S-layer secretion Independent gene knockouts were created and levels of RsaA secreted by the rsaF knockout strains were analyzed. Neither single knockout led to a complete loss of S-layer secretion, but levels of RsaA secretion did decrease in the two single knockouts. Because S-layer secretion was not completely abolished, a mutant was created with both rsaFa and rsaFb knocked out, resulting in what appeared to be an S-layer negative strain. When low pH extracted protein levels were compared between the knockout and wild type strains, there was a progressive decrease in levels of S-layer secretion as rsaFa and rsaFb were lost (Fig. 4-8a and b.). Disrupting rsaFa decreased S-layer secretion to a greater extent than loss of rsaFb, but both single rsaF mutants were still capable of secreting RsaA. Levels of S-layer secretion could not be easily determined through coomassie stained SDS-PAGE, as small amounts of S-layer secretion are not easily seen, and therefore western blotting was performed. 49 wt (rsaA') AFa/Fb AFa AFb wt wt(rsaA) AFa/Fb AFa AFb wt U 6 k D a -96 k D a -66 k D a -44 k D a --116 k D a -98 k D a -54 k D a Figure 4-8. a- b. Effect of disruption of the rsaF genes on S-layer secretion. Levels of RsaA as determined by low pH extraction on wild type and rsaF knockouts. Normalized levels of cells were used for preparations and were run on a 7.5% SDS-PAGE gel. Coomassie stained SDS PAGE (a) and chemiluminescence western blot (b) using anti-188/784 shows the effect of rsaF gene disruption on levels of RsaA. S-layer protein (98 kDa) can be clearly seen due to its high level of production, marked by arrows. No detectable S-layer is observed in the S-layer negative JS1003 strain (wt-rsaA). Minor levels of S-layer can be seen in JS1009 (rsaFa /rsaFb') caused by burst cells during low pH extraction, whereas definite levels of S-layer can be seen in the JS1007 (rsaFa) and JS1008 (rsaFb') strains. Levels in both single rsaF knockouts do not secrete as much S-layer protein as that seen in the wild type NAI000 strain. Quantification of S-layer secretion levels by chemiluminescence western blotting showed that disruption of rsaFb decreases RsaA secretion by 24% whereas loss of rsaFa decreases S-layer secretion by 46% from those of wild type NAI000 levels (Table 2.). This suggested that RsaFa is more important to S-layer secretion as loss of rsaFa led to a more significant decrease in RsaA secretion. Since Coomassie stained gels were not able to show small amounts of S-layer protein, western blotting was able to reveal a small amount of RsaA in the JS1009 (rsaFa/ rsaFb) strain. S-layer protein (4% of wild type) seen in the double rsaF knockout strain appeared to be due to release of RsaA from cells burst during the low pH extraction and further experiments were carried out to determine if the levels of S-layer were internal RsaA. 50 Strain %RsaA to wild type Low pH extracted Whole culture NA1000 100% 100% JS1008 76% 78% JS1007 54% 56% JS1009 4% 9% JS1003 0% 0% JS1009: rsaFa 78% 80% JS1009: rsaFb 56% 57% JSlOOl -na- 95% Table 2. Comparison of RsaA levels as determined by whole culture preparations or low pH extraction. Spot densitometry of chemiluminescence western blots using polyclonal anti-188/784 were used to determine relative levels of RsaA produced by cells. Levels determined by densitometry were compared to wild type NA1000 levels. All low pH and whole culture protein preparations were normalized prior to running samples. To confirm that levels of S-layer secretion in the double rsaF knockout were due to burst cells during low pH extraction, whole-cell preparations were performed to determine levels of S-layer inside the cells. To make sure that only internal RsaA was analyzed, certain strains were subjected to other protein extraction methods (outlined in materials and methods) before whole-cell-protein preparations were done. Colorimetric western blots showed levels of internal RsaA (Fig. 4-9.). Levels of RsaA produced in the double rsaF knockout were higher than those seen by low pH extraction, however the levels did appear very similar to levels seen in the filtered JSlOOl strain. Both the JS1009 and the JSlOOl internal RsaA levels were less than those seen in the NA1000 strain. The levels of RsaA in the JSlOOl strain were only internal levels of RsaA, as all transported protein was shed (S-LPS") and removed (filtration and washing) suggesting that RsaA seen in the double rsaF knockout was also held internally. 51 w t rsaFa/rsaFb' S - L P S - w t (rsaA) -116kDa -98 kDa -54 kDa Figure 4-9. Determination of internal levels of RsaA in the rsaF double knockout Whole cell preparations of wild type, JSlOOl, JS1003 and JS1009 strains were run on 7.5% SDS-PAGE gel and transferred to nitrocellulose membrane for colorimetric western blot. NA1000 was subjected to low pH extraction prior to whole cell preparation, removing RsaA that was crystallized on the cell surface, to ensure that only internal RsaA levels would be observed. Similarly, JSlOOl strain was poured through fine mesh filter to remove aggregated RsaA before whole cell preparation. Equal amounts of whole cell protein preparations were loaded onto the gel polyclonal anti 188/784 RsaA antibody was used for western blotting. The Arrow indicates full length RsaA. Wild type NA1000 levels (wt) appear much greater than those seen in both the knockout JS1009 (rsaFa/rsaFb) and JSlOOl (S-LPS") strains. The JS1003 strain (wt (rsaA')) shows no visible S-layer protein. The JSlOOl strain has no S-LPS, and the shed aggregates were strained off, levels of RsaA observed should be totally internal. Therefore, since the level of RsaA in the JS1009 strain is similar to the JSlOOl strain it would suggest that RsaA observed is internal. 4.05- Complementation of the secretion deficient JS1009 strain To further demonstrate that the rsaF knockouts were responsible for reduction or loss of the S-layer secretion, I complemented the rsaF knockouts in trans using a multiple copy broad host range plasmid. In both cases, complementation restored partial secretion (Fig. 4-10.). Interestingly, /raws-complementation of the rsaF genes only restored S-layer secretion to levels similar to that seen in the single rsaF knockouts (Table 2). Levels of RsaA secretion in the JS1009: rsaFa and JS1009: rsaFb strains were restored to levels - 2 % greater than those seen in the single rsaF knockouts. Due to the lack of additional antibiotic markers, complementation of both rsaF genes into the double knockout was not carried out. 52 wt rsaFb' rsaFa' Fa'/Fb -130 kDa -100 kDa -72 kDa Figure 4-10. Complementation of the rsaF genes in trans recovers S-layer secretion. JS1009 strain was complemented with the either rsaFa or rsaFb using the medium copy pBBR4 vector. Levels of RsaA were determined by low pH extraction and run on 7.5% SDS-PAGE gel. Chemiluminescent western blotting using anti-188/784 RsaA antibodies allowed for quantification of RsaA. RsaA secretion is recovered to similar levels as those seen in the single rsaF knockouts with the trans-complemented JS1009 strains having fractionally higher levels of RsaA secretion. The rsaFa complemented strain (lane 2) has 78% of wild type levels, and the rsaFb complemented strain (lane 3) at 56% of wild type levels. The JS1009 strain (lane 4) was run to show effect of complementation. The trans complementation of the rsaF genes does suggest that the rsaF gene products are involved in RsaA transport. Strains were then assessed to directly determine the extent to which the plasmid borne copies of rsaF were expressed. Culture preparations were done, and using polyclonal antibodies against RsaFa, expression of both plasmid borne rsaF genes was determined by chemiluminescence western blotting (Fig. 4-11.). Expression of RsaFa was 9.72 ± 0.71 times greater, and RsaFb 8.01 ± 0.66 times greater, than wild type levels. Independent knockouts suggested that there is a greater requirement for RsaFa, as loss of the protein lowers RsaA secretion by 46% where as loss of RsaFb only leads to a 24% decrease (Fig. 4-8.). For this reason one would expect that i f RsaFa is more capable of handling secretion of the S-layer protein, then increasing its levels in the bacterium should lead to greater RsaA secretion. However, despite significantly increasing RsaFa levels, when RsaFb is not present, recovery of wild type levels of RsaA secretion cannot be achieved. This suggested that although RsaFa appears to be more important, RsaFb is essential to achieving wild-type levels of RsaA secretion. If the RsaF proteins formed independent homotrimeric units, then a large increase in either protein should lead to recovery of wild type S-layer secretion levels in the 53 JS1009 strain. Since neither complemented strains produced levels significantly above those seen in the single rsaF knockouts, this suggests that instead of forming independent homotrimeric units, the two OMPs might form a heterotrimeric unit that is required for maximal secretion (Fig. 4-12.). wt Fa /Fb" F b + F a + 55 kDa-40 kDa-RsaFa RsaFb Figure 4-11. Expression of the RsaF proteins in the complemented JS1009 strain. rsaFa and rsaFb were transformed into the JS1009 strain using the pBBR4 plasmid and whole-culture preparations were run on 12% SDS-PAGE gel. Chemiluminescence western blot using the anti-RsaFa antibodies showed recovery of RsaFa and RsaFb in the respective complemented JS1009 strain. The wild type NA1000 strain (wt) showed presence of both RsaFa and RsaFb. JS1009 (rsaFa/rsaFb') had neither RsaF protein. RsaFb was present in the JS1009: rsaFb complement strain (rsaFb+) and similarly RsaFa is present in the JS1009: rsaFa strain (rsaFa+). Western blotting revealed that not only were the plasmid borne rsaF genes transcribed, but that levels of RsaFa were 9.72 ± 0.72 times greater than wild type, and RsaFb was 8.01. ± 0.66 fold more than wild type levels as determined through densitometry. 54 RsaA monomer Figure 4-12. Cartoon depiction of heterotrimer formation. A combination of three RsaF monomers would come together to form one hetertrimeric unit which would associate with the A B C transporter and MFP. The heterotrimeric unit would be some combination of the two RsaF proteins (may not be the combination depicted) with one being the more frequently utilized protein. 4.06- Production of RsaA appears to be regulated when secretion is impeded Since the type I apparatus is efficient I sought to determine i f levels of RsaA secretion and RsaA production were similar. I determined that the production of RsaA appeared to be regulated by the amount that could be transported out of the cell. Densitometry performed through chemiluminescence western blotting revealed levels of RsaA from whole-culture-protein preparations were almost identical to those observed through low pH extraction (Fig. 4-13.). Western blots showed the effect of knocking out rsaFb reduces S-layer production to 78% of wild type levels, and destruction of rsaFa reduces RsaA production to 56% of wild type levels (Table 2). Levels of RsaA seen with the double knockout strain were somewhat higher than those seen by low pH extraction at 9% of wild type N A I 000 levels. The levels of RsaA found by whole-culture preparation were much more representative of total RsaA produced in the cell than those obtained through low pH extraction. wt rsaFb' rsaFa' rsaFa'/rsaFb' -130 kDa -100 kDa -72 kDa Figure 4-13. RsaA production and RsaA secretion levels are comparable suggesting that little residual S-layer is left inside of the cell. Chemiluminescent western blot analysis of whole-culture preparations using anti 188/784 RsaA antibodies allowed comparison of RsaA levels in the rsaF knockouts. Progressive decrease in RsaA levels was seen as the rsaF genes were knocked out. NAI 000 (wt) RsaA levels were not seen in the JS1008 (rsaFb) or JS1007 (rsaFa') strains. Comparable levels of S-layer were seen in whole-culture preparations, to those observed by low pH extraction. The JS1009 (rsaFa'/rsaFb') strain shows levels of internal RsaA at 9% of NAI00 levels. Whole-culture preparations may be the best estimate for RsaA production levels, since internal and external levels of RsaA can be analyzed in the same preparation. 56 Whole-culture and low pH extracted levels of RsaA were only fractionally different, varying approximately 2%. The ability to readily compare different methods of protein extraction is not only valuable from an experimental perspective, but it also shows there is almost no level of RsaA built up inside of the cell, and that the levels seen on the surface of the cell are representative of the level of total RsaA produced. These results suggest that the level of RsaA produced in the cell may be regulated by the amount of protein that can be secreted, as there was a definite down-regulation of RsaA production in the RsaF mutants. I decided to pursue possible RsaA regulation by using the JS1009 strain and a strain which had an inserted charged a.a. region in RsaA. Heterologous proteins with charged regions have been found to have problems being transported through the RsaA secretion apparatus, as they likely interact with charged residues in the outer membrane component (47). A plasmid borne copy of rsaA was modified by insertion of nucleotides coding a 4 amino acid PJOCR furin cleavage site at the 723 amino acid site and inserted into the JS1003 S-layer negative strain. Protein levels of the transporter mutant and the modified RsaA mutant were analyzed. Levels of RsaA produced by the JS1003: Hpsl2furin strain are similar to that of the RsaF double knockout strain (Fig. 4-14.). Internal levels of RsaA were 5% in the modified RsaA strain, whereas levels in the RsaF double knockout strain were 9% of wild type levels. If RsaA is expressed constitutively then one might expect that internal RsaA would be degraded by internal proteases. Cells which can not transport the S-layer protein, would then either degrade the accumulated RsaA or down-regulate its production. However, no breakdown products of RsaA were present in the bacteria and so I presumed the latter option is the case. 57 The JS1009 (rsaFa/ rsaFb) and JS1003: Hpsl2furin strains grew much poorer than wild type strains suggesting that accumulation of RsaA inside the cell leads to some degree of metabolic imbalance (Table 3.). In addition to poor growth, there were very few motile cells and most cells were misshapen and elongated. Cultures grew at normal rates until exponential phase, and then cell growth rate slowed (Fig. 4-15 a-b.). When rsaFa is inserted into the JS1009 strain, growth rates increase towards wild type levels. These results would suggest that poor growth is due to accumulation of RsaA inside of the cell. Taken together, these results suggest that there may be a 'feedback' loop for RsaA production, and that buildup of RsaA inside the cell leads to stoppage of rsaA transcription or translation. wt FaVFb" RsaA:furin wt (rsaA-) -130 k D a -100 k D a -72 k D a Figure 4-14. Impeded RsaA transport in rsaF (JS1009) mutant and RsaA (11 ps 121 in\\\) mutant. Whole-cell preparations were loaded on 12% SDS-PAGE gels and compared by chemiluminescent western blotting using anti 188/784 RsaA antibodies. The NAI000 strain was subjected to low pH extraction before whole-cell preparations were done to minimize levels of S-layer. No RsaA breakdown products were present in the mutants. Wild type NAI000 was used as a positive S-layer control (wt). Both the JS1009 strain (rsaFa'/rsaFb') and the JS1003: Hpsl2furin strain (RsaA:furin) showed no evidence of breakdown products. All RsaA protein was in a non-degraded form, with levels of RsaA appearing similar for both mutant strains. The JS1003 strain showed no RsaA (wt (rsaA")). These results suggested that when RsaA begins to accumulate inside of the cell, no breakdown occurred, and that some type of RsaA regulation was present. 58 Figure 4-15 a. Exponential growth curve of knockout and modified RsaA strain. The JS1009 and JS1003: Hpsl2furin strains grow significantly slower than wild type strains. Introduction of rsaFa to the JS10Q9 strain recovers growth rates to a degree. Plots shown are averages of three runs Logarithmic growth curve 1000 < o v E 3 C O - • - J S 1 0 0 9 - • — J S 1009: rsaFa NA1000 - K - J S 1 0 0 3 - * - J S 1 0 0 3 : Hps12furin Figure 4-15 b. Logarithmic growth curve of knockout and modified RsaA strain. All strains were grown at 30 °C at 200 rpm and OD600 was taken every hour. Three runs were done and average generation times were calculated for the strains. 59 Strain Generation time (min) N A I 000 JS1009 78 114 96 79 108 JS1009: rsaFa JS1003 JSl003:Hpsl2furin Tab le 3: Genera t ion t imes of the C. crescentus mutant strains. rsaA is like most other S-layer genes and is transcribed continuously throughout the cell cycle(24). In Lactobacillus brevis the S-layer gene (slpA) is transcribed during stationary phase even when the S-layer protein is not produced (34). If transcription occurs continuously, then it is likely that regulation would occur at the translational level. The S-layer of Thermus thermophilus HB8 has been shown to autoregulate the translation ofslpA by binding of a C-terminal SlpA fragment to the 5'end of the mRNA (23). Whether a similar situation occurs in C. crescentus is still unknown and further examination is needed, however it is likely that some sort of S-layer regulation exists. It is possible that built up RsaA inside of the cell binds to mRNA transcripts and halts production of RsaA (Fig. 4-16.). 60 Figure 4-16. Cartoon depiction of hypothesized autoregulation of the rsaA gene. RsaA production may be affected by autoregulation, where RsaA binds to mRNA transcripts inhibiting translation. 1- Since no OMP is present (or charged proteins interfere with secretion as in Hps 12 furin clone) RsaA is blocked from secretion and thus RsaA sits in the cytoplasm. 2- Free RsaA finds the rsaA mRNA transcript and binds. 3- Ribosomes moving along the mRNA transcript encounter bound RsaA and are blocked or dislodged. 61 4.07- Coordinate Overexpression of RsaA and RsaF Since the RsaA secretion system has been modified for use in recombinant protein production, optimal efficiency of the system is important. Therefore, the effect of additional transporter units in wild-type (rsaFa+, rsaFb+) bacteria was examined to determine i f the RsaA machinery could be induced to secrete more RsaA than normal. Vector borne copies of a single rsaF were inserted into S-layer positive C. crescentus. A S-LPS negative strain, JSlOOl, was used instead of the wild type NA1000 strain. This strain was used to ensure that there is no possibility of RsaA regulation by surface crystallization, or in other words, inhibition of transport when the bacterial surface is "full". Levels of RsaA were initially determined by collection of aggregated protein at first, and later whole-culture-protein preparations were examined. The latter proved to be easier or more valuable since it ensured that any micro-aggregates were included in the sample. Protein aggregates were collected and both wet and dry weights of the RsaA were taken. Ratios of protein to culture density (OD600) were compared to determine if over expression was occuring. JSlOOl strains containing plasmid borne copies of rsaFa or rsaFb showed little or no increase in aggregated RsaA (Fig. 4-17 a - b.). These results suggested that despite increased OMP gene copy RsaA secretion could not be increased. I then inserted additional copies of rsaD and rsaE into the JSlOOl strain. These results showed similar variability, with the JSlOOl: pRAT9 clone actually showing less S-layer secretion than wild type JSlOOl (Fig. 4-17 c) . Since levels of aggregates frequently varied despite controlled conditions, I decided to determine levels of RsaA secretion using an alternate method. 62 rsaFa overexpression O.D. Dry weight (g) Dry/O.D. rsaFb overexpression • J S 1 0 0 1 H J S 1 0 0 1 : rsaFb O.D. Dry weight (g) Dry/O.D. O.D. Dry weight (g) Dry/O.D. I Figure 4-17. a-c. Levels of aggregate production in the JSlOOl as compared to strains with additional copies of the transporter components. a. rsaFa overexpression in JSlOOl does not lead to significantly higher levels of RsaA. Comparison of dry weight/ OD does not show much difference as levels for JSlOOl are 0.262 g/O.D60o± 0.039 and JS\00l:rsaFa values are 0.279 g/O.D 6 0 0 ± 0.009. b. rsaFb overexpression in JSlOOl appears similar to levels in JSlOOl. Comparison of dry weight/ OD does not show significant difference. JSlOOl levels are 0.1 g/O.D6oo± 0.08 and ]S\00l:rsaFb values are 0.110 g/O.D 6 0 0 ± 0.009. c. pRAT9 (rsaD/rsaE) overexpression in JSlOOl appears deleterious to RsaA secretion and levels are lower than those seen in the rsaFa and rsaFb overexpressors. Dry weight to O.D. for JSlOOl was are 0.262 g/O.D 6 0 0 ± 0.039 and for JS1001:pRAT9 was 0.1627g/O.D 6 0 0± 0.104. High fluctuation in the OD and dry weights of the JS1001:pRAT9 led to erratic values. These fluctuations may be due to toxicity of extra RsaD and RsaE. Simultaneous runs were carried out for the JSlOOl, JS1001:reaFa and JS1001:pRAT9 strains. The JS\00l:rsaFb runs were done separately with a new set of JSlOOl controls. Fluctuation of OD values and processing of aggregates for all runs led to varying values. Changes in O.D. values, etc. may be due to stability of media, starting culture, and other variable factors. For this reason, other methods were evaluated. 64 Whole culture protein preparations became the dominant method as it allowed for comparison of both S-LPS mutants and wild-type cells, since surface bound and/ or secreted protein was included in the preparation. In order to ensure that all secreted protein was solubilized, urea was added to 2M (final concentration). Levels of RsaA production were not significantly increased when additional copies of rsaFa or rsaFb were expressed in JS1001 (Fig. 4-18.) (Table 4). Levels of RsaF were also determined to confirm that the rsaF genes were expressed, and levels of the proteins were found to be higher than in JS1001 strain (Fig. 4-19). The increased levels of RsaFa and RsaFb appeared similar to the complementation strains with RsaFa at levels 10.6 fold greater than wild type, and RsaFb levels 8.4 fold greater. wt F a Fb Figure 4-18. Effect of RsaF overexpression in the JS1001 strain. Introduction of plasmid borne copies of either rsaFa or rsaFb in the JS1001 strain led to a slight increase in the secretion of RsaA. Chemiluminescent western blot using anti 188-784 RsaA antibodies of normalized whole culture extracts showed similar levels of RsaA for all strains. Spot densitometry revealed that levels were only increased by 3-4% for the JS1001: rsaFa (rsaFa++) and JS1001: rsaFb (rsaFb*) above wild type JS1001 (wt (S-LPS")) levels. Figure 4-19. Levels of RsaFa and RsaFb in JS1001 strain. Plasmid borne copies of rsaFa and rsaFb result in increased levels of RsaFa (10.6 X wild type) and RsaFb (8.4 X wild type). Wild type JS1001 (wt (S-LPS")) has normal levels while JS1001: rsaFa (rsaFa++) and JS1001: rsaFb (rsaFb++) have elevated levels of their respective overexpressed RsaF. 65 Strain %RsaA to JS1001 JS1001 JS1001: rsaFa JS1001: rsaFb JS1001:pWB9r^ZlP JS1001: rsaFa: pWB9rsaAAP 100% 103% 102% 100% 128% Table 4. Levels of RsaA determined by whole culture preparations. Spot densitometry of chemiluminescence western blots using polyclonal antibodies against 188/784aa RsaA, were used to determine relative levels of RsaA produced by cells. Levels determined by densitometry were compared to JS1001 levels. All whole culture protein preparations were normalized prior to running samples. To be assured that additional RsaFa was properly targeted to the outer membrane, protein A-colloidal gold labeling with anti-RsaF antibody was used to assess levels of RsaF detectable on the outer membrane surface (experiments carried out by Dr. J. Smit). A uniform low-level label was noted with JS1001 (Fig. 4-20.), indicating that some portion of the RsaF OMP was surface exposed when the oligosaccharide chains of the S-LPS fraction of total LPS were eliminated (see below). Label of JS 1001: rsaFa and JS1001: rsaFb (not shown) showed two major classes of cells: those labeled at levels similar or slightly greater than that seen with JS1001 and a fraction (approximately 20% of the total) where a dense label was noted. I interpret that as an indication that plasmid copy numbers for the moderate copy number pBBR4 plasmid vary significantly from cell to cell, suggesting it is not a stably maintained plasmid. Nevertheless it appeared that some cells were expressing much higher levels of RsaFa and that it was targeted correctly to the outer membrane. 66 Figure 4-20. Colloidal gold labeling of surface displayed RsaF. Surface displayed RsaFa was determined for the JSlOOl strains overexpressing rsaFa by electron microscopy using anti-RsaFa and colloidal gold labeling (carried out by Dr. J. Smit). JSlOOl (a) shows wild type levels of RsaF with moderate labeling. The JSlOOl: rsaFa strain (b) shows a significant increase in surface display of RsaF. Note that only about 20% of the JSlOOl: rsaFa cells showed significant increase in RsaF display. Interestingly, in contrast, to JSlOOl (which has no S-LPS), there was no detectable label with strain JS1003, which has no S-layer but does have a normal complement of S-LPS (not shown). Presumably this smooth form of LPS effectively blocks antibody access to the OMPs, perhaps indicating their exposure on the surface is minimal. 67 When both rsaF genes are present and additional copies of rsaFa are introduced, secretion of RsaA is increased only slightly. Levels of RsaA were approximately 3% greater than in wild-type JSlOOl (Fig. 4-18.). These results are similar to experiments in which additional plasmid borne copies of tolC were expressed in Gram-negative bacterial strains containing the HlyA secretion apparatus (64). It was found that despite additional copies of TolC neither enhancing nor deteriorating effects occurred. The authors suggested that the HlyB and HlyD protein levels might be the limiting factor as only the outer membrane protein levels were increased. However, since aggregate experiments showed no significant change in secretion by over expressing rsaD and rsaE and likely led to deleterious effects, I thought other factors might be at play. I speculate that instead of the A B C transporter and MFP levels being the limiting factor, that the transported protein may be the limiting factor. Since JSlOOl has only the single chromosome resident copy of rsaA I considered whether RsaA transcription or translation (and not the OMP levels) might now determine the maximum levels of RsaA secretion. To address this possibility a multi-copy plasmid borne rsaA gene was introduced into the JSlOOl:rsaFa strain. The resulting strain was grown and whole-culture-protein levels were compared (Fig. 4-21.). The resulting strain JSlOOl: rsaFa: rsaA produced -28% more S-layer protein than that seen in the JSlOOl strain. Since the JSlOOl: rsaA strain did not produce more RsaA than both the wild type JSlOOl and the JSlOOl: rsaFa strain, secretion must be a function of both available outer membrane proteins as well as RsaA copies. This is a remarkable increase in RsaA secretion as wild-type levels of RsaA already represent 10-12% of total cell protein and with this increase could represent upwards of 15% of the total cellular protein. I interpret 68 this as an indication that elevated secretion of RsaA is possible but requires both overexpression of RsaA and at least the RsaFa OMP. It is likely that RsaA secretion is dependent on a number of factors; the type and level of both RsaF proteins, and the number of rsaA copies (Fig. 4-22.). wt (S-LPS) rsaA* rsaFa* rsaX^/F*" M M J | ' Figure 4-21. Effect of RsaFa and rsaA overexpression in the JS1001 strain. Increasing RsaA secretion is a function of amount of RsaA to transport and the number of holes in the outer membrane. Chemiluminescent western blots revealed that increased copy numbers of rsaA and rsaFa in the JS1001 strain led to increased RsaA levels (marked by arrow). The JS1001 (wt (S-LPS')), JS100T: rsaA (rsaA++) and JS1001: rsaFa (rsaFa++) appear to have similar levels of RsaA secretion with fractional increase of 3% seen in the JS1001: rsaFa strain. The JS1001: rsaFa: rsaA strain (rsaA++/rsaFa++) had a definite increase in RsaA secretion with levels 28% greater than JS1001. The type I secretion apparatus is able to transport normal levels of RsaA produced by the cell, but cannot accommodate increased RsaA levels unless OMP levels are increased. I speculate that the ability to increase RsaA secretion may be a function of increasing the number of 'holes' in the bacteria. It has been shown that the HlyB and HylD proteins exist in a pre-formed complex in the inner membrane and when HlyA is engaged, the complex recruits the TolC protein (66). The RsaA transport complex is likely analogous, and thus increasing the number of OMPs in the outer membrane may make it easier for the secretion apparatus components to find each other and transport RsaA. 69 Figure 4-22. Cartoon of RsaA secretion and transport system in the overexpressing strains Increase in rsaFa gene (JSlOOl: rsaFa) copy leads to more OMP units in the outer membrane, only slightly increasing RsaA secretion. When rsaFa and rsaA are increased, more RsaA is produced and can be transported due to higher numbers of OMP's in the outer membrane. 7 0 5. DISCUSSION AND CONCLUSION This study reports further characterization of the type I RsaA secretion apparatus, showing that two outer membrane proteins, RsaFa and RsaFb, are involved in RsaA transport. As well, it shows that unlike previously hypothesized, the type I transporter components are transcribed separately from rsaA. These findings may help elucidate how the RsaA secretion apparatus attains such high levels of protein secretion. Type I organization has been based on early findings of the HlyA system. The hlyABCD genes are located together and were found to be co-transcribed (75). Most type I systems have been assumed to be organized and transcribed in a similar manner. To date, only the S. marcescens (Lip) and C. fetus (SapA) systems have been shown to use separate promoters for the S-layer gene and its respective transporter genes. A putative rsaD promoter has been predicted using in-silico methods and consensus promoter sequences. I determined that the -10 site predicted by the B P R O M program was likely correct, but that an alternate -35 site was likely correct based on its location and similarity to a predicted -35 consensus sequence. Analyzing the rsaADE gene set using molecular methods revealed that rsaD and rsaE were co-transcribed using a separate promoter from rsaA. This showed that the RsaA secretion system was not transcribed like the HlyA system, and was more like the Lip system, having a separate promoter for the A B C transporter and MFP. A mechanism where all transporter components are transcribed separately makes sense for a system secreting high levels of protein. If transcription of rsaD and rsaE relied on leakiness of the r/jo-independent terminator, it may be that not enough copies of the transport apparatus would be present to support wild-type S-layer levels. The amount of protein 71 secreted by the E. coli HlyA system is about 0.5% of the total cell protein where as the S-layer of C. crescentus accounts for 10-12% of the total cell protein. Separate transcription of the RsaA secretion apparatus may allow for higher levels of secretion. C. crescentus may have modified the type I secretion gene organization to ensure that the bacterial surface is covered at all times during the cell cycle. In addition to gene organization, C. crescentus may have also adapted to accommodate high levels of protein secretion by duplicating or acquiring an additional OMP. Having two OMPs in RsaFa and RsaFb allows for increased levels of RsaA secretion. Both rsaFa and rsaFb are located away from the type I system and appear to be transcribed irrespective of the transport system. Their isolated location and expression likely allows for increased OMP units in the outer membrane, increasing the chances of complete transporters associating and in turn allowing high levels of secretion. The two OMPs for the RsaA secretion system had been previously identified in the lab using in-silico methods and rsaF knockouts and complementation experiments were attempted, but did not provide any definite results. When this study started, I wanted to knockout rsaFa and rsaFb by gene replacement with internal deletion forms of the two genes. Hundreds of clones were screened by PCR, using a variety of internal deletion sizes unsuccessfully, so new methods to knockout the genes were undertaken. A double knockout was made in the NA1000 strain using antibiotic insertion, and A N AC gene insertion of the rsaF genes. Independent knockouts of rsaFa and rsaFb did not stop S-layer secretion. There was a noticeable decrease in the rsaFa knockout, but the rsaFb knockout appeared only slightly lower than wild type levels. Quantification of these levels gave us a better 72 understanding of the possible importance of the two OMPs. Destruction of rsaFa and rsaFb led to loss of S-layer secretion. Complementation of either gene could not restore RsaA secretion to wild type levels, despite significant increases in the respective RsaF. Indeed, when overexpressed the individual OMPs could only restore secretion to the level of their apparent contribution in the normal, single gene copy situation. This suggests that it may not be simply a matter of a limit on the number of active transporter complexes (when only one OMP is expressed) but on the transporter composition; I tentatively suggest that the trimeric OMP complex may function most effectively when assembled as a heterotrimer of RsaFa and RsaFb and that this may be the native situation. In this scenario, heterotrimers are technically not required for RsaA secretion, though survival in a natural environment may require maximal secretion of RsaA to maintain surface coverage of this protective device. The complemented strains, and RsaA production data suggested a mechanism involving feedback down-regulation of RsaA production. I noted in such situations the cells grew much slower and there were few motile cells and most cells were misshapen and elongated. It may be that i f such an autoregulation is occurring it may not be specific, impacting the synthesis of other proteins as well. If autoregulation is occurring, the levels of RsaA actually secreted and RsaA production should be quite similar. Interestingly I found that levels of RsaA production in the rsaF mutants were similar to the levels of RsaA secretion. Despite RsaA secretion decreasing with the loss of the rsaF genes, there was no accumulation of RsaA inside of the cell. Whole-culture and low pH extracted levels of RsaA protein were only fractionally different, varying by about 2%. Taken together with the effects of complete blockage of protein export, these results suggest that 73 the level of RsaA produced in the cell may be regulated by the amount of protein that can be secreted. This, in turn, may be set by the amount of RsaA that can remain in the cytoplasm as a result of autoregulation. Because I had indications that RsaA autoregulated the amount of protein produced I believed it might be possible to increase expression levels by coordinate increases in RsaA translation rates mated to an increase in the number of transporters. Overexpression of an individual rsaF gene in the absence of the other rsaF gene was presumably not an appropriate course since it did not even lead to normal levels of RsaA secretion, so I instead began overexpressing individual transporter components in the presence of a normal complement of remaining transporter proteins. The effect of additional transporter units in wild-type (rsaFa+, rsaFb+) bacteria was examined to determine i f the RsaA machinery could be induced to secrete more RsaA than normal. By using the S-layer shedding strain JSlOOl, which produces the same amount of RsaA as the parental NA1000, any RsaA feedback regulation due to the presence of a "full" S-layer on the bacterial surface would not affect the results. When both rsaF genes are present and additional copies of rsaFa were introduced, secretion of RsaA is increased only slightly. Similarly, a strain containing additional vector borne copies of the rsaA gene with a wild type complement of rsaFa and rsaFb genes did not exceed wild type RsaA levels. However, the JSlOOl: rsaFa: rsaA strain produced 28% more protein than wild type levels. Moreover, the RsaA overexpression may well be ascribed to the 20% fraction of cells that were truly overexpressing RsaFa from an unstable vector. Presumably stable expression of RsaFa by all cells may lead to still higher levels of RsaA secretion. 74 These experiments have led to a better understanding of the type I secretion system of C. crescentus and of type I systems in general. Little is known about type I secretion system organization and expression, and especially in high level secreting type I systems. I have been able to further characterize an extremely interesting transport apparatus, identifying potential bottlenecks in S-layer secretion as well as suggest possible modifications that may increase the transporter's capabilities. In doing so, I have also created new methods such as whole-culture preparations which allow for comparison of shedding and crystallizing strains, as well as internal levels of RsaA. I propose future experiments to increase gene copies of both rsaF genes so that expression is stable. Finding the optimal combination of the RsaFs as well as determining i f coordinate up-regulated expression of various combinations of all the transporter elements may lead to still greater levels of RsaA expression or whether other factors, such as membrane stability or available ATP to drive the transport process will set limits. 75 REFERENCES 1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman 1997. 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