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Regulation of S-layer synthesis and secretion in Caulobacter crescentus Lau, Janny Ho Yu 2007

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Regulation of S-layer Synthesis and Secretion in Caulobacter crescentus by Janny Ho Yu Lau B.Sc, The University of British Columbia, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in Faculty of Graduate Studies (Microbiology and Immunology) THE UNIVERSITY OF BRITISH C O L U M B I A September 2007 ©Janny Ho Yu Lau, 2007 ABSTRACT Caulobacter crescentus is a non-pathogenic, Gram negative bacterium that forms a surface layer (S-layer) that is composed exclusively of the protein RsaA in a crystalline array. RsaA, which constitutes 10-12% of total cellular protein, is secreted by a robust type I protein transport system in C. crescentus. These features have made this bacterium an attractive candidate for biotechnology applications that require the production of proteins of interest at high levels and purity. In a previous study aimed at determining the maximal protein secretion capability of the type I system in C. crescentus, the overexpression of rsaA did not result in the increased production and secretion of RsaA. However, upon further investigation it was determined that the plasmid used to overexpress rsaA included an extended 5' untranslated region (UTR). The results presented herein suggest that this extended 5' UTR caused a decrease in the half-life of rsaA mRNA to -19 minutes compared to the -36 minutes half-life of wild type rsaA mRNA which may in turn explain the lack of increased RsaA production. By contrast, production and secretion of RsaA was significantly increased (2.2 ±0.1 fold) in C. crescentus transformed with a plasmid containing rsaA without an extended 5' UTR when compared to wild type. Deletion of the outer membrane transporters, RsaFa and RsaFb, prevented the secretion of RsaA and resulted in a significant down-regulation of RsaA production. By using quantitative reverse-transcriptase PCR (qRT-PCR) it was determined that the amount of rsaA mRNA in the transporter deletion mutant was similar to wild type (0.9 ± 0.1-fold of wild type). This suggests that the down-regulation of RsaA observed in these mutants occurred at a posttranscriptional level. Previous experiments showed that recombinant forms of RsaA containing an abundance of positively charged amino acids were not detected at the cell surface, indicating a complete inhibition of secretion. Three dimensional models of RsaFa and RsaFb using the Swiss Model program placed twelve negatively charged amino acids near the entrance of the predicted pore structure on the periplasmic side. In order to test the hypothesis that these negatively charged amino acid residues were inhibiting the secretion of recombinant RsaA, site-directed mutagenesis was used to alter them. However, none of the mutants relieved the inhibition of recombinant RsaA secretion. Moreover, three of the mutants, RsaFb-D395A, RsaFb-E185A/D395A, and RsaFb-D395A/E402A, also inhibited the secretion of wild type RsaA. Taken together, these results demonstrate that RsaA expression can be upregulated and that the type I secretion system of C. crescentus can facilitate this increase. In addition, regulation of RsaA can occur at a posttranscriptional level when its secretion is blocked, as is the case in the outer membrane transporter deletion mutants. Furthermore, site-directed mutagenesis suggests a role for negatively charged amino acids in the secretion of S-layer protein in RsaFb but not RsaFa. TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS ix ACKNOWLEDGEMENTS x 1 INTRODUCTION 1 1.1 Caulobacter crescent us 1 1.2 S-layers of Bacteria 1 1.3 S-layer of Caulobacter crescent us 2 1.4 Type I protein secretion 3 1.5 Secretion of RsaA 4 1.6 Regulation of RsaA secretion 5 1.6.1 Overexpression 5 1.6.2 Null expression 6 1.6.3 Negatively charged amino acids 6 1.7 Biotechnology applications of the RsaA system 11 1.8 Thesis Objectives 12 2 MATERIALS AND METHODS 14 2.1 Bacterial strains, plasmids and growth condition 14 2.2 Plasmid and DNA manipulations 14 2.3 Caulobacter crescentus expression vectors 18 iv 2.4 Construction of plasmid used for gene disruption 20 2.5 Construction of plasmid used to introduce BAC genes 21 2.6 Construction of plasmids for mutagenesis study of RsaFa and RsaFb 22 2.7 Construction of strains with gene disruptions 26 2.8 Construction of strains with the BAC genes 27 2.9 Protein techniques 28 2.10 RNA isolation and cDNA synthesis.... 30 2.11 Determination of mRNA half-lives 31 3 RESULTS 32 3.1 A non-specific extension of the 5' untranslated region of rsaA resulted in decreased mRNA stability 32 3.2 RsaA production and secretion can be increased by overexpression of rsaA alone 36 3.3 Expression of RsaA is dependent to the steady-state mRNA level of rsaA.... 42 3.4 Overexpression of RsaA did not lead to increased expression of RsaA-specific transporters 42 3.5 Overexpression of rsaFa resulted in decreased expression of RsaA 46 3.6 Deletion of rsaFa and rsaFb resulted in decreased RsaA production at the posttranscriptional level 46 3.7 Deletion of rsaD and rsaE led to decreased steady-state mRNA levels of rsaA. 48 3.8 The inhibition of secretion of RsaA recombinant proteins containing a cluster of positively charged amino acids was not reversed by the removal of specific negatively charged amino acids on RsaFa and RsaFb 53 4 DISCUSSION AND CONCLUSION 60 4.1 S-layer production can be increased in Caulobacter crescentus 60 4.2 S-layer-specific transporters can down-regulate the expression of RsaA 62 v 4.3 Certain negatively charged amino acids on RsaFa and RsaFb were not involved in the inhibited secretion of RsaA:RKKR fusion proteins 64 4.4 Conclusion 66 REFERENCES 68 vi LIST OF TABLES Table 2-1: Bacterial Strains and Plasmids used 15 Table 2-2: List of Primers used 23 Table 3-1 Relative levels of RsaA and rsaA m R N A expression in different strains of Caulobacter crescentus 41 Table 3-2 Levels of RsaA and rsaA m R N A expressed in JS2003 cells with rsaA encoded on plasmids that are maintained at different copy numbers inside Caulobacter crescentus 44 Table 3-3 Relative levels of RsaA and rsaA m R N A expression in different RsaA transporter deleted strains of Caulobacter crescentus 50 Table 3-4 Secretion of R saA :RKKR and wi ld type RsaA by the mutated forms of RsaFa and RsaFb 56 L I S T O F F I G U R E S Figure 1-1 Surface electrostatic potentials of the outer membrane proteins RsaFa and RsaFb 9 Figure 1-2 Predicted 3D structure of RsaFa and RsaFb 10 Figure 3-1 Schematic representation of the regulatory regions upstream of wild type rsaA and rsaAASD which includes an extended 5' untranslated region 33 Figure 3-2 Predicted secondary folding of the 5' untranslated region of rsaA mRNA with Mfold 34 Figure 3-3 Effect of extended 5' untranslated region of rsaA on mRNA stability 35 Figure 3-4 Effect of S-layer on increased RsaA secretion 37 Figure 3-5 Expression of RsaA in cells transformed with the p4BrsaAASD construct.... 40 Figure 3-6 Expression of RsaA in cells transformed with plasmids maintained at different copy numbers 43 Figure 3-7 Expression of RsaA-specific transporters in RsaA-overexpressed cells 45 Figure 3-8 Effect of rsaFa and rsaA overexpression on RsaA production 47 Figure 3-9 Effect of rsaFa and rsaFb deletion on RsaA production and secretion 49 Figure 3-10 Effect of increased transcript levels of rsaA on the production of RsaA in rsaFa and rsaFb deleted cells 51 Figure 3-11 Effect of rsaD and rsaE deletion on RsaA secretion and production 52 Figure 3-12 The effect of site-directed mutagenesis of negatively charged amino acids on RsaFa on the secretion blockage of RsaA:RKKR proteins 54 Figure 3-13 The effect of site-directed mutagenesis of negatively charged amino acids on RsaFb on the secretion blockage of RsaA:RKKR proteins 55 Figure 3-14 Effect of site-directed mutagenesis on RsaFb on the secretion of RsaA to the cell surface 58 Figure 3-15 Effect of site-directed mutagenesis on localization of mutant RsaFb to the outer membrane 59 LIST OF ABBREVIATIONS aa Amino acid ABC ATP binding cassette Amp Ampicillin Ampr Ampicillin resistant BAC Replication genes (repB, repA, repC) Cm Chloramphenicol Cmr Chloramphenicol resistance C-terminus carboxy terminus DNA Deoxyribonucleic acid Dnase Deoxyribonuclease EDTA Ethylene diamine tetracetic acid EtBr Ethidium Bromide GSP General secretory system TM Inner Membrane kDa kilodalton Km Kanamycin Kmr Kanamycin resistance MCS Multiple cloning site MFP Membrane fusion protein mg milli-gram ml milli-liter Pg micro-gram Ml micro-liter mRNA Message RNA N-terminus Amino terminus OD6 0 0 Optical density at 600nm OM Outer Membrane OMP Outer membrane protein PCR Polymerase Chain Reaction PYE Peptone Yeast Extract RNA Ribonucleic acid Rnase Ribonuclease RT-PCR Reverse transcription polymerase chain reaction S-layer Surface layer SD Shine Dalgarno S-LPS Smooth lipo-polysaccharide Sm Streptomycin Smr Streptomycin resistance Tris Trishydroxymethylaminomethane UTR Untranslated region 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 guidance and support throughout my project. His patience and understanding is greatly appreciated. I would also like to thank Dr. John Nomellini for his encouragement and his generosity in helping me throughout my stay in the Smit lab. I would like to thank the lab members including Matthew Ford, Corin Forrester, and Sadeem Fayed for their assistance in many aspects of my project. I thank my fellow graduate students whom have offered help in many technical aspects throughout the years. Lastly, I would like to thank my parents, my brother and all my family and friends. Their love and support had carried me through my entire masters thesis. 1 INTRODUCTION 1.1 Caulobacter crescentus Caulobacter crescentus are crescent-shaped, Gram negative, non-pathogenic bacteria found mainly in freshwater and soils [1]. They lead a dimorphic life cycle and are often studied in regards to their development from the swarmer to a stalked cell. They have distinct cell surface organelles including a flagellum, holdfast, stalk and pili that are formed at different times during their development [2]. The surface layer (S-layer) protein RsaA covers the entire cell surface throughout its entire life cycle. 1.2 S-layers of Bacteria S-layers are 2 dimensional crystalline arrays of proteinaceous subunits that are found on the outermost surface of over 400 Gram-positive and Gram-negative archeabacteria and eubacteria [3, 4]. At present, there is limited data on the exact function of an S-layer, although most researchers speculate that it serves as a protective coating [3]. In some specific cases they had been shown to be molecular sieves [5, 6], attachment sites for exoprotein [7, 8], extracellular virulence factors [9, 10] or simply to provide rigidity to the cell [11]. The S-layer is usually composed of a single protein 40-200 kDa in size and that can sometimes be glycosylated. The protein subunits are arranged in a tetragonal or hexagonal lattice with pore sizes ranging from 2-8 nm. Surface layer proteins are usually one of the most abundant proteins in the cell, often making up 10-15% of the total amount of cellular protein [12, 13]. In Gram-positive bacteria, the S-layer is bound to the surface through a peptidoglycan, but in Gram-negative bacteria its attachment involves components of the outer membrane such as lipopolysaccharide (LPS). Most S-layers of Gram-negative bacteria are secreted through the signal peptide (sec)-dependent General Secretory Pathway (GSP) by a type II secretion system [4, 12]. In the type II secretion system, the S-layer protein is transported through the inner membrane (IM) and the outer membranes (OM) in 2 separate processes that require many accessory proteins [14]. An N-terminal secretion signal is used to translocate the S-layer protein from the cytoplasm through the IM to the periplasmic space. This is followed by cleavage of the signal peptide, and transport of the S-layer through the OM [15]. While the last step usually occurs by simple diffusion through a pore, some bacteria such as Aeromonas salmonicida use specialized OM transporters [16]. The S-layer can also be secreted through the type I sec-independent system, but this has only been found to occur in C. crescentus, Campylobacter fetus, and Serratia marcescens [17-19]. 1.3 S-layer of Caulobacter crescentus The S-layer of C. crescentus is made up of a single 98 kDa protein, RsaA [20]. Secreted at high levels and covering the entire cell surface, RsaA can account 10-12% of total cellular protein [18]. Approximately 40,000 monomers of RsaA are formed into a hexagonal crystalline array [21] that may function as a protective coat against viruses, proteases, and parasites such as bdellovibrio-like organisms [4, 22]. The first 225 amino acids at the N-terminus of RsaA are responsible for mediating attachment of the protein to the smooth LPS (S-LPS) present on the OM [23,24]. In mutants lacking S-LPS, RsaA is secreted into the medium and does not form an S-layer [25]. In addition calcium has been shown to be essential for the secretion and proper assembly of RsaA [25]. An uncleaved secretion signal at the C-terminus of RsaA directs the protein to its type I secretion apparatus on the IM [24, 26, 27]. 1.4 Type I protein secretion Type I secretion requires only 3 proteins and involves a sec-independent pathway. It is found in many Gram-negative bacteria including Escherichia coli, Erwinia chrysanthemi, Pseudomonas fluorescens, Serratia marcescens, and Pseudomonas aeruginosa where it is used for the secretion of lipases, toxins and proteases [28, 29]. The transport apparatus includes an ATP binding cassette (ABC) transporter, a membrane fusion protein (MFP) and an outer membrane protein (OMP)[30]. The genes encoding the ABC transporter and the MFP are usually clustered with the target protein. The OMP gene can, on the other hand, be clustered together with the target protein or be found elsewhere on the chromosome. The secreted protein is transported directly from the cytoplasm to the extracellular space in one step with no periplasmic intermediate. Energy for transport is provided from the hydrolysis of ATP by the ABC transporter [30]. Type I-transported proteins all have a C-terminal secretion signal 30-60 amino acids in length and often have glycine rich repeats flanking the secretion signal that are thought to aid in the presentation of the secretion signal to the ABC transporter [28, 31]. Many of the characterized type I secretion systems are found in pathogenic bacteria [29]. The secreted proteins are usually involved in its pathogenesis such as the adenycyclase-hemolysin from Bordetella pertussis, the RtxA toxin from Vibrio cholerae and colicin V from E. coli and they rarely constitute more than 2-3% of the total cellular protein [29]. The most characterized type I secretion system is that of alpha-hemolysin (HlyA) in E. coll HlyA accounts for 2-3% of total cellular protein [32] and the gene is clustered together with its ABC transporter {hlyB) and MFP (hlyD). Transcription of hlyB and hylD occurs as a readthrough product of the hlyA promoter, but the OMP gene tolC is found elsewhere on the chromosome and is transcribed independently. During protein transport the ABC transporter is formed as a homodimer embedded in the IM and assembles into a stable IM complex with the homotrimeric MFP spanning into the periplasm. Engagement of the translocating substrate induces the IM complex to recruit the OMP to form a bridge between the membranes allowing a one step translocation of HlyA into the extracellular space [33, 34]. 1.5 Secretion of RsaA Secretion of RsaA in C. crescentus occurs via a type I system that includes an ABC transporter (RsaD), a membrane fusion protein (RsaE), and the outer membrane protein (RsaF). The C-terminus of RsaA contains an 82 arnino acid long secretion signal and a repeating region of glycine and aspartic acid residues [35]. The RsaD and RsaE were found to be involved in the secretion of RsaA by screening a Tn5 transposon library for the loss of RsaA transport [18]. Although, the OMP of RsaA secretion could not be located using this technique, a more recent study identified 2 putative OMP genes, termed rsaFa and rsaFb that were 39% identical at the amino acid level [36]. Both RsaFa and RsaFb were found to be necessary for the optimal secretion of RsaA [18, 36]. The organization of the genes for RsaA secretion in C. crescentus is different from HlyA in E. coll. RsaA secretion has more in common with other type I secreted S-layers such as those found in C. fetus or S. marcesens where transporter genes are transcribed separately from the target protein [17, 19]. Although rsaD and rsaE are immediately downstream of rsaA, they are co-transcribed from a separate promoter [37]. Downstream of rsaE, and separated by 5 S-LPS genes is rsaFa, which has its own promoter [36]. Meanwhile, rsaFb is located 322 kbp downstream of rsaA and is flanked by unrelated genes [36]. It has been speculated that rsaFb may have been acquired to ensure the high level of RsaA secretion. 1.6 Regulation of RsaA secretion 1.6.1 Overexpression The finer details of RsaA secretion, its regulation and its limitations, are not yet fully understood. For example, the introduction of a plasmid construct encoding an extra copy of rsaA into C. crescentus did not result in increased RsaA secretion. Yet, with the concomitant overexpression of the outer membrane transporter, RsaFa, the secretion of RsaA did increase by 28% over wild type [36]. However, the overexpression of RsaFa alone had no significant effect on the amount of RsaA secreted. These results suggested that the availability of the outer membrane transporter, RsaFa, can be a limiting factor in cells expressing multiple copies of rsaA, but not in wild type cells. To complicate matters, it was later discovered that the plasmid construct used to overexpress rsaA had an extended region in the 5'untranslated region (UTR) immediately upstream of the Shine Dalgarno (SD) site. Several studies have shown that changes in the 5' UTR of the mRNA can affect its stability [38, 39]. Therefore, it is important to ascertain if the lack of increased RsaA secretion in cells overexpressing multiple copies of rsaA observed previously by Toporowski et al. [36] is due to decreased mRNA stability or due to other regulatory mechanisms. 1.6.2 Null expression The deletion of rsaFa and rsaFb blocked all RsaA secretion, but very little RsaA was accumulated inside the cell [36]. The same phenotype was observed when RsaD or RsaE was deleted. It was suggested that RsaA production was not regulated at the transcriptional level since rsaA is normally transcribed throughout the life cycle [40]. But there were no direct studies done to determine if transcription of rsaA was sustained when S-layer secretion is blocked. 1.6.3 Negatively charged amino acids Previously, a recombinant form of RsaA (RsaA:RKKR) was engineered to include a short peptide sequence containing the furin cleavage site; a cluster of positively charged amino acids consisting of RKKR [36]. Strains of C. crescentus that were negative for wild type RsaA and positive for RsaA:RKKR expression had undetectable levels of secreted RsaA:RKKR, as determined by surface protein extraction, and minimal amounts of intracellular RsaA:RKKR, as determined by whole cell protein preparations [36]. Similar results were also observed in rsaA deletion strains expressing recombinant forms of RsaA containing other short peptide sequences with a cluster of positively charged amino acids or RsaA fused to proteins such as lysozyme that contain many positively charged amino acids [41]. This suggests that either an abundance of, or a cluster of positively charged amino acids on the secreted protein can inhibit its ability to translocate through the outer membrane of C. crescentus. A likely mechanism of inhibition may involve the interaction of the positively charged amino acids on the secreted protein with negatively charged amino acids present on the outer membrane transporters, RsaFa and RsaFb. Although no experimental 3D structure of either transporter is currently available, the crystal structure of TolC, the OMP transporter of E. coli has been solved by Koronakis and colleagues [42]. The structure revealed 2 distinct sections in the trimeric TolC pore, a B-barrel portion embedded in the OM and an a-helical domain which forms a channel through the periplasm [42]. Since RsaFa was 23% and RsaFb was 25% identical to TolC at the amino acid level, it was possible, with the aid of Dr. Michael Murphy (University of British Columbia), to thread their sequence through the TolC structure using the tertiary protein structure prediction program Swiss Model [43]. Figure 1-1 shows that RsaFa and RsaFb can fold into a 3D structure forming a channel similar to TolC. Furthermore, several negatively charged amino acids were mapped to the inner wall of the channel near the entrance of the predicted pore structure on the periplasmic side of both RsaFa and RsaFb (Figure 1-2). It is possible that the interactions between these charges are sufficient to anchor the protein to the transporter resulting in a physical block of secretion. As tested with TolC reconstituted in black lipid membrane, conductance through the transport pore can be blocked by the addition of trivalent cations, such as chromium. It was suggested that these cations were bound to the negatively charged aspartic acid residue present near the perisplasmic entrance of TolC [44]. In C. crescentus, the divalent cation calcium was shown to be necessary for the secretion of RsaA [25]. These negatively charged amino acids near the periplasmic entrance in RsaFa and RsaFb might, on the other hand, be responsible for the accumulation of calcium to facilitate RsaA secretion. Therefore, it may also be possible that secretion of the recombinant RsaA containing an abundance of or a cluster of positively charged amino acids can indirectly inhibit secretion by interfering with calcium binding. RsaFa RsaFb TolC 0 Positive Figure 1-1 Surface electrostatic potentials of the outer membrane proteins RsaFa and RsaFb Predicted structures of the outer membrane proteins RsaFa and RsaFb from C. crescentus and the solved structure of TolC from E. coli, with their surface electrostatic potentials shown. A number of negatively charged amino acid residues were mapped to the interior periplasmic entrance of the transporters. Electrostatic potential surfaces of the OMPs at electrostatic potentials from -3.5 to 3.5 were generated by the Swiss PDB viewer. Negative potential is colored red and positive potential is colored blue. Figure generated by Dr. John Smit, used with permission. D188 Fa Figure 1-2 Predicted 3D structure of RsaFa and RsaFb Ribbon structures of RsaFa and RsaFb were predicted using the Swiss Model program and the structure of TolC as the starting template. The P-barrel regions are colored yellow while the a-helical regions are colored red and blue for RsaFa and RsaFb, respectively. Several negatively charged amino acid residues were mapped to the inner wall of the channel near the entrance of the putative pore structure on the periplasmic side of RsaFa and RsaFb. 10 1.7 Biotechnology applications of the RsaA system At present, the bacterial systems used for the secretion of heterologous proteins involve either the sec-dependent pathway or the type I sec-independent pathway. They have served as invaluable tools in the production of vaccines, hormones, antibodies and are useful for other therapeutic purposes. Examples of recombinant proteins that have been produced for therapeutic uses include rh-insulin analogs, h-parathyroid hormones (rPTH), and r-Cholera toxin B subunit [45]. However, they do have several limitations including low yields of secretion of the protein of interest that are also often in the presence of contaminants such as endotoxin and proteases whose separation require laborious and costly purification steps. By contrast, C. crescentus secretes only its S-layer constituent, RsaA, and at extremely high levels. And although it is Gram-negative, studies have shown that its form of LPS has a low endotoxin potential [41]. Therefore, the secretion system of C. crescentus may offer researchers an upgrade over previous systems for the expression of heterologous proteins in terms of efficiency, scale, purity and cost. Heterologous protein production by the RsaA system of C. crescentus can be utilized as a secretion system or as an antigen display system. As a secretion system, proteins of interest are fused to the C-terminus of RsaA. By eliminating the N-terminus of RsaA, the secreted RsaA fusion protein of interest can no longer attach to the cell surface or crystallize. Instead it is secreted into the growth medium and aggregates to form a mucin-like mass that is 95% pure and can easily be isolated by filtration and solublized in urea [41]. Soluble RsaA fusion proteins of interest can be isolated with other techniques such as affinity or size exclusion chromatography. Proteins that have been secreted using the RsaA system include those from Salmonid fish viruses and domains II and IV of anthrax protective antigens [41]. Alternatively, proteins of interest can be displayed on the surface of C. crescentus when their sequences are inserted into the full length RsaA. Previous studies have identified sites in the full length RsaA that can tolerate insertion of foreign peptide sequences containing 20-600 amino acids [46] such as the MUC1 conserved sequence of mucins and cadmium-binding peptides [41]. The ability to manipulate C. crescentus to display a variety of proteins and peptides can be developed into powerful biotechnology and research tools. For example, various viral and bacterial antigens can be displayed on the cell surface of C. crescentus and examined for their potential as a whole cell vaccine by measuring their ability to induce an immune response [41]. Moreover, researchers are pursuing the possible anti-tumor effect of displaying tumor-targeting antibodies on C. crescentus (Smit and colleagues, unpublished results). In addition, efforts are under way to develop C. crescentus displaying the Fc binding domain of Streptococcal protein G as a low cost immuno-precipitation tool [47]. More recently, this system has been advanced to allow the simultaneous display of two different peptides on the cell surface. 1.8 Thesis Objectives The S-layer component RsaA of Caulobacter crescentus is secreted by a robust type I protein transport system. It holds the potential of being an efficient method of generating large amounts of heterologous proteins at high purity and at low cost. However, many details of RsaA secretion, such as its regulation and its limitations, are not yet fully understood. Previous studies have suggested that RsaA secretion can be increased, and that the availability of the OMP transporter, RsaFa, and not the expression of RsaA, was the limiting factor. However, stability of rsaA mRNA may also have played a role. Other results suggest that RsaA may also be regulated at the posttranscriptional level, at least in mutants deficient in both OMP transporters, where secretion of RsaA does not occur. In addition, it has also been noted that several RsaA fusion proteins containing an abundance of positively charged amino acids were not able to be secreted. Therefore the study of the regulations of this high level protein secretion system continues to be intriguing. Listed below are the objectives of this thesis: la) To determine if secretion of S-layer can be increased by overexpressing rsaA alone. lb) To determine if the lack of increased RsaA secretion in cells overexpressing multiple copies of rsaA observed in a previous study was due to decreased mRNA stability caused by an extended 5' untranslated region. 2) To ascertain whether the down-regulation of RsaA occurs at the posttranscriptional level in mutants deficient in transporters specific for RsaA secretion. 3) To determine if specific negatively charged amino acids present on RsaFa and RsaFb play a role in inhibiting the secretion of certain RsaA fusion proteins containing a cluster of positively charged amino acids. 2 MATERIALS AND METHODS 2.1 Bacterial stains, plasmids and growth condition Strains and plasmids used in this study are listed in Table 2-1. E. coli ToplO F' and DH5a cells are used for E. coli cloning manipulations. E. coli was grown in Luria Broth (1% tryptone, 0.5% NaCI, 0.5% yeast extract) at 37°C with 1.3% agar for plates. C. crescentus was grown in PYE medium (0.2% peptone, 0.1% yeast extract, 0.01% CaCb, 0.02% MgS04) at 30°C with 1.3% agar for plates. Ampicillin, kanamycin, and streptomycin were used at 50 pg/ml, and chloramphenicol was used at 20 pg/ml in E.coli cultures. Kanamycin and streptomycin were used at 25 pg/ml and chloramphenicol was used at 2 pg/ml in C. crescentus cultures. Antifoam 204 was added to 0.02% in cultures grown for whole culture protein preparations to prevent the formation of RsaA aggregates. 2.2 Plasmid and DNA manipulations Standard methods of DNA manipulations were used [48]. Isolation of plasmid DNA was performed using the Qiaprep spin mini prep (Qiagen) system eluting in elution buffer or water. Restriction enzyme digestions were performed with Invitrogen or New England Biolabs Inc. enzymes and buffers specified by the manufacturers. Plasmid DNA was separated and analyzed by electrophoresis on 0.9% TBE agarose gels with 0.5 pg/ml EtBr running at 80-120V. DNA fragments were isolated and excised from gels to be purified using the Qiaex II gel extraction kit (Qiagen) following the manufacturer's protocol. Ligations were done with T4 DNA ligase from Invitrogen according to manufacturer's Table 2-1: Bacterial Strains and Plasmids used Bacterial Strain Relevant properties Reference C. crescentus NA1000 Ap r syn-1000; variant of wild type strain CB15 that synchronizes well A T C C 19089 A RsaA CB15, rsaA gene knocked out deleting rsaA promoter and portion of rsaA gene [36] JS1001 S-LPS mutant of N A 1000, sheds S-layer into medium [25] JS1010 NalOOO, rsaFa negative strain 1231 JS1018 N A 1000, rsaFa and rsaFb negative strain This study JS1019 JS 1001 with B A C replication genes inserted in xylX gene This Study JS1020 NA1000 with B A C replication genes inserted in xyIX gene This Study JS1021 NA1000, rsaFa and rsaFb negative strain, with B A C replication genes inserted in the xy/Xgene This Study JS2003 A RsaA with B A C replication genes inserted in xylX gene This study JS2006 A RsaA, rsaFa and rsaFb negative strain This study B15 Km', Smr, NA1000 Tn5 insertion in the rsaD gene [18] E. Coli DH5a F-cp80IacZAMl 5A(lacZYA-argF)U 169 recA 1 endAl, gyrA96 thi-\ hsdRll supE44 relA\ phoA Invitrogen Top 10 Y-mcrA A(mrr-hasRMS-mcrBC) (pSQlacZAMl5MacX74 recAX araD\39A(araleu) 7696 galU gal) rpsL (StrR) endA] nupG Invitrogen Plasmids pBSKII ColEl cloning vector, lacZ; Ap r Stratagene pBSKIIEEH Modified pBSKII cloning vector with modified MCS; Ap r [36] pBSKIIESH Modified pBSKII cloning vector with modified MCS; Ap r [36] pUC8CVX pUC based cloning vector; Cm r [471 . pTZ18UB:rsa^AP A promoterless version of rsaA flanked by £coRI and Sstl inside pTZ18UB [46] pTZ19R Cloning vector; Ap r Fermentas pTZ\9R:rsaA600 rsaA gene and wild type rsaA promoter inserted into Hindlll and Sst\ of pTZ19R; Ap r This study pH45Q Source of SM cassette removed as Sma\ fragment; Smr [49] pBBR3 Board host range vector derived from p B B R l ; Sm r This study pBBR3:rsaA600 rsaA gene and wild type rsaA promoter inserted into Hindlll and EcoRl of pBBR3; Sm r This study pBBR4 . Broad host range vector; Km' [36] pBBR4:rsaFa rsaFa gene inserted in EcoRl and BamlAl of pBBR4; Km' [36] Plasmids Relevant properties Reference pBBR4:rsaFb rsaFb gene inserted in iscoRI and BamHl of pBBR4; Km' [36] pBBR4:rsaD/rsaE rsaD and rsaE gene with it native promoter inserted into the Spel and Kpn\ site of pBBR4; Km r This study p4A E. coli and C. crescentus shuttle vector with modified rsaA promoter; Cm r [23] p4B Derivative of p4A; Cm r This study p4B:rsaA600 rsaA gene and wild type rsaA promoter inserted into HindUl and EcoRl of p4B; Cm' This study p4B:rsaAASD rsaA gene inserted after the modified rsaA promoter as EcoRl-Hindlll fragment; Cm' This study pWB9 pKT215 derived expression vector incorporating the modified rsaA promoter; Cm r Smr [24] pWB9:rsaAAP rsaA gene and modified rsaA promoter; Cm' Sm r [461 pWB9:rsaA600 rsaA gene and wild type rsaA promoter inserted into HindlU and EcoRl of pWB9; Cm r Sm r This study pWB9:723/furin rsaA containing a furin cleavage site (RKKR) at aa723; Cm r Sm r [36] pkl8mobsacB E. coli based suicide vector, OriT sacB; Km' Sues [50] pkl 8mobsacB:r,raFaAKP pkl8mobsacB containing rsaFa with an internal deletion; Km' Sues [23] pk 18mobsacB :rsaFb A220 pkl8mobsacB containing rsaFb with an internal deletion of 230 bp; Km' Sues This study pkl8mobsacB:xy/A2BAC AES pkl8mobsacB containing the B A C genes within the xy/Xgene; Km' Sues This study pBSKII:rsaFa-PA Subclone fragment of rsaFa from Pst\ XoApal for site directed mutagenesis; Ap' This study pBSKII:rsaFa-AK Subclone fragment of rsaFa from Apa\ to Kpn\ for site directed mutagenesis; Ap' This study pBSKII:rsaF6-EVN Subclone fragment of rsaFb from EcoRV to Notl for site directed mutagenesis; Ap' This study pBSKII:r«jFZ>-NE Subclone fragment of rsaFb from Notl to EcoRl for site directed mutagenesis; Ap' This study p B B R 4 : « a F a - D 1 7 4 A Expression vector of rsaFa with alanine replacement at aa 174; Km' This study pBBR4 :rsaFa-D 176 A/E177 A Expression vector of rsaFa with alanine replacement at aa 176 and 177; Km' This study pBBR4 :rsaFa-D 188A Expression vector of rsaFa with alanine replacement at aa 188; Km' This study p B B R 4 : « a F a -E368A/E369A/E370A Expression vector of rsaFa with alanine replacement at aa 368, 369 and 370; Km' This study pBBR4:ra«Fa-E383A/E384A/E385A Expression vector of rsaFa with alanine replacement at aa 383, 384, and 385; Km' This study pBBR4:r.saFa-E394A Expression vector of rsaFa with alanine replacement at aa 394; Km' This study p B B R 4 : « a F a - E 4 0 1 A Expression vector of rsaFa with alanine replacement at aa 401; Km' This study pBBR4:ra3Fa-E3 68A/E3 69A/E3 70A/E401A Expression vector of rsaFa with alanine replacement at aa 368, 369, 370, and 401; Km' This study pBBR4 :rsaFb-E 171 A / E 172 A Expression vector of rsaFb with alanine replacement at aa 171 and 172; Km' This study Plasmids Relevant properties Reference pBBR4:rsaFZ>-D185A Expression vector of rsaFb with alanine replacement at aa 185; Km ' This study pBBR4:rsaF6-E385A Expression vector of rsaFb with alanine replacement at aa 385; Km ' This study pBBR4:rajF6-D395A Expression vector of rsaFb with alanine replacement at aa 395; Km r This study pBBR4:«aF6-E402A Expression vector of rsaFb with alanine replacement at aa 402; Km r This study pBBR4:r.saF6-D395A/D185A Expression vector of rsaFb with alanine replacement at aa 395 and 185; Km ' This study pBBR4:/-saF6-D395A/E402A Expression vector of rsaFb with alanine replacement at aa 395 and 402; Km ' This study protocol. Fill-in of DNA overhang from digestion was performed with the large fragment Klenow polymerase from Invitrogen. All PCR products were generated using Platinum Pfic DNA polymerase (Invitrogen). Electroporation of C. crescentus was performed as previously described [51]. 2.3 Caulobacter crescentus expression vectors pBBR3 Dr. John Nomellini (University of British Columbia) constructed the plasmid pBBR3 from plasmids pBBRlMCS and pH45Q [49]. The Q streptomycin resistance cassette was removed from pH45Q as a Hindlll fragment and the ends were blunted by Klenow large fragment polymerase (Invitrogen). A 300 bp fragment of the Cmr encoding gene in pBBRlMCS was removed by cutting with Dral and replaced with the blunted Q streptomycin fragment to produce a Smr broad host range vector that replicates in C. crescentus. pBBR3:rsaA600 This plasmid was constructed by Dr. John Nomellini (University of British Columbia) by PCR amplification of the rsaA and 630 bps 5' of the gene including a Hindlll site from the chromosome of NA1000. This PCR product called rsaA600 includes the rsaA and the native promoter region of rsaA. The flanking sites of rsaA600 are Hindlll and EcoW. vTZ\9R:rsaA600 The fragment of rsaA and its promoter was removed from pBBR3:rsaA600 by-treatment with Hindlll and Sstl. The fragment was ligated into pTZl 9R plasmid. p4B This is a derivative of the p4A plasmid [47] modified by digesting at the BamHl just upstream of the EcoRI site and filling in the ends with Klenow polymerase. The plasmid was then blunt ligated resulting in a plasmid without a BamHl site. p4B:rsaA600 The rsaA600 fragment was removed from pTZl9R:rsaA600 by treatment with Hindlll and EcoRI and ligated into p4B plasmid. p4B:rsaAASD This construct was made by Dr. John Nomellini (University of British Columbia), where the rsaA without its promoter was excised from pTZ18U-APrsaA and ligated into the p4B plasmid next to the modified rsaA promoter region as an EcoRI and Hindlll fragment. pWB9:rsaA600 The rsaA600 fragment was removed from pTZ19R:rsaA600 by Hindlll and Sstl. The modified rsaA promoter region in pWB9 was excised and replaced by the rsaA600 fragment which has the native promoter region. 2.4 Construction of plasmid used for gene disruption pKl 8mobsacB:rsaFbA220 This construct was used for making internal deletions to rsaFb. 220 bps of rsaFb between bases 646 to 866 was deleted. The 5' portion rsaFbA220(l) containing 84 bps 5' to rsaFb and the first 645 bps of rsaFb was amplified by PCR using the following primers, FbXHl-F (5'-CCC AAG CTT AAC TGG CGA CGA CTG GCG G-3') and FbSpl-R (5'-GGA CTA GTG TAG GAG GCG CGG ATG ACT TCC AA-3') which will amplify a 728 bps product that is flanked by Hindlll and Spel (shown in bold). The back portion of rsaFbA220(2) was a 881 bps product flanked by Spel and BamHl (shown in bold) amplified by PCR using the following primers, FbSp2-F (5'-GGA CTA GTG TTC TGG CGT CGG CAG CCA GTT TGA-3') and FbXB2-R (5'-CGG GAT CCG TCA ATT CCA GAA CCT CGT-3') which contains the 3' 576 bps of rsaFb and 304 bps downstream of rsaFb. The two PCR products amplified were blunt ligated into pBSKIIESH [36] cut with Stul resulting in pBSKIIESH:728 and pBSKIIESH:881. pBSKIIESH:881 was then cut with EcoRl and Hindlll to release the Fb881 fragment and cloned into pUC8CVX [47] yielding pUC8CVX:Fb881. pBSKIIESH:728 and pUC8CVX:Fb881 were then digested at the Spel site and ligated together. The ligation was transformed into E. coli and plated on LB ampicillin and chloramphenicol to select for pBSKIIESH:728-pUC8CVX:Fb881. This plasmid was then digested with Hindlll and BamHl to release the internally deleted rsaFb fragment of 1609 bps and ligated into the Hindlll and BamYil sites of pK18mobsacB. 2.5 Construction of plasmid used to introduce BAC genes pKl 8mobsacB:jcv/X2BACAES This construct was made by Louis Lam (University of British Columbia) to insert the BAC replication genes into C. crescentus at the xylX gene that is needed for xylose utilization. Within the xylX there are unique Pstl and EcoRI sites. The xyLX region with lkb of flanking sequence at each end were amplified using the primers LLxylX2F (5'-ACG ACG TCG TTG GTG TTG GAC GGG-3') and LLxylX2R (5'-GCG GAT CCG GCA TTC GCC GGG GAG GTC GG-3') from NA1000 (wild type strain) which would add a BamHl site (shown in bold) at the end of the PCR product. The PCR product was amplified using Taq polymerase (New England Biolabs) and was cloned into pTOPO using the TA cloning method (Invitrogen). The PCR product was then excised as a Hindlll and BamHl fragment and cloned into the same site in the MCS of pBSKII to make pBSKII:xy/X2. To isolate the BACAES replication genes from pKT215 which also had the OriV genes, the plasmid was linearized with Pstl and fused with pBSKII and transformations with this plasmid was selected on ampicillin and streptomycin. This fusion plasmid was then cut with Eco0\09 and re-ligated to excise out the 2745bp of pKT215 that held the OriV, while leaving the 5023 bp of the BAC genes in pBSKII creating pBSKILBAC . The BAC genes from pBSKILBAC were then moved into pUC18 as a Kpnl and Pstl fragment to add Hindlll and EcoRI flanking sites to the BAC genes. The BAC genes were then cut from pUCl 8 first with Hindlll which was filled in with Klenow followed by a digest with EcoRI. This blunted Hindlll - EcoRI BAC fragment was cloned into the pBSKII:jcy/X2 that was first cut with Pstl and filled in with Klenow and then cut with EcoRl. This resulted in the BAC genes cloned into the middle of the xy/X gene while also removing a small portion of the xylX. The xylX2 BAC fusion segment was then ligated into pK18mobsacB as a Hindlll and BamHl fragment. 2.6 Construction of plasmids for mutagenesis study of RsaFa and RsaFb pBSKII:^ aFfl-PA, pBBR4:rsaFa-D174A. pBBR4:rmFa-D176A/E177A. pBBR4:rmFa-D188A The 316 bps segment between the Pstl(502) and Apal(%\8) sites in rsaFa were digested with the respective enzyme from pBSKIIEEH:/-5,aFa and cloned into the MCS of pBSKII resulting in pBSKII:rsaFa-PA. This plasmid was used to perform site-directed mutagenesis of rsaFa for amino acids D174, D176 and D188. These were all changed to alanine residues. Mutations were achieved using the Quikchange® method (Stratagene) using the Platinum Pjx DNA polymerase (Invitrogen) and the respective primers listed in Table 2-2. The 316 bps rsaFa segment carrying the mutations were then transferred back into pBSKIIEEHirsoFa at the Pstl and Apal sites. The full rsaFa now carrying the mutations were excised with BamHl and EcoRl and ligated into pBBR4 to be expressed in C. crescentus. Table 2-2: List of Primers used Primer name Sequence JL174Fa-F 5'-CAG A A G C A A T T G A A G GCG A C T G A G G A C A A G T A C AGC-3' JL174Fa-R 5'-GCT GTA C T T GTC CTC A G T CGC C T T C A A T T G CTT CTG-3' JL176Fa-F 5'-CAA TTG A A G G A C A C C GCG GCT A A G T A C A G C GTC CGT C-3' JL176Fa-R 5'-GAC GGA CGC TGT A C T T A G CCG CGG TGT CCT T C A A T T G-3' JL188Fa-F 5'-CAG GTG A C C T T G A C C GCG GTG C A G C A G G C C AAG-3 ' JL188Fa-R 5'-CTT GGC CTG C T G C A C CGC GGT C A A GGT C A C CTG-3' JL368Fa-F 5'-CGC T G G T C A GCC T C G C A G C G G C A A T G A A G G C C A A C A CG-3' JL368Fa-R 5'-CGT GTT GGC CTT CAT TGC CGC TGC G A G GCT G A C C A G CG-3' JL383Fa-F 5'-CTA TGG GGT GCG CGC A G C TGC GCG T T T CGC GCT TCG-3' JL383Fa-R 5 ' -CGAAGC GCG A A A CGC GCA GCT GCG CGC A C C CCA TAG-3' JL394Fa-F 5'-CGC TTC GCA GCA CGA T A G C T G TGC T G A A C G CCC AAG-3 ' JL394Fa-R 5'-CTT GGG CGT T C A G C A C A G C T A T C G TGC TGC G A A GCG-3' JL401Fa-F 5'-GAG CGC C C A A G C C G C A T T GCA G A A CGC C-3' JL401Fa-R 5'-GGC GTT CTG C A A TGC GGC T T G GGC GTT C-3' JL171Fb-F 5'-CTG C A G CGC C A G C T G GCC GCC TCG A A C GCT CGC TTC-3' JL171Fb-R 5'-GAA GCG A G C GTT CGA GGC GGC C A G CTG GCG CTG CAG-3' JL185Fb-F 5'-GAG A T C A C C CGG A C C GCG GTC GCC C A G T C T CAG-3' JL185Fb-R 5'-CTG A G A CTG GGC G A C CGC GT CCG GGT G A T CTC-3' JL385Fb-F 5'-GAA GGC GTG CGT C A G GCT C A G C A G GTC GGC C T G CGG-3' JL385Fb-R 5'-CCG C A G GCC GAC C T G CTG A G C CTG A C G C A C GCC TTC-3' JL395Fb-F 5'-GCG G A C G A C GCT GGC A G T A C T G A A CGC C C A G A T G-3' JL395Fb-R 5'-CAG CTG GGC GTT C A G T A C TGC C A G CGT CGT C C A C-3' JL402Fb-F 5'-CTG A A C GCC C A G C T A GCT CTG TCC A A C GCC G A A C-3' JL402Fb-R 5'-GTT CGG CGT TGG A C A G A G C T A GCT GGG CGT T C A G-3' FbXHl-F 5'-CCC A A G C T T A A C TGG CGA C G A C T G GCG G-3' FbSpl-R 5'-GGA C T A GTG T A G G A G GCG CGG A T G A C T TCC AA-3 ' FbSp2-F 5'-GGA C T A GTG TTC TGG CGT CGG C A G CCA GTT TGA-3' FbXB2-R 5'-CGG G A T CCG T C A A T T CCA G A A CCT CGT-3' RsaA5P-F 5'-TGT TGC C A T C C A G C A C T A C C A GTT-3' RsaA5P-R 5'-TGG C C A GGT TGA T C G A G A A G T TGA-3' 16Srna-F 5'-TAA TTC G A A G C A A C G CGC A G G ACC-3 ' 16Sma-R 5'-TGC GGG C A T T A A CCC A A C A T C TCA-3' pB SKII: rsaFa-AK, pBBR4:r5aFa-E368A/E369A/E370A,pBBR4:r5aFa-E383A/E384AyE385A, pBBR4:rsaFa-Ei94A, pBBR4:rsaFa-E401A The 537 bps segment between the Apal{%\%) and Kpnl{\?>55) sites in rsaFa were digested with the respective enzymes from pBSKIIEEHirsoFa and cloned into the MSC of pBSKII resulting in pBSKII:raii<a-AK. This plasmid was used to perform site-directed mutagenesis of rsaFa for amino acids E368, E383, E394 and E401 using the method mentioned above. The 537 bps rsaFa segment carrying the mutations was ligated back into pBSKJIEEHirmFa at the Apal and Kpnl sites as above and also moved into pBBR4. pBSKII:rsaF6-EVN. pBBR4:rsaFb-E 171A/E172A, pBBR4:rsaFb-Dl85A The 558 bps segment between the £coRV(229) and NotI(787) sites of rsaFb were digested with the respective enzymes from pBSKIIEEH:rsaF6 and cloned into the MCS of pBSKII resulting in pBSKJI:rsaF6-EVN. This plasmid was used to perform site directed mutagenesis of rsaFb for amino acids D171 and D185 using the methods mentioned above, and this 558 bps rsaFa segment carrying the mutations was also moved into pBBR4. pBSKJI:reaFft-NE. pBBR4:rsaFo-E385A. pBBR4:rsaF6-D395A. pBBR4:rmFo-E402A The 665 bps segment between the Notl(l%l) and £coRI(1452) sites of rsaFb were digested with the respective enzymes from pBSKIIEEH:rmF6 and cloned into the MCS of pBSKII resulting in pBSKII:r,saFb-NE. This plasmid was used to perform site directed mutagenesis of rsaFb for amino acids E385, D395 and E402 using the methods mentioned above, and these rsaFb mutants were also moved into pBBR4. pBBR4:rsaFa-E368A/E369A/E370A/E401A The pBSKIIEEH: rsaFa-E368A/E369A/E370A was used as the template for site directed mutagenesis for position E401 in rsaFa using the JL401Fa primers from Table 2-2. The full rsaFa now carrying the 2 mutations were excised with BamHl and £coRI and ligated into pBBR4 to be expressed in C. crescentus. pBBR4:rsflF6-D395A/E402A The pBSKIIEEH: rsaFb-E395A was used as the template for site directed mutagenesis for position E402 in rsaFb using the JL402Fb primers from Table 2-2. The full rsaFb now carrying the 2 mutations were excised with BamHl and EcoRI and ligated into pBBR4 to be expressed in C. crescentus. pBBR4:rsaFo-D395A/D185A The EcoRV to Notl segment of pBSKII:rsaF&-EVN-D185A was excised and ligated into the EcoRV/ Notl site of pBSKIIEEH: rsaFb-E395A. The full rsaFb now carrying the 2 mutations were excised with BamHl and EcoRl and ligated into pBBR4 to be expressed in C. crescentus. 2.7 Construction of strains with gene disruptions JS10-18 The pKl 8mobsacB :rsaFbA220 was used to knockout rsaFb in JS 1010 (a knockout strain of rsaFa in NA1000). Primary recombination of the plasmid was selected by Km-resistance, followed by three consecutive sub-culturing events to allow the second recombination. Cells were then selected on 3% sucrose PYE plates and subsequent replica plating on PYE and PYE Km plates were used to confirm the occurrence of the second recombination event. The colonies growing on PYE were screened by a colony western lift for the loss of S-layer secretion due to the deletion of both rsaFa and rsaFb. Colonies not secreting S-layer were grown and whole cell protein preparations of them were run on SDS-PAGE (sodium dodecyl sulfate poly-acrylamide gel electrophoresis) and western blotted to verify RsaFa and RsaFb protein were not being made. A strain confirmed to be rsaFa and rsaFb negative was designated JS1018. JS2006 The plasmid, pKmobsacB.rsaFa&KP, was used to make an internal deletion of rsaFa in CB15ARsaA. Primary and secondary recombination of the plasmid with the chromosome was done as described above. Colonies were then screened by PCR using the primers 5'-CGC CGG CTT CGC AGC GAT GA CCC-3' and 5'-CCC GGA GGC CTC CCA GGC GGC GTA-3' to confirm that the appropriate gene replacement occurred and rsaFa was internally deleted. To introduce an internal deletion of rsaFb, pKmobsacB :roaFM220 was then introduced into this strain. Primary recombination and secondary recombination was screened using methods as described above. PCR was performed with the primers FbXHl-F (5'-CCC AAG CTT AAC TGG CGA CGA CTG GCG G-3') and FbXB2-R (5'-CGG GAT CCG TCA ATT CCA GAA CCT CGT-3')- The production of a PCR product 220 bps smaller in size confirmed the internal deletion of rsaFb. Whole cell protein preparations were run on SDS-PAGE and western blotting was done to verify that no RsaFa, RsaFb and RsaA were being made. A strain confirmed to be rsaFa, rsaFb, and rsaA negative was designated JS2006. 2.8 Construction of strains with the BAC genes JS1019.JS1020. JS1021, JS2003 The BAC replication genes necessary for the propagation of plasmids with the OriV vegetative origin was introduced into the JS1001, NA1000, JS1018 and ARsaA strains. The plasmid pK18mobsacB:xy/X2BACAES was electroporated into these cells and selected on PYE Km plates for the primary recombination. The p4B plasmid, which can only replicate if the BAC genes were integrated into the chromosome at the xylX, was then eletroporated, as described in [51], into the cells and selected for on PYE Cm plates. The Cm resistant cells were then sub-cultured three times with Cm to allow a second recombination at xylX and plated on 3% sucrose Cm PYE plates. Subsequent replicate plating on PYE Cm and PYE Cm/Km plates were used to confirm the second recombination event. Colonies which would grow only on Cm and not on Km were picked and sub-cultured in PYE four times to eliminate the p4B plasmid from the cells by removing the Cm selection. After plating on PYE, colonies were replicate plated on PYE and PYE Cm to confirm that the p4B plasmid had indeed been eliminated from the cells. The resulting strains would have the BAC genes introduced into the xylXand could support replication of plasmids with an OriV. 2.9 Protein techniques Low pH extraction The S-layer of C. crescentus was extracted by low pH extraction as previously described using 100 mM HEPES pH2 solution [52]. Cells were grown to log phase and normalized amount as determined by OD600, was pelleted for S-layer extraction. Equal amounts of extracted protein samples were loaded onto SDS PAGE gels for analysis. Whole cell protein preparations. Equal amount of cells determined by ODgoo growing at log phase were pelleted and resuspended in lOmM Tris -EDTA. Lysozyme (lOOpg/ml) was added to the cell and incubated at 37°C for 30 minutes. RNaseA (50 pg/ml) and DNasel (1 pg/ml) were added and incubated for 1 hour at 37°C. Powdered urea was added to a final concentration of 3M urea. Equal amounts of whole cell protein preparations were loaded onto protein gels. Whole Culture protein preparations Equal volumes of cell culture grown to log phase were collected and lysozyme was added to 300ug/ml and incubated for 30 minutes at 37°C. RNaseA (60pg/ml) and DNasel (6u.g/ml) were added and incubated for 1 hour at 37°C. Powdered urea was added to a final concentration of 3M urea. Volumes of whole culture protein preparations loaded on SDS-PAGE gels were normalized according to spectrometry at OD600. Culture Supernatant Equal volume of culture media from different strains of C. crescentus at the log phase of growth was collected and centrifuged for 2 minutes at 13000K. The supernatant was then recovered and resolved by SDS-PAGE. . Volumes of culture supernatant loaded on SDS-PAGE gels were normalized according to spectrometry at ODgoo-Outer membrane protein preparations Cells grown to log phase were harvested and 3.9x1011 cells were centrifuged at 8000g for 10 minutes. Cells were washed 2 times with 50mM Tris-HCl pH8. The cell pellet was resuspended in 15ml of Tris-HCl and sonicated for 5 minutes. Cell debris was removed by centrifuging twice at 8000g for 10 minutes. The supernatant was removed and spun at 100,000g for 40 minutes in an ulfracentrifuge at 4°C. Pellets were resuspended in 10ml of 2% Sodium lauryl sarcosinate. After 1 hour of incubation at room temperature, the resuspended solution was centrifuged at 100,000g for 40 minutes in an ultracentrifuge at 4°C. The outer membrane protein from the pellet was resuspended in 8M urea. SDS-PAGE and Western blot analysis SDS PAGE using 5% stacking, and 7.5% separating gels were run at 200 Volts. Coomassie staining of gels and western immunoblotting were done following standard methods [48]. Protein was transferred onto 0.2 pm BioTrace NT nitrocellulose membrane (Pall Biosciences) and blocked by 3% skim milk, 0.9% NaCI, and 20mM Tris-HC1 pH8. Western blots were probed with primary rabbit polyclonal antibodies (University of British Columbia). RsaAl 88-784 antiserum was used at a 1/15,000 dilution. RsaFa antiserum was used at a 1/6,000 dilution. RsaE antiserum is used at a 1/10,000 dilution. Infrared secondary antibody, Alex Fluor 680 goat anti rabbit IgG (Invitrogen), was used at 1/50,000 dilutions and detected and quantified by the Odyssey Infrared Imaging System using the Odyssey 2.0 program (Licor Biosciences). To validate the infrared signals detected were in the linear range for protein quantification, multiple loadings of protein were quantified. 2.10 RNA isolation and cDNA synthesis RNA from C. crescentus was isolated from lxl08 cells using the RNeasy kits (Qiagen). Cells were pelleted and resuspended in 100 pi of TE pH 7.5 with 1 pg/ml lysozyme and incubated for 5 minutes at room temperature to lyse cells prior to RNA extraction. Any contaminating DNA was removed by using the DNA-free™ kit (Ambion). Synthesis of cDNA and real time PCR were done by using the SYBR® GreenER™ Two-step qRT-PCR Universal kit (Invitrogen) following the manufacturer's protocol. All Real-Time PCR reactions were performed in a 25 ul mixture. Real-Time PCR was performed using Stratagene Mx3000P® (Stratagene) and fluorescence thresholds were calculated with the Stratagene MxPro system software. Relative quantities were determined using the qBASE software [53]. Primers used for qPCR are listed in Table 2-2. The 16S RNA was used as the reference gene to normalize the relative quantities of rsaA mRNA in all the qPCR experiments. 2.11 Determination of mRNA half-lives Rifampicin was added to growing cultures at an OD600 of 0.7 to a final concentration of 200 u.g/ml to stop any further transcription. Ix 10 cells were removed at time 0, 5, 15 and 30 minutes after addition of rifampicin. Cells were pelleted and 100 pi of 1 mg/ml lysozyme was used to resuspend the pellet that was then sonicated with 10 one second pulses on high to lyse the cells in a mechanical fashion before RNA was extracted using RNeasy kits (Qiagen). Following RNA isolation, cDNA synthesis and real time qPCR were performed as described above. The primers used to amplify near the 5' coding region was named RsaA5P-F/R. The primers used to amplify 16S RNA were named 16Srna-F/R. 3 RESULTS 3.1 A non-specific extension of the 5' untranslated region of rsaA resulted in decreased mRNA stability In a previous study, C. crescentus transformed with a plasmid encoding an extra copy of rsaA did not result in increased production and secretion of RsaA [36]. However, it was later revealed that a non-specific 51 bps extension was inadvertently added to the 5' UTR of rsaA in the construct (rsaAASD) due to the cloning method (Figure 3-1). The sequence of the 5'UTR of the wild type rsaA mRNA and the modified rsaA mRNA (rsaAASD) was analyzed by the Mfold program [54] predicting the secondary folding of these regions. With the additional nucleotides in the 5'UTR, the secondary structures were quite different, inducing changes to the conformation of the region for ribosome binding (Figure 3-2). Since changes in the 5' UTR of mRNA can affect its stability [39, 55], total RNA was collected and subjected to qRT-PCR to compare the half-life of rsaA transcripts isolated from wild type C. crescentus and rsaA deficient cells that only expressed plasmid-encoded rsaA transcripts with the extended 5' UTR (clone JS2003:rj,a^ ASD). As shown in Figure 3-3 the half-life of rsaA transcripts containing the extended 5' UTR was 19 minutes, significantly shorter than the 36 minute half-life of wild type rsaA transcripts. This result suggests that decreased rsaA mRNA stability may have contributed to the lack of increased RsaA expression observed in a previous study [36] that attempted to boost RsaA secretion. rsaA with wild type promoter Transcriptional start site 1 -35 -10 SD ATG rsaA Promoter region rsaA with 5' UTR lengthened ( A S D ) Transcriptional start site I ^ ^ • Q t J ^ ^ ^ ^ ^ u ^ ^ i m ^ ^ ^ ^ B m i 1111 • ! 111 i » r o m ! . M . i i y j . m u m -35 -10 Extra 51 bps SD ATG rsaA Promoter region Lengthened 5 ' Untranslated Region Figure 3-1: Schematic representation of the regulatory regions upstream of wild type rsaA and rsaAASD which includes an extended 5' untranslated region The wild type 5'UTR of rsaA is 61 bases long. An additional 51 bps extension was identified in the rsaAASD construct used previously to overexpress RsaA. SD site WT-rsaA H i i O-U I I U-A I I -U-A^ A A A / rsaAASD G C \ SDsite U I A / G G - A ' Figure 3-2: Predicted secondary folding of the 5' untranslated region of rsaA mRNA with Mfold The secondary folding of the 5'untranslated region of rsaA mRNA and rsaAASD mRNA was predicted using the Mfold program. While the SD site in the wild type 5'UTR of rsaA mRNA is exposed in a loop, the SD site in the rsaAASD version is involved in binding with other sections of the 5' untranslated region. Decay of S -layer mRNA c O D_ 10 0 • W T r s a A o r s a A A S D I' ' ' ' I ' 1 " I ' " ' I 10 15 3D 25 30 35 40 Time (min) Figure 3-3: Effect of extended 5' untranslated region of rsaA on mRNA stability An additional 51 bps extension was present at the 5'untranslated region of the rsaAASD mRNA. Rifampicin was added to NA1000 (WT strain) and JS2003:pWB9:raa4ASZ), to stop further mRNA transcription and total RNA was isolated from cells at time 0, 5, 15 and 30 minutes. Total RNA was subjected to real time quantitative RT-PCR to determine the amount of rsaA mRNA remaining at each time point. 16sRNA was used as the internal control (reference gene) to normalize the level of rsaA mRNA detected. The Real time RT-PCR experiments were repeated 3 times from separate RNA isolation. Half-life was determined by the regression line calculated on a semi-logarithmic graph. 3.2 RsaA production and secretion can be increased by overexpression of rsaA alone In order to properly address the issue of whether RsaA levels can be increased by simple overexpression of rsaA, a new plasmid construct containing a faithful copy of the coding and regulatory regions of wild type rsaA was generated, as described in materials and methods, and verified by nucleotide sequencing. This plasmid, termed p4B:r,s,a/4600, was subsequently transformed into C. crescentus strain JS1019 (rsaA+ and S-LPS"). A C. crescentus strain deficient in S-LPS was used for these experiments since secreted RsaA is anchored to the cell surface by S-LPS. Thus, in the absence of S-LPS, secreted RsaA will be present in the culture medium. To determine the total amount of RsaA produced, whole culture protein preparations (cells plus medium) were resolved by SDS-PAGE and blotted against RsaA. Figure 3-4A shows that JS1019 cells transformed with p4B:A,5,o4600 produced significantly higher amounts of RsaA (2.2 ± 0.1 fold) compared to wild type JS1019 cells. In addition, protein preparations from culture supernatant alone showed a similar increase in RsaA levels, indicating that secretion of intracellular RsaA was not noticeably inhibited (Figure 3-4B). Taken together, these results demonstrate that the overexpression of rsaA resulted in a ~2-fold increase in RsaA production and secretion. Another advantage of using the S-LPS deficient strain, JS1019, was that it avoided the possibility that a crystallized S-layer on the surface of the cell may interfere with RsaA secretion in situations where RsaA expression may be upregulated. In order to test this possibility, the same experiments were repeated in JS1020 cells, a C. crescentus strain with a wild type S-LPS. Figure 3-4A shows that RsaA production, as determined S-LPS + v e S-LPS " v e p4BrsaA600 + - + Figure 3-4: Effect of S-layer on increased RsaA secretion The plasmid pABrsaA600 was transformed into C. crescentus strains JS1020 (rsaA+, S-LPS+) and JS1019 (rsaA+, S-LPS"). A) Infrared western blot analysis of whole culture protein preparations probed with polyclonal anti-188/784 RsaA antibodies reveals similar level of RsaA overexpression in both JS1020 and JS 1019. B) RsaA western blots of culture supernatant protein preparations indicate that portions of the overexpressed RsaA of JS1020 is shed into the growth media. C) Surface bound RsaA was extracted using the low pH method and analyzed by western blotting. Approximatly 30% more RsaA is found bound to the surface of JS1020 overexpressing RsaA. A, B and C were probed with polyclonal anti-188/784 RsaA antibodies. by western blots of whole culture protein preparations, was increased to similar levels in both S-LPS deficient and wild type S-LPS strains when transformed with p4B:rsaA600. However, of the increased amount of RsaA produced by cells of the JS1020 strain transformed with p4B:rsaA600 (~2.2 ± 0.2 fold) approximately 65% of this amount was present in the culture medium (Figure 3-4B). By contrast, RsaA is nearly undetectable (~1%) in the culture medium of parental JS1020 cells. This suggests that the surface of C. crescentus can accommodate a modest increase in S-layer accumulation. Also, because a previous study had noted that a blockage in RsaA secretion resulted in decreased production of RsaA [36], it can be reasonably inferred that the larger S-layer did not interfere with RsaA secretion. To confirm that the overexpression of RsaA, 2.2 ±0.1 fold compared to wild type, observed in JS 1019 transformed with p4BrsaA600 was due to the introduction of an authentic version of rsaA with its regulatory regions and not because of the vector backbone p4B, the DNA fragment containing rsaAASD was subcloned into p4B and tested for its ability to overexpress RsaA. Figure 3-5 shows that the introduction of the construct p4B:rsaAASD into JS1019 did not increase RsaA production, since only wild type level of RsaA (1.0 ± 0.1-fold) was detected. To verify that RsaA could be produced by the plasmid p4B:rsaAASD alone, the construct was introduced into the C. crescentus strain JS2003 (rsaA') and tested. The amount of RsaA produced by p4B:rsaAASD alone was 0.9 ± 0.1-fold compared to wild type, which indicates that functional RsaA proteins could be made by the plasmid construct. However when p4B:rsaA600 was transformed into JS2003, RsaA level was increased to 2.0 ± 0.1-fold compared to wild type. The steady state level of rsaA mRNA were also determine for these strains by quantitative RT-PCR as listed in Table 3-1. 1 2 3 4 Figure 3-5: Expression of RsaA in cells transformed with the p4BrsaAASD construct Infrared Western blot analysis of whole culture protein preparations of JS2003 (rsaA') and JS 1019 (rsaA+) with p4B:rsaAASD or p4B:rsaA600 introduced. RsaA was detected with polyclonal anti-188/784 RsaA antibodies. Lanes: 1, NA1000; 2, JS2003; 3, JS2003:p4B:rao4ASD; 4, JS2003:p4B:rsaA600; 5, JS1019; 6, JS1019:p4B:raa^A5D; 7, JSlO\9:p4B:rsaA600. Table 3-1: Relative levels of RsaA and rsaA mRNA expression in different strains of Caulobacter crescentus Strain Relative levels of RsaA Relative levels of rsaA compared to wild type mRNA compared to wild type NA1000 1 1 JS1019 1.0 ±0.0 1.2 ±0.2 JS2003:rsaA600 2.0 ±0.1 2.8 ±0.1 3S2003:rsaAASD 0.9 ±0.1 0.4 ±0.1 mO\9:rsaA600 2.2 ±0.1 3.2 ±0.9 JSl0l9:rsaAASD 1.0 ±0.1 1.0 ±0.1 RsaA levels were determined by infrared western blot analysis of whole culture protein preparations from the respective strains. Total RNA isolation was performed on the strains followed by quantitative RT-PCR to detennine the static level of rsaA mRNA. 16sRNA was used as the internal control (reference gene) to normalize the level of rsaA mRNA detected. The Real time RT-PCR experiments were repeated 3 times. NA1000 (wild type); JS1019 (rsaA+, S-LPS"); JS2003 (rsaA') 3.3 Expression of RsaA is dependent on the steady-state mRNA level of rsaA To determine i f the increased RsaA expression was regulated at the transcriptional level, the D N A construct containing unaltered rsaA (rsaA600) was inserted into plasmids that maintain a low (1-2), medium (5-6), or high (20-22) copy number [56]. Figure 3-6 illustrates that in C. crescentus transformed with these vectors, the expression level of RsaA was increased with the number of plasmid copies present in the cell. With the low, medium and high copy number plasmids, the level of RsaA produced was 58%, 122% and 202% of wi ld type, respectively (Table 3-2). Quantitative RT -PCR revealed that the cells with low, medium and high plasmid copy numbers had transcript levels of rsaA that were 52%, 229% and 2 80% of wi ld type levels, respectively (Table 3-2). These results demonstrate that ectopic expression of RsaA is dependent on the steady-state level of its transcript. 3.4 Overexpression of RsaA did not lead to increased expression of RsaA-specific transporters To determine i f increased RsaA secretion was accompanied by upregulated expression of O M P transporters RsaFa and RsaFb and the IMP transporter RsaE, whole cell protein preparations were resolved by SDS -PAGE and western blotted. A s shown in Figure 3-7 the levels of each transporter was similar in the two strains, less than 1 0% change in transporter levels were observed in RsaA-overexpressing cells when compared to wi ld type. This indicates that elevated production of RsaA does not result in increased expression of either O M P transporter or the IMP transporter RsaE and that their normal levels can sustain the secretion of RsaA at a level that is about double that of wi ld type. 1 2 3 4 5 F i g u r e 3-6: E x p r e s s i o n o f R s a A i n cells t r a n s f o r m e d w i t h p l a s m i d s m a i n t a i n e d at d i f f e r e n t c o p y n u m b e r s The D N A fragment containing rsaA and its promoter region, rsaA600, was cloned into 3 different plasmid vectors that are maintained at low, medium or high copies when transformed into JS2003 (rsaA'). Infrared western blot analysis was performed on the whole culture protein preparations from these strains and JS1020 (rsaA+) probing with polyclonal anti-188/784 RsaA antibodies. JS1020 produces wi ld type levels of RsaA. Lanes: 1, JS1020; 2, JS2003: pWB9:rsaA600 (low copy); 3, JS2003: pBBR3:rsaA600 (medium copy); 4, JS2003:p4B:rsaA600 (high copy); 5, JS2003. Table 3-2: Levels of RsaA and rsaA mRNA expressed in JS2003 cells with rsaA encoded on plasmids that are maintained at different copy numbers inside Caulobacter crescentus Strain Plasmid Relative RsaA Level compared to wild type Relative levels of rsaA mRNA compared to wild type NA1000 1 1 JS2003 0 0 JS2003 1-2 copies (Low) 0.6 ±0.1 0.5 ±0.1 JS2003 6-7 copies (Medium) 1.2 ±0.2 2.3 ± 0.3 JS2003 20-22 copies ( High) 2.0 ±0.1 2.8 ±0.1 RsaA levels were determined by infrared western blot analysis of whole culture protein preparations from the respective strains (n=3). Total RNA isolation was performed on the strains followed by quantitative RT-PCR to determine the static level of rsaA mRNA. 16sRNA was used as the internal control (reference gene) to normalize the level of rsaA mRNA detected. The Real time RT-PCR experiments were repeated 3 times from separate RNA isolation. The relative levels of rsaA mRNA are significantly different with a 90% confidence intervals in a Student T-test. 44 p4BrsaA600 + Figure 3-7: Expression of RsaA-specif ic transporters in RsaA-overexpressed cells (A) Infrared western blot with polyclonal anti-188/784 RsaA antibodies in whole culture protein preparations of wild type JS1019 cells (rsaA+, S-LPS") or cells transformed with the plasmid encoding rsaA (p4BrsaA600). Whole cell protein preparations from wild type JS 1019 cells or cells transformed with p4BrsaA600 were incubated with polyclonal anti-RsaFa antibodies (B) or polyclonal anti-RsaE antibodies (C) and evaluated by infrared western blot analysis. 3.5 Overexpression of rsaFa resulted in decreased expression of RsaA This above result, however, still does not eliminate the possibility that the native level of RsaA-specific transporters were limiting factors in the production and/or secretion of S-layer in RsaA-overexpressed cells. As noted before, blockage in RsaA secretion can lead to lower synthesis of RsaA [36]. Therefore, it is possible that the introduction p4B:rsaA600 drove expression of RsaA to levels much greater than the ~2-fold increase that was measured, at least initially, and that its secretion through the inner and outer membranes was not quick enough to handle these levels of RsaA. This could lead to an accumulation of intracellular RsaA that then initiates a mechanism to down-regulate RsaA expression similar to that observed in cells where RsaA secretion was blocked. Therefore, it was worth investigating whether increased expression of the RsaA-specific transporters concomitant with rsaA overexpression could lead to increased RsaA production and secretion beyond 2-fold. A plasmid containing rsaFa, pBBR4:rsaFa, was transformed into a C. crescentus that showed ~2-fold increase in RsaA expression. An infrared western blot revealed that the level of RsaFa increased 5.4 ± 0.2-fold in these cells (Figure 3-8). Unexpectedly, the expression of RsaA decreased to 0.6-fold of wild type levels in cells transformed with both rsaA and rsaFa. 3.6 Deletion of rsaFa and rsaFb resulted in decreased RsaA production at the posttranscriptional level Deletion of the OMP transporters for the RsaA secretion system resulted in a dramatic drop in S-layer production (11% of wild type) [36]. This observation was Figure 3-8: Effect of rsaFa and rsaA overexpression on RsaA production (A) Infrared quantitative western blot analysis with polyclonal anti-188/784 RsaA antibodies of whole culture protein preparations from JS1019 (rsaA+, S-LPS") with or without additional plasmid born copies of rsaA and rsaFa were probed with polyclonal anti-188/784 RsaA antibodies. B) Whole cell protein preparations of these strains probed with anti-RsaFa antibodies. verified and shown in Figure 3-9. To determine if the down-regulation of RsaA synthesis was at the transcriptional or posttranscriptional level, the steady-state level of rsaA mRNA in wild type cells (NA1000) was compared by qRT-PCR to an rsaF knockout strain (JS1018) where both OMP transporter genes, rsaFa and rsaFb were internally deleted. As shown in Table 3-3, rsaA transcripts in the knockout strain remained at 0.9 ±0.1- fold of wild type levels. This suggests that in the absence of OMP transporters RsaFa and RsaFb, the expression of RsaA is down-regulated at the posttranscriptional level. Furthermore, when cells from the rsaF knockout strain were transformed with the plasmid encoding rsaA, the dramatic reduction in RsaA production was not reversed. This indicates that the posttranscriptional regulation of rsaA mRNA in rsaF knockout strain was robust enough to handle elevated levels of rsaA transcripts (Figure 3-10). 3.7 Deletion of rsaD and rsaE led to decreased steady-state mRNA levels of rsaA The deletion of rsaD and rsaE in the B15 strain abolished S-layer secretion and intracellular RsaA level was only 3% of wild type (Figure 3-11). The amount of rsaA mRNA in B15 dropped to 0.4 - fold of wild type level (Table 3-3). The decrease in rsaA mRNA is much more significant in the RsaD and RsaE deletion strain compared to the OMP deletion strain. Since steady-state level of transcript is dependent on two major factors, the rate of transcription and the rate of degradation of the mRNA, preliminary experiments to determine the half-life of rsaA mRNA in B15 were done, but they did not indicate a decrease in rsaA mRNA stability. This implies the rate of rsaA transcription was decreased in the absence of RsaD and RsaE. 1 2 Figure 3-9: Effect of rsaFa and rsaFb deletion on RsaA production and secretion (A) Whole culture protein preparations of NA 1000 (wild type) and JS1018 (rsaFa, rsaFb') cells were analyzed by infrared western blots probed with polyclonal anti-188/784 RsaA antibodies. (B) Whole cell protein preparations analyzed by western blots probed with anti-RsaFa antibodies. Lanes: 1, NA1000; 2, JS1018. This experiment was done to verify previous findings. Table 3-3: Relative levels of RsaA and rsaA mRNA expression in different RsaA transporter deleted strains of Caulobacter crescentus Strain Relative levels of RsaA compared to wild type Relative levels of rsaA mRNA compared to wild type NA1000 1 1 JS1018 0.1±0.0 0.9 ±0.1 B15 <0.1 0.4 ± 0.0 RsaA levels were determined by infrared western blot analysis of whole culture protein preparations from the respective strains. Total RNA isolation was performed on the strains followed by quantitative RT-PCR to determine the static level of rsaA mRNA. 16sRNA was used as the internal control (reference gene) to normalize the level of rsaA mRNA detected. The Real time RT-PCR experiments were repeated 3 times from separate RNA isolation. NA1000 (wild type); JS1018 (rsaA+, rsaFa', rsaFb'); B15 (rsa/C, rsaD', rsaE) Figure 3-10: Effect of increased transcript levels of rsaA on the production of RsaA in rsaFa and rsaFb deleted cells (A) Infrared western blot analysis of whole culture protein preparations of JS1020 (rsaA+) and JS1021 (rsaA+, rsaFa, rsaFb') strains with or without the introduction of extra copies of rsaA introduced on p4B:rsaA600 was probed with polyclonal anti-188/784 RsaA antibodies. (B) Whole cell protein preparations probed with anti-RsaFa antibodies. Figure 3-11: Effect of rsaD and rsaE deletion on R s a A secretion and production (A) Infrared western blot analysis of whole culture protein preparations of C. crescentus strains NA1000 (wild type strain) and B15 (rsaA+, rsaD', rsaE') probed with polyclonal anti-188/784 RsaA antibodies. (B) Whole cell protein preparations probed with anti-RsaE antibodies. Lanes: 1, NA1000; 2, B15. This experiment was done to verify previous findings. 3.8 The inhibition of secretion of RsaA recombinant proteins containing a cluster of positively charged amino acids was not reversed by the removal of specific negatively charged amino acids on RsaFa and RsaFb It was observed in previous studies that secretion of RsaA fusion proteins containing an abundance of, or a cluster of positively charged amino acids was severely inhibited [36]. Threading of the OMP transporter sequences to that of TolC in E. coli revealed that RsaFa and RsaFb can form a channel similar to TolC. In addition, it predicted the presence of several negatively charged amino acids at the inner wall of the channel near the entrance of the pore structure on the periplasmic side of both RsaFa and RsaFb, specifically D174, D176, D188, E368, E383, E394 and E401 on RsaFa and E171, D185, E385, D395 and E402 on RsaFb (see Figure 1-2). Therefore, site-directed mutagenesis of these amino acids to alanine was performed to determine if they were involved in the inhibition of secretion of RsaA:RKKR fusion proteins. These experiments also required the construction of a new C. crescentus strain, termed JS2006, that was negative for wild type rsaFa, rsaFb and rsaA. The rsaA.RKKR construct and then each mutant rsaFa or rsaFb construct was electroporated into JS2006 cells. Western blotting of whole cell protein preparations verified that each rsaFa and rsaFb construct resulted in high levels of expression of its mutant protein (Figures 3-12A and 3-13A). However, the expression of RsaA:RKKR fusion proteins was below detectable levels (Figures 3-12B and 3-13B and Table 4-4). These results suggest that none of the mutations of the negative residues on RsaFa and RsaFb were able to reverse the inhibition of secretion of RsaA:RKKR fusion proteins, and thus, its subsequent down- regulation of expression. To verify that Figure 3-12: The effect of site-directed mutagenesis of negatively charged amino acids on RsaFa on the secretion blockage of RsaA:RKKR proteins Alanine scanning of specific negatively charged residues at the periplasmic entrance of RsaFa was performed, producing the mutated versions of RsaFa. The plamid pWB9rsaARKKR was first transformed into JS2006 (rsaA', rsaFa', rsaFb'). The different mutated rsaFa were then introduced on pBBR4 into JS2006:pWB9rsaARKKR. A) Infrared of JS20Q6pWB9rsaARKKR strains with the different RsaFa mutants detected with anti RsaFa antibodies. B) Western blots analysis of whole cell protein preparations from JS2006pWB9rsaARKKR with the different RsaFa mutants probed with polyclonal anti-188/784 RsaA antibodies. C) Western blot analysis of whole cell protein preparations from JS1018 (rsaFa', rsaFb') with the different RsaFa mutants probed with polyclonal anti-188/784 RsaA antibodies. (Not all mutants created are shown on this figure). Figure 3-13: The effect of site-directed mutagenesis of negatively charged amino acids on RsaFb on the secretion blockage of RsaA:RKKR proteins Alanine scanning of specific negatively charged residues at the periplasmic entrance of RsaFb was performed, producing the mutated versions of RsaFb. The plamid pVJB9rsaARKKR was first transformed into JS2006 (rsaA', rsaFa', rsaFb'). The different mutated rsaFb were then introduced on pBBR4 into JS2006:pWB9rsaARKKR. A) Infrared of JS2006pWB9rsaARKKR strains with the different RsaFb mutants detected with anti RsaFa antibodies. B) Western blots analysis of whole cell protein preparations from JS2006pWB9rsaARKKR with the different RsaFb mutants probed with polyclonal anti-188/784 RsaA antibodies. C) Western blot analysis of whole cell protein preparations from JS 1018 (rsaFa', rsaFb') with the different RsaFa mutants probed with polyclonal anti-188/784 RsaA antibodies. Table 3-4: Secretion of RsaA:RKKR and wild type RsaA by the mutated forms of RsaFa and RsaFb. Mutations in RsaFa Secretion of RsaA RKKR Secretion of wild type RsaA D174A Not detected ~WT level E176A,D177A Not detected ~WT level D188A Not detected ~WT level E368-388A Not detected ~WT level E383-385A Not detected ~WT level E394A Not detected ~WT level E401A Not detected ~WT level E368A, E401A Not detected ~WT level Mutations in RsaFb Secretion of RKKR Secretion of RsaA E171A,E172A Not detected ~WT level D185A Not detected ~WT level E385A Not detected ~WT level D395A Not detected ~80%ofWT E402 Not detected ~WT level D395A,D185A Not detected ~40%ofWT D395A, E402A Not detected ~45%ofWT the mutated RsaFa and RsaFb proteins did not lead to general deficiencies in S-layer formation, their respective constructs were transformed into an rsaFa and rsaFb' strain (JS1018) and evaluated for their ability to secrete wild type RsaA. All of the mutated rsaFa- and rsaF^ -transformed cells produced S-layer at levels similar to wild type cells (Figures 3-12C and 3-13C), except those with RsaFb mutated at D395, D395/E402 and D395/E185, which resulted in decreased total RsaA levels to -80%, -40% and -45% of wild type (Table 4-4). In addition, decreased amounts of RsaA was also observed after low pH extraction that evaluated RsaA levels present as an S-layer (Figure 3-14). Outer membrane protein preparations were performed to confirm that these RsaFb mutants were properly directed to the outer membrane (Figure 3-15). Therefore, none of the negatively charged amino acids mutated on RsaFa and RsaFb were involved in the inhibition of S-layer secretion as postulated, whereas El 85, D395, and E402 on RsaFb appeared to promote the secretion of S-layer. Moreover, the addition of calcium to the culture medium (up to 25 mM) did not alleviate the inhibition of S-layer secretion observed in the D395, D395/E402 and D395/E185 RsaFb mutants. 1 2 3 4 5 6 < RsaA Figure 3-14: Effect of site-directed mutagenesis on RsaFb on the secretion of RsaA to the cell surface Surface attached RsaA from JS1018 (rsaFa, rsaFb') expressing different mutants of RsaFb was extracted by the low pH method and resolved by SDS-PAGE and stained with Commassie blue. 1 2 3 4 5 6 RsaFa RsaFb Figure 3-15: Effect of site-directed mutagenesis on localization of mutant RsaFb to the outer membrane Infrared western blots of the outer membrane protein preparations from NA1000 (WT strain) and JS 1018 (rsaFa, rsaFb') expressing different RsaFb mutants probed with anti RsaFa antibodies. Lanes: 1, NA1000; 2, JS1018; 3, JS1018:rsaFb; 4, JS1018:D395A rsaFb; 5, JS1018:D395A/D185A rsaFb; 6, JS1018:D395A/E402A rsaFb. 4 DISCUSSION AND CONCLUSION 4.1 S-layer production can be increased in Caulobacter crescentus Previous attempts at exogenous overexpression of rsaA did not lead to increased S-layer production in Caulobacter crescentus [36]. However, a closer inspection of the plasmid construct used revealed a 51 bps extension to the 5' UTR of rsaA. Structures in the 5' or 3' UTR had been known to affect mRNA stability. In E. coli, hairpin structures at the 5'UTR of ompA are responsible for its long half-life [39] while the addition of extra nucleotides at the 5' UTR ompA transcripts can lower its stability [57]. In Rhodobacter capsulatus, a hairpin structure at the 3' UTR stabilizes pufBA transcripts [58, 59]. The addition of 51 nucleotides to the 5'UTR of rsaA transcripts resulted in changes to its secondary folding, as predicted by Mfold analysis, particularly in the area surrounding the Shine-Dalgarno site. In this thesis, it was determined that the half-life of rsaA mRNA with this 5' UTR extension was considerably shorter at 19 minutes, compared to 36 minutes for rsaA transcripts isolated from wild type cells. Although the half-life of rsaA mRNA derived from the new plasmid construct, p4B:rsa/4600, that contained a faithful copy of wild type rsaA and its regulatory regions was not tested, rsaA negative cells transformed with this vector did result in a 2.8-fold increase in steady-state rsaA transcript levels and a 2.0-fold increase in RsaA expression. However, the introduction of p4B:rsaA600 into cells with a chromosomal copy of rsaA only resulted in a 3.2-fold increase in steady-state rsaA transcript levels and a 2.2-fold increase in RsaA expression. Therefore, it is possible that increases in rsaA mRNA levels of ~3-fold approached the upper limit of the transcriptional machinery of C. crescentus. Nonetheless, the long half-life of the rsaA transcript is a crucial factor in the robust expression of RsaA. In contrast, the average half-life of E.coli transcripts is about 3-8 minutes [60]. Transcripts with long half-lives include the ompA mRNA in E. coli at 15 minutes [38], the puf operon in R. capsulatus where pufBA mRNA is 30 minutes [61] and the hrpA mRNA from Pseudomona syringae at 37 minutes [62]. S-layer proteins with long mRNA half-lives include vapA oiAeromona salmonicida at 22 minutes [63] and the slpA of Lactobacillus acidophilus at 15 minutes [64]. All of these stable transcripts encode for membrane associated proteins that tend to be expressed at very high levels. The S-layer of C. crescentus consists of RsaA proteins that are captured on the cell surface by S-LPS. The results presented herein demonstrated that RsaA production and secretion can be increased in a strain that is negative for S-LPS. The advantages of using this strain for biotechnology applications are that the isolation of secreted RsaA recombinant proteins would be very simple and free of any LPS contamination. On the other hand, there are other applications for C. crescentus, such as antigen display, in which the incorporation of RsaA recombinant proteins into the S-layer is required. It was speculated that a fully crystallized S-layer on the surface of S-LPS positive cells could limit the overexpression of RsaA by acting as a physical barrier to the secretion of the increased RsaA produced, which can in turn down-regulate the synthesis and overexpression of RsaA. This report revealed that RsaA can be overexpressed even in the presence of a fully crystallized surface layer and of the increased RsaA present in RsaA-overexpressed cells, roughly 30% went into the S-layer while 65% was present in the culture medium. While the cell surface of C. crescentus has been shown to be completely covered with the S-layer [20], it has also been observed that negative staining of the hexagonal array structure was uneven (Smit, unpublished results), and this unevenness has been interpreted as limitations of the negative staining technique. Knowing that extra RsaA monomers could attach to the surface of C. crescentus, the unevenness observed in the negative staining could likely represent missing monomers on the hexagonal S-layer array. Therefore, overexpression of RsaA resulted in a modest increase to the existing S-layer and that this increase did not inhibit the production or secretion of RsaA. 4.2 S-layer-specific transporters can down-regulate the expression of RsaA Although an approximate doubling of RsaA expression has now been demonstrated, it remains to be seen if this is the maximal output possible. Of course, there are several factors that can limit the amount of RsaA production and secretion, such as the availability of RsaA-specific IMP transporters RsaD and RsaE and RsaA-specific OMP transporters RsaFa and RsaFb. Overexpression of RsaA in C. crescentus did not result in increased levels of RsaE, RsaFa and RsaFb as determined by infrared western blot analysis. And although no RsaD-specific antibody was available, it can be reasonably inferred that its expression level also did not increase, as it is co-transcribed with rsaE. Experiments using strains of C. crescentus with deletions of RsaA-specific IMP or OMP transporters indicated that RsaA secretion was blocked and that this was accompanied by a severe reduction in RsaA production [18, 36]. However, qRT-PCR revealed that the transcript level of rsaA in cells lacking both OMP transporters was not significantly different from wild type. This suggests that the downregulation of RsaA production occurred at the posttranscriptional level. A similar phenomenon was observed in Campylobacter fetus with the expression of its S-layer protein SlpA [17]. When the SlpA-specific transporters were deleted, intracellular SlpA was reduced to undetectable levels while slpA transcript levels remained comparable to wild type. Therefore, posttranscriptional regulation may be a common and efficient mechanism used to prevent the intracellular accumulation of unwanted S-layer proteins with long mRNA half-lives. However, the posttranscriptional regulation of RsaA expression is one of the few to be identified in a type I secretion system. Other examples where posttranscriptional regulation of secreted proteins occurs are often found in type Ill-transported flagella proteins or in the translocation apparatus for the type III transport systems [65-67]. In Yersinia enterocolitica the YopQ protein secreted by a type III system is only synthesized when its secretion is not blocked. Posttranscriptional regulation can involve a rapid degradation of previously synthesized RsaA or a decreased translation of the rsaA transcript. Since degraded RsaA products have not been observed, the former possibility is not likely. On the other hand, translation can be inhibited by repressors or by antisense RNA [55, 68]. In addition, the transcript itself can fold and form structural motifs to block translation [55]. In Thermus thermophilus B8, the C-terminus of its S-layer protein, SlpA, can bind to the 5' untranslated leader mRNA region and block translation [69]. Trapped intracellular RsaA may function in a similar fashion where the protein binds its own transcript and prevents its translation when its levels exceed a certain threshold level. Increased intracellular levels of RsaA could also signal other molecules to bind the rsaA transcript to halt translation. Further experiments are required to determine if RsaA can bind its own transcript and what region is responsible for this binding. Insufficient levels of RsaA-specific transporters, as is possible when RsaA is overexpressed, may also mask a subtler down-regulation of RsaA. For instance, the ~2-fold increase in total RsaA observed in cells transformed with p4B:rsaA600 may have been greater, but was attenuated due to an inability to secrete these proteins in a timely manner. However, cells transformed with plasmids encoding rsaA and RsaFa, resulted in a decrease in RsaA levels to 0.6-fold of wild type. A previous study reported that RsaFa was sufficient for the secretion of RsaA, as cells lacking RsaFb still secreted RsaA at 80% of wild type levels [36]. The results presented in this report showed that the levels of RsaFa increased 5-fold while the levels of RsaA decreased. It may be possible that rsaFa transcripts are preferentially transcribed and/or translated over rsaA transcripts and that the overexpression of RsaFa reduced the availability of cellular components required for the synthesis of RsaA [70]. However, confounding this interpretation is the previous observation that RsaA levels did increase 28% in cells transformed with plasmids encoding rsaFa and rsaA (with the 5' extended UTR) [36]. This result suggested that the levels of RsaFa and rsaA transcription can be limiting the amount of RsaA produced, even in cells expressing these proteins at levels similar to wild type. 4.3 Certain negatively charged amino acids on RsaFa and RsaFb were not involved in the inhibited secretion of RsaA:RKKR fusion proteins. Previous studies showed that RsaA recombinant proteins containing an abundance of, or a cluster of positively charged amino acids on the secreted protein were not secreted from C. crescentus. Although the crystal structure has not been resolved for any of the RsaA-specific transporters, seven and five negatively charged amino acids were predicted to be exposed on the inner wall of the channel near the entrance of the pore structure of RsaFa and RsaFb, respectively. This led to the hypothesis that interactions between the negative charges and positive charges were sufficient to anchor RsaA recombinant proteins such as RsaA:RKKR to the OMP transporter, thereby preventing its translocation to the cell surface. However, site-directed mutagenesis of one or two of these negatively charged amino acids into a neutral amino acid did not result in any secretion of RsaA:RKKR. Although this does not exclude the possibility that the negatively charged amino acids were preventing the secretion of RsaA:RKKR, it does suggest that not all residues were required to achieve full inhibition. The simultaneous mutagenesis of 3 or more negatively charged amino acids on RsaFa and RsaFb may resolve this issue. In addition, the role of RsaD and RsaE in the inhibition of RsaA:RKKR was not examined and cannot be ruled out. Further experiments that verified the ability of mutant RsaFa and RsaFb to form functional transporters revealed that the presence of D395 on RsaFb promoted the normal secretion of wild type RsaA. In addition, amino acids El 85 and E402 on RsaFb may also play a beneficial role in the secretion of wild type RsaA in conjunction with D395. It is possible that these negatively charged amino acids are involved in the proper pore formation of RsaFb. Moreover, the addition of calcium to the culture medium did not reverse the inhibition of S-layer secretion observed in the D395, D395/E402 and D395/E185 RsaFb mutants. While calcium has been shown to be required for the secretion of RsaA, this result suggests that these amino acids are not critically involved in this aspect. 4.4 Conclusion Bacterial secretion systems have been vital for biotechnology and research purposes as a means to produce recombinant proteins, hormones, antibodies and other proteins of interest [41, 45]. However, the type I secretion system of RsaA in Caulobacter crescentus has several advantages over the current systems including a higher yield and higher purity at lower cost. In addition, efforts are under way to test the efficacy of the C. crescentus S-layer as an antigen display system in a number of diverse applications [41]. The main goals of this thesis were to increase the expression of RsaA and to identify mechanisms that control its production and secretion. To this end it was demonstrated that RsaA expression can be increased 2.2-fold with the ectopic expression of rsaA encoded on a plasmid construct. The addition of 51 bps to the 5' UTR of rsaA on a previous vector used to transform C. crescentus did not result in increased RsaA expression [36], and this may be due to the decreased stability of this rsaA transcript. It was observed that the production of RsaA increased when the steady-state level of its transcript was increased. However, ectopic overexpression of rsaA in wild type and rsaA negative cells produced similar transcript and protein levels of RsaA. This suggests that the maximum steady-state level of rsaA may have been achieved, and consequently, the maximum level of RsaA production as well. Results from additional experiments were not conclusive as to whether the wild type levels of the RsaA-specific transporters were limiting factors in situations where RsaA was overexpressed. Previous studies showed that RsaA expression can be reduced in circumstances where its secretion is inhibited or blocked. The results presented in this report revealed that the decrease in RsaA synthesis in cells with deletions of the OMP transporter genes rsaFa and rsaFb [36] occurred at the posttranscriptional level. In cells with deletions of the IMP transporter genes rsaD and rsaE, RsaA down-regulation occurred at both the transcriptional and posttranscriptional level. However, site-directed mutagenesis failed to determine whether negatively charged amino acids present at the inner wall of the channel near the entrance of the pore structure on the periplasmic side of RsaFa and RsaFb were involved in the inhibition of secretion of RsaA recombinant proteins that contained an abundance of, or a cluster of positively charged amino acids. Taken together, the results demonstrate that RsaA production can be increased in C. crescentus by the ectopic overexpression of its gene. However, several regulatory mechanisms and factors may limit the degree of RsaA overexpression. In particular, special attention should be paid to ensure that any interference of RsaA secretion is avoided if the goal is to maximize RsaA production in the future. 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