,2 H . n ¢ ft -1$=‘ 5; 2113‘: a”; * is: J . , ‘ :_- l . i h“ 21 K .3: ”’.\\*5 {'1 33 fig“ .. V‘ v .211“ "2 J- . 1 a‘éggfig ”1" '4‘ 1 ‘q 0 ‘M‘a h r-x‘. ' “33 ,- .1 u i"? 1‘ ‘ aye w . 9h. , 16:14 '8" 1' {M «WWW?!ifliiilfiiifliii'lil'liflmfiifiil 3 1293 01048 8462 This is to certify that the dissertation entitled Identification and Characterization of the General Secretion Pathway Genes in Vibrio cholerae presented by Linda Joanne Overbye has been accepted towards fulfillment of the requirements for Ph.D. degree in Microbiology Mag/professor MSU is an Affirmative Action/Equal Opportunity Institution 042771 LIBRARY Michigan State University PLACE II RETURN summoned-mum yum TOAVOIDFINEB Munonorbdonddoda. DATE DUE DATE DUE DATE DUE rp_-4 MSU ION! Afflnnltlvo WOMEN Opporlunfly Immon ____. __—.____ _ ..__ _ .__ ___.___. Wanna-9.1 IDENTIFICATION AND CHARACTERIZATION OF THE GENERAL SECRETION PATHWAY GENES IN VIBRIO CH OLERAE By Linda Joanne Overbye A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1994 ABSTRACT IDENTIFICATION AND CHARACTERIZATION OF THE GENERAL SECRETION PATHWAY GENES IN VIBRIO CHOLERAE By Linda Joanne Overbye Pleiotropic transposon insertion mutants of V. cholerae that cannot secrete protease, chitinase, and E. coli heat-labile enterotoxin (LT) B subunit pentamer through the outer membrane were isolated in order to identify the genes involved in this process. A cosmid from a V. cholerae gene library restored protein secretion to the mutants. DNA sequence analysis of the insert of this cosmid identified twelve open reading frames, epsC-N, which are similar to general secretion pathway (GSP) genes of Aeromonas, Erwinia, Klebsiella, and Pseudomonas which are required for extracellular protein secretion in these Gram‘ bacteria. Two of the V. cholerae mutants were found to have Tn5 inserted into the identical location in the epsC gene and the other two had insertions in the epsM gene. These genes are therefore known to be required for extracellular protein secretion. The remaining eps genes of V. cholerae were identified by the similarity of their sequence to the sequence of genes known to be required for secretion in other Gram“ bacteria. Strains with mutations in the epsC and epsM genes exhibit aberrant outer membrane profiles indicating a requirement for the GSP in proper assembly of the outer membrane. Results showing that several ORFs of the eps cluster overlap, that the secretion defect in an epsC mutant is not complemented by the epsC gene alone but is by a cosmid containing the entire eps gene cluster, and that the insertion of Tn5 into the epsC gene reduces the expression of epsE indicate that the epsC-N genes may be arranged into an operon. Although the GSP seems to be conserved in many Gram' bacteria and the proteins required for its function exhibit sequence similarities in different genera, high specificity of the pathway has been observed. To see how prevalent the ability to recognize the B subunit pentamer of LT as a secretable protein is among Gram' bacteria was, eth, which encodes the B subunit, was introduced into different Gram“ bacteria. All species tested which are able to secrete the B subunit belong to or are proposed members of the families Vibrionaceae or Aeromonadaceae. To Frederick Michel and Vern and Dorothy Overbye. Thank you for all your love and support. iv ACKNOWLEDGMENTS Special thanks go to the following people for their assistance: Dr. Michael Bagdasarian, my advisor, for his patience and guidance, for tolerating all my complaints about sequencing 12 kb, and for letting me use his computer when mine died. Dr. Wendy Champness, Dr. Rawle Hollingsworth, Dr. Martha Mulks, and Dr. Pat Oriel, my guidance committee members, for their advice and time. Chi J u Chen, for running my last few sequencing reactions for me. The DuVall family for their generous assistance through the DuVall scholarship. The NSF Center for Microbial Ecology for providing me with a stipend. Vern and Dorothy Overbye, for all the support they have given me in everything I have done. Dr. Maria Sandkvist for teaching me so much and for all her help, even after she left Michigan State. Dr. Suzanne Thiem for offering a lot of advice and for teaching me an easy way to pour sequencing gels. Dr. Mike Winfrey for inspiring me to become a microbiologist. Finally, special thanks go to Fred Michel for standing by me through everything. I don't know what I would have done without you. PREFACE This thesis is divided into four parts, a general introduction and three chapters presenting the results of the dissertation work in manuscript form. The second chapter was published in Gene 132 (1993) 101-106 with coauthors Maria Sandkvist and Michael Bagdasarian. Maria Sandkvist's contribution to this paper was the isolation of a DNA fragment containing the epsM gene which was found to complement two of my mutants. The third chapter will be submitted to Molecular Microbiology with coauthors Maria Sandkvist, Victor Morales and Michael Bagdasarian. Maria Sandkvist is a coauthor since she isolated the fragment which contains the eps genes, sequenced the epsE gene, and identified that an epsC mutant expressed less EpsE than wild type cells (Figure 3.6). Victor Morales did a portion of the DNA sequencing that is presented in this chapter. The fourth chapter will be submitted to Gene with Michael Bagdasarian as coauthor. TABLE OF CONTENTS Page LIST OF TABLES ......................................... ix LIST OF FIGURES ......................................... x ABBREVIATIONS ......................................... xii CHAPTER 1 General Introduction ....................... 1 References ............................... 24 CHAPTER 2 Genes Required for Extracellular Secretion of Enterotoxin are Clustered in Vibrio cholerae 31 Abstract ................................. 32 Introduction .............................. 33 Materials and Methods ..................... 35 Results and Discussion ..................... 39 Acknowledgments ......................... 51 References ............................... 53 CHAPTER 3 Organization of the General Secretion Pathway Gene Cluster of Vibrio cholerae --------------- 56 Abstract ................................. 57 Introduction .............................. 53 Materials and Methods ..................... 60 Results and Discussion ..................... 64 References ............................... 92 CHAPTER 4 CHAPTER 5 Secretion of the E. coli Heat-Labile Enterotoxin B Subunit by Bacteria Other ThanVibrio cholerae - - 97 Abstract ................................. 93 Introduction .............................. 99 Materials and Methods ..................... 100 Results and Discussion ..................... 105 References ............................... 111 Conclusions and Recommendations for Future Research .......................... 115 References ............................... 119 viii 1.1 2.1 3.1 3.2 3.3 3.4 4.1 4.2 4.3 LIST OF TABLES The GSP and other related proteins of bacteria and phage. Complementation of the LT secretion defect in mutants PU3 and PU5 by chromosomal fragments of V. cholerae TRH7000. Strains and plasmids used in this study. Location and characteristics of deduced Eps gene products of the epsC - N gene cluster. Comparison of deduced Eps proteins with proteins from other bacteria involved in protein secretion and processing and assembly of type IV pilin. Complementation of the secretion defect in epsC mutant PU6 by the eps gene cluster. Strains used in this study. Plasmids used in this study. Secretion of LT B subunit pentamer (Eth) and [3- lactamase (file) through the outer membrane of Gram' bacteria. ix Page(s) 11-12 40 61 77 81 91 102 103 107 1.1 1.2 1.3 1.4 1.5 2.1 2.2 2.3 2.4 2.5 2.6 LIST OF FIGURES Secretion of IgA protease through the outer membrane of Neisseria gonorrhoea. The signal peptide independent pathway of protein secretion through the outer membrane. The general secretion pathway of protein secretion through the outer membrane. The organization of the pul gene cluster of Klebsiella oxytoca. Biogenesis of E. coli heat-labile enterotoxin. Plate assays for protease and chitinase secretion in V. cholerae TRH7000 derivatives. Secretion defective mutants of V. cholerae exhibit a change in morphology which disappears when secretion is restored. DNA fragments carrying the epsM gene of V. cholerae strain TRH7000. Expression of eps genes under the control of bacteriophage T7 promoter. Nucleotide sequence of the insert from pMMB524 (Gene Bank accession number L13660) and the deduced aa sequences. Amino acid alignments of EpsM from V. cholerae, OutM from E. chrysanthemi, PulM from K. oxytoca, and chZ from P. aeruginosa. Page(s) 10 15 22 41 42 44 45 48-49 50 2.7 3.1 3.2 3.3 3.4 3.5 3.6 4.1 Physical and genetic map of the EcoRI fragment from the V. cholerae TRH7000 chromosome containing the eps genes present in pMMB339. Nucleotide sequence of the eps genes. Putative prepilin cleavage sites of TcpA, EpsG, EpsH, EpsI, Esz, and EpsK. Physical map of the eps genes showing subclones cloned into pT7-5/6 used for deletion mapping of the genes. Expression of eps genes under the control of bacteriophage T7 ¢10 promoter. Outer membrane fractions isolated from wild type Vibrio cholerae TRH7000 and epsM and epsC mutants grown in LB medium or LB medium containing 0.4% maltose. Production of EpsE from wild type Vibrio cholerae TRH7000 and epsE and epsC mutants. The construction of plasmid vector pMMB503EH. 52 65-76 79 84 85 87 90 104 aa Ap(R) BCIP Bla BSA Cm(R) CT ctxA cth E. coli eth GCG GEP GMI Gram” GSP HA/protease IPTG kb ABBREVIATIONS absorbance (1 cm) amino acid(s) ampicillin (resistance) 5-bromo-4-chloro-3-indolyl-l-phosphate base pair(s) B—lactamase bovine serum albumin chloramphenicol (resistance) cholera toxin gene encoding subunit A of CT gene encoding subunit B of CT Escherichia coli gene encoding subunit B of LT Genetics Computer Group (Madison, WI, USA) general export pathway galactosyl-N-acetylgalactosaminyl-(N- acetylneuraminyl)-galactosylglucosylceramide Gram-negative general secretion pathway V. cholerae haemagglutinin/protease isopropyl-B-D-thiogalactopyranoside kilobase(s) or 1000 bp xii kDa Km(R) ORF PAGE PBS PU Px(R) RBS RiflR) Sm(R) SDS Tc(R) V. cholerae [ ] kilodalton kanamycin (resistance) Luria-Bertani medium Escherichia coli heat-labile enterotoxin relative molecular mass nitro blue tetrazolium nucleotide(s) open reading frame polyacrylamide-gel electrophoresis 10 mM phosphate buffer pH 7.3 / 150 mM NaCl mutant secreting less protease than wild type polymixin (resistance) ribosome binding site rifampicin (resistance) streptomycin (resistance) sodium dodecyl sulfate tetracycline (resistance) Vibrio cholerae denotes plasmid carrier state xiii CHAPTER 1 GENERAL INTRODUCTION 2 Protein Secretion Mechanisms Protein secretion through the outer membrane is an important process for Gram' bacteria. They secrete a wide variety of proteins into their external environment including toxins such as hemolysin and degradative enzymes such as proteases, many of which play significant roles in the pathogenesis and dissemination of these organisms (Hirst and Welch, 1988). In order for proteins to be secreted outside the organism, they must pass through both the cytoplasmic and outer membranes. Some secreted proteins, such as Neisseria gonorrhoeae IgA protease (Pohlner et al., 1987), appear to carry all the information for secretion within the protein itself (Figure 1.1) since this protein is secreted from E. coli carrying the iga gene. It is possible, however, the E. coli encodes gene products which have not been identified but which are required for secretion of IgA protease. iga, the IgA protease structural gene, encodes a precursor protein with a N-terminal signal peptide and a large C-terminal helper domain. The precursor crosses the cytoplasmic membrane via the general export pathway (GEP). In E. coli, the GEP is known as the sec system, and, since most members of Enterobacteriaceae have proteins which cross-react with polyclonal antibodies directed against SecA and SecB (de Cock and Tommassen, 1991), a similar pathway likely exists in other bacteria as well. In the sec system, as the precursor protein emerges {Tom the ribosome, it binds to SecB or a similar chaperone protein to prevent folding from occurring. The SecB-precursor complex then interacts with SecA in the vicinity of the cytoplasmic membrane. SecA and a cytoplasmic membrane complex consisting of SecE and SecY then interact, initiating translocation of the protein. Two other proteins, SecD and SecF, presumably act in later translocation steps. Translocation is completed when 3 leader peptidase removes the signal peptide and the protein is released into the periplasm (for review see Schatz and Beckwith, 1990) Once in the periplasm, the C-terminal helper domain inserts itself into the outer membrane forming a pore through which the the N-terminal domain can pass. Autoproteolysis then occurs and the mature protein is released into the extracellular medium. Further autoproteolysis yields the mature form of the protein (Pohlner et al., 1987). The self-promoted secretion mechanism of IgA protease is an exception rather than the rule since the majority of proteins require extragenic factors to be secreted from Gram' bacteria. There are three pathways of secretion which seem to be highly conserved among Gram' bacteria: 1) the signal peptide independent pathway; 2) the general secretion pathway (GSP), and; 3) the hybrid pathway. @ Step 3: Auto- wrotiolysis Step 2: Pore \ formation Periplasm W10 Leader peptidase A Step 1: General Export Pathway -:-:-'-'-"z-:'z-:-:-:-:':-:-:-:-:':-:-.Signal.-:- £1I'I'I'I'I‘I'I‘I'I'I'I'I'I'I‘I'I'I'I‘I'I'I'I‘I'I'I] I-I-I-QMI-I-I-I-I-I-I-I-I-I-LI-I-I-Ipelp'tiHEI -I-I ~I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I N C Cytoplasm A Figure 1.1 Secretion of N. gonorrhoeae IgA protease through the outer membrane. The protein probably encodes all information necessary for secretion. After being translocated through the CM, the C-terminal domain likely forms a pore in the outer membrane through which the protease is secreted. Autoproteolysis releases the mature protein into the milleu. CM is the cytoplasmic membrane, OM is the outer membrane. N is the N-terminus of the protein, C is the C-terminus. 5 l. The Signal Peptide Independent Pathway In the signal peptide independent pathway, the protein to be secreted crosses both membranes in a single step and is not found in the periplasm during secretion (Delepelaire and Wandersman, 1989; Koronakis et al., 1989). This pathway is used to secrete a variety of proteins including RTX (repeats in toxins) toxins such as the hemolysins of Escherichia coli, Proteus vulgaris, Morganella morganii and Actinobacillus pleuropneumoniae, as well as Bordetella pertussis cyclolysin, Pasteurella hemolytica leucotoxin (see review by Fath and Kolter, 1993) and proteases including Erwinia chrysanthemi proteases A, B, and C (Ghigo and Wandersman, 1992), Pseudomonas aeruginosa alkaline protease (Tommassen et al., 1992), and Serratia marcescens SM protease (Letoffé et al., 1991). None of these proteins contain an N-terminal signal peptide which is typical of proteins that cross the cytoplasmic membrane via the GEP (Schatz and Beckwith, 1990). E. coli oc- hemolysin is secreted independently of SecA (Gray et al., 1989; Gentschev et al., 1990) and, similar to the other proteins secreted by this mechanism, has its secretion signal located in the a C-terminal portion of the protein (Nicaud et al., 1986; Delepelaire and Wandersman, 1990). These proteins show little sequence similarity in their C-termini (Kenny et al., 1992) and therefore the secretion signal was believed to be based on structural features rather than sequence of the protein. A glycine-rich motif (GGXGXD) is repeated several times close to the C-terminus in most of these proteins (Ghigo and Wandersman, 1992). E. chrysanthemi protease B is still secreted when only the last 39 amino acids of the protein (which does not have this motif) are present (Delepelaire and Wandersman, 1990) so the motif is not involved in secretion. Ghigo and Wandersman (1992) postulated that an amphiphilic helix (with hydrophobic residues clustered on one face and polar residues on 6 the other), which is predicted to occur in the C-termini of the metalloproteases of E. Chrysanthemi, S. marcescens, and P. aeruginosa and also a-hemolysin (Koronakis et al., 1989), could be the secretion signal of these proteins. Kenny et al. (1992), however, found that hemolysin was still secreted from E. coli when this helix was destroyed by site-directed mutagenesis. This group discovered that although the C-terminus of hemolysin was quite tolerant of mutations, there were several important residues scattered about which would decrease secretion of hemolysin if mutagenized. Consequently, they believed that these residues formed the "secretion signal" and were required for interaction with the other components of the secretion system. The extragenic secretion factors of this system include a cytoplasmic membrane ATP binding protein, a second protein which is likely anchored in the cytoplasmic membrane but spans the periplasm and an outer membrane protein. The proposed mechanism of secretion by the signal peptide independent pathway is as follows (Figure 1.2): 1) the C-terminus of the protein to be secreted, for example E. coli (at-hemolysin (HlyA), associates with the cytoplasmic membrane; 2) it then comes in contact with HlyB (the ATP binding component of the system) which had formed a channel-like complex with HlyD (the membrane spanning component) and TolC (the outer membrane component) that spans both membranes; 3) HlyA is then transported across both membranes in a single step, with ATP hydrolysis by HlyB supplying the energy required for this process (Fath and Kolter, 1993). Hemol sin 225542222222;sasszssssssssssssss; mo ]s2222522ssssszsssssssssssssss r a r W Periplasm H] D H] D _ y y Hemolysin N Figure 1.2 The signal peptide independent pathway of protein secretion through the outer membrane. With the aid of three extragenic factors, the protein (in this case hemolysin) crosses both membranes in a single step. CM is the cytoplasmic membrane, OM is the outer membrane. N is the N- terminus of the protein, C is the C-terminus. 8 Channels bridging the cytoplasmic and the outer membrane such as the one described above have not been proven to exist. Kellenberger (1990) demonstrated that adhesion zones between the two membranes seen in electron micrographs of E. coli were artifacts due to the chemical fixation used in preparation of the cells rather than junctions between the membranes. He proposed that secreted proteins must cross the periplasm, either by diffusion or through thin lipidic bridges. While the accessory factors of the hemolysin secretion system must play some role in the transport through both membranes since mutants defective in one of the hemolysin secretion accessory factors accumulate hemolysin in the cytoplasm (Gray et al., 1989), it is possible to imagine that they are responsible for the generation of an energy gradient to facilitate transport of the secretable protein across the periplasm by some other means. The identification of adhesion zones as artifacts does not however exclude the existence of temporary structures which form between the two membranes to allow secretion of hemolysin and other proteins. Some cross complementation exists between the factors responsible for secretion of proteins by this pathway. The protease B secretion system can also secrete the SM protease (Letofl‘é et al., 1991) and the hemolysin secretion apparatus can secrete protease B (Delepelaire and Wandersman, 1990), albeit with less efficiency than the host proteins. 9 2. The General Secretion Pathway (GSP) Proteins secreted by the general secretion pathway (GSP), cross the cytoplasmic and outer membranes in distinct steps (Figure 1.3). They require the products of the GEP to cross the cytoplasmic membrane as well as up to fourteen gene products to cross the outer membrane (for a recent review see Pugsley 1993) GSP genes have been isolated from a wide variety of Gram' bacteria including Aeromonas (Jiang and Howard, 1992; Howard et al. , 1993), Erwinia (He at al., 1991; Condemine et al., 1992; Lindeberg and Collmer, 1992; Reeves et al., 1993), Klebsiella (d'Enfert et al., 1987), Pseudomonas (Tommassen et al., 1992), Vibrio (Sandkvist et al., 1993; Chapters 2 and 3), and Xanthomonas (Dums et al., 1991; Hu et al., 1992) and are required for the secretion of a number of proteins from these bacteria. GSP gene products exhibit similarity amongst themselves as well as to proteins involved in the biogenesis of type 4 pili, proteins required for DNA uptake in the Gram positive organisms Bacillus and H aemophilus and filamentous phage proteins required for phage assembly (Table 1.1). 10 ..... Tf.r..za..r...i. 'I-IOM-I-LI-I-I-I-I-I-I-I-I-I-C-I-I-,:I:I:I:I: .......‘.....-......-...r.fi.] Step 2: General Secretion Pathway Folding of protein _ inperiplasm Periplasm Step 1: General Leader peptidase Export Pathway fl 'w'”HI:2:31:2323212311252323351371331.}. :III 23333131312:Iilililiiililiiziilii1323321223212 [ ..... 9954::::I::::::::::::::::::P€H¥i€ie.::1:::“z::::::::::::::r::::::::::::::::::::::::::::::::::: N C Cytoplasm mRNA Figure 1.3 The general secretion pathway of protein secretion through the outer membrane. The protein to be secreted crosses the CM via the general export pathway. After folding occurs in the periplasm, it crosses the OM with the aid of up to 14 extragenic factors. CM is the cytoplasmic membrane, OM is the outer membrane. terminus. N is the N-terminus of the protein, C is the C- 11 Table 1.1 The GSP and other related proteins of bacteria and phage. The Pul proteins of K. pneumoniae are listed across the top; similar proteins from other organisms are listed below. References: (1) Pugsley, 1993 (2) Jiang and Howard, 1991; Howard et al. , 1993 (3) Salmond and Reeves, 1993 (4) He et al., 1991; Condemine et al., 1992; Lindeberg and Collmer, 1992 (5) Tommassen et al., 1992 (6) Sandkvist et al., 1993; Chapters 2 and 3 (7) Dums et al., 1991; Hu et al., 1992 (8) Michiels et al., 1991 (9) Pasloske et al., 1985; Nunn et al., 1990 (10) Faast et al., 1989; Kaufman et al., 1993; Ogierman et al., 1993 (11) Albano et al., 1989 (12) Tomb et al., 1991 (13) Russel, 1991 Organism K. oxytoca A. hydrophila E. carotovora E. chrysanthemi P. aeruginosa“ V. cholerae X. compestris Y. enterocolitica P. aeruginosa“ V. cholerae B. subtilis H. influenzae Filamentous phage Role Secretion: Pullulanase Secretion: Aerolysin Secretion: Cellulase Pectinase Secretion: Cellulase Pectinase Secretion: Exotoxin A Protease Secretion: Toxin Protease Chitinase Secretion: Cellulase Pectinase Secretion: YOPs Piliation Pilialion Transfor- mation Transfor- mation Morpho- genesis 12 Ref 10 ll 12 Name Pul S B C D Exe D Out C D Out S B C D ch P Q Eps C D Xps D Ysc C Pil Q Tcp ComG ORF E gp IV 13 *The chA and PilD proteins of Pseudomonas aeruginosa are homologous (Tommassen et al. , 1992) 13 The GSP gene products required for the secretion of proteins consist of cytoplasmic, inner membrane, pilin-like and outer membrane proteins as well as a prepilin peptidase/N-methyl transferase. In Klebsiella oxytoca (Pugsley et al., 1990), the genes are organized close to pulA, the structural gene for pullulanase, the protein whose secretion they assist (Figure 1.4). pulA and pulB form an operon just upstream of pulS, which is transcribed in the opposite direction. Upstream ofpulA, pulC-O form a long operon. pulB is the only one of these genes whose inactivation does not inhibit pullulanse secretion by K. oxytoca (Pugsley, 1993). The pulB homologue in Erwinia chrysanthemi, outB, is required for secretion however (Condemine et al., 1992). PulE is the only one of these gene products found in the cytoplasm. A feature common to PulE and its homologues in other bacteria, is the presence of Walker boxes A and B which are believed to be involved in ATP binding (Walker et al., 1982). The conserved motif of the Walker Box A is G-X-S-G-X—G-G-K-T/S. When the fourth glycine residue of this motif was mutated to serine in chR, the PulE homologue of P. aeruginosa, the bacteria expressing the mutant protein was no longer able to secrete exotoxin A (Turner et al., 1993). Additionally, when the lysine of the Walker box A of V. cholerae EpsE was mutated to alanine, secretion of cholera toxin was abolished (M. Sandkvist, personal communication). An intact Walker box A must therefore be required for protein secretion. ATP hydrolysis by PulE and its homologues may therefore be supplying energy for the secretion process or perhaps activating other required products by phosphorylation. It should be noted however, that although PulE is located in the cytoplasm and other GSP proteins are in the cytoplasmic membrane, they are not directly involved in transport of secretable proteins across the cytoplasmic membrane. epsE mutants of V. cholerae accumulate cholera 14 toxin in the periplasm rather than the cytoplasm (Sandkvist et al., 1993); so the toxin is still crossing the cytoplasmic membrane even in their absence of EpsE. Some of the other proteins required for pullulanase secretion, PulC, PulF, PulK, PulL, PulM and PulN and their homologues, all have a hydrophobic region which could span the membrane and act as a membrane anchor. Several have also been shown to associate with the cytoplasmic membrane in cellular fractionation studies (Pugsley and Reyss, 1990; Possot et al., 1992). PulD, which is believed to associate with the outer membrane, is similar to the bacteriophage f1 gpIV protein which is an outer membrane protein required for filamentous phage assembly and secretion (Possot et al., 1992). PulS is a lipoprotein which is also likely located in the outer membrane (d'Enfert and Pugsley, 1989). The remaining proteins, PulG, PulH, PulI and PulJ, all possess signal peptides which are similar to those of type IV pilin precursors, six to eight amino acids followed by the consensus sequence Gly-Phe-Thr-Leu-(Leu or Ile)-Glu (Reyss and Pugsley, 1990). This is the site of the processing carried out by another Pul protein, PulO, a prepilin peptidase/N-methyl transferase which has been shown to cleave the signal peptide and methylate the N-terminal Phe of PulG and likely acts on the other pilin-like proteins as well (Pugsley and Dupuy, 1992). PilD (chA), the PulO homologue in P. aeruginosa, has been shown to process all of the PulG-J homologues, chT-W, by cleaving off the signal peptide and methylating the N-terminal phenylalanine of the pilin-like proteins which is a characteristic of most type IV pilins. This modification could be required for export or it could protect the proteins from protease degradation (Nunn and Lory, 1993). 15 pul SB A C D E FGHIJKLMNO Figure 1.4 The pul gene cluster of Klebsiella oxytoca 16 Little is known about the actual mechanism of secretion through the outer membrane and what role the GSP gene products play in the process (Figure 1.3). As stated earlier, the cytoplasmic and cytoplasmic membrane components of the GSP are not responsible for translocation of the exoprotein across the cytoplasmic membrane. When Pugsley et al. (1991) expressed the put secretion genes in wild type E. coli, they found that pullulanse was transported to the cell surface. When the genes were expressed in various sec mutants however, pullulanase was not secreted, implying the requirement for the GEP. The GEP may be required not only for the translocation of pullulanase across the cytoplasmic membrane, but also for the transport of GSP components, such as PulD, which are located in the periplasm or outer membrane. Potential functions of the PulE protein include acting as a protein kinase to phosphorylate and activate other proteins required in pullulanase secretion or to provide a source of energy to drive pullulanase secretion (Possot et al., 1992). Another possible function of PulE is to provide ATP for the translocation of other GSP proteins, likely the pilin like proteins, across the cytoplasmic membrane. The cytoplasmic proteins of the GSP may be involved in formation of an energy transducing system to couple secretion across the outer membrane to the electrochemical gradient across the cytoplasmic membrane (Tommassen et al., 1992). Once the proteins to be secreted have crossed the cytoplasmic membrane via the general export pathway, they transiently reside in the periplasm before being secreted through the outer membrane (Hirst and Holmgren, 1987b). There is evidence that folding of the exoproteins occurs here before secretion to the external environment. Cloned E. coli heat-labile enterotoxin (LT) appears to assemble into a holotoxin consisting of a pentamer of B subunits and a single A subunit in the periplasm of V. cholerae 17 before being secreted. By pulse-labeling V. cholerae cells with radioactive methionine and then isolating periplasmic contents after various time intervals, Hirst and Holmgren (1987a) were able to observe the assembly of B subunit pentamers in the periplasm. Additional evidence indicating that LT assembles in the periplasm is that in a dsbA- mutant of V. cholerae, where disulfide bonds are not formed, Eth is transported only to the periplasm (Findlay et al., 1993). The dsbA gene encodes a periplasmic disulfide oxidoreductase which aids in the formation of disulfide bonds, hence this result suggests that LT must fold in the periplasm in order to be secreted. Pullulanse, which has at least one intramolecular disulfide bond, also shows reduced secretion to the outer membrane when it is expressed in a dsbA mutant of E. coli carrying the put secretion genes (Pugsley, 1992). Disulfide bond formation is also a necessary step in the secretion of cellulase EGZ of E. Chrysanthemi (Bortoil-German et al., 1994). The cellulase is not secreted in the absence of disulfide bond formation but it is able to fold into a functional conformation. It is possible, therefore that disulfide bond formation creates a three-dimensional motif in the cellulase which is recognized by the secretion machinery. After protein folding occurs, it has been suggested that the pilin- like proteins form a pseudopilus which extends across the periplasm to guide the secretable proteins to the outer membrane (Pugsley, 1993). Pugsley and Possot (1993) however, found no evidence for the formation of such a structure when the pul genes were expressed in E. coli. In fact, the pilin like proteins of P. aeruginosa have been found to associate predominantly with the cytoplasmic rather than the outer membrane (Nunn and Lory, 1993). It is possible that some type of pseudopilus structure does form but collapses under the conditions used to study its formation. The folded exproteins may cross the outer membrane through a pore created by the D and S proteins, 18 the only GSP proteins believed to be present in the outer membrane. Another function of these proteins could be to secure other secretion proteins to the outer membrane. 3. The Hybrid Pathway The hybrid pathway is the third protein secretion pathway which has recently been identified in pathogenic bacteria and has features in common with each of the first two pathways. In Yersinia enterocolitica, Yops, plasmid encoded virulence factors which are essential for the pathogenicity of the organism, are secreted by this pathway (Michiels et al., 1991; Woestyn et al., 1994). Comparable to hemolysin and other proteins secreted by the signal peptide independent pathway, the Yops do not possess signal peptides; unlike this pathway though, the secretion signal is located in the N-terminus of the protein rather than the C-terminus. None of the ysc gene products, which are required for secretion of the Yops, are similar to any gene products of the signal peptide independent pathway. A characteristic of the hybrid pathway that is shared with the GSP is that they both require a number of extragenic factors to be secreted from the cell. One of the genes required for Yop secretion, yscC, shows similarity to the pulD gene (Table 1.1), however other ysc genes do not show similarity to other GSP genes. This also indicates that the hybrid pathway is distinct from the other two pathways. Genes similar to the ysc genes of Y. enterocolitica have also been identified in other human pathogens including Shigella flexneri and Salmonella typhimurium (Wei and Beer, 1993). Similar genes have also been identified in plant pathogens including Pseudomonas solanacearum (Salmond and Reeves, 1993), P. syringae pv. syringae, Erwinia amylovora, and Xanthomonas campestris pv. vesicatoria (Wei and Beer, 1993). 19 Protein secretion in Vibrio cholerae The purpose of this research was to identify the genes responsible for the secretion of proteins across the outer membrane of V. cholerae. This organism, an inhabitant of the gut and the causative agent of the disease cholera (Finkelstein, 1973), is also autochthonous to estuarine and marine waters, where it may be present in a viable but non-culturable state (Colwell et al., 1985). It is often found attached to chitin-containing waterborne particles such as the phytoplankton Volvox and zooplankton molts (Tamplin et al., 1990). In order to survive in these two distinct niches, the bacteria must be able to adapt and respond to changes in their environment. Chitinase is known to be secreted from V. cholerae (Chapter 2). In the aquatic environment, it is possible that this secreted chitinase helps V. cholerae attach to chitin particles. Once ingested by a host, the acid resistant chitin may also protect the bacteria from the low pH of the human stomach (N alin et al., 1979). After the Vibrio reaches the small intestine, secretion of various proteins such as mucinase, neuraminidase, and toxin coregulated pili (TCP) aids in colonization of the gut and nutrient acquisition (Miller et al., 1989). Mutants of V. cholerae deficient in the secretion of another extracellular enzyme, hemagglutinin-protease (HA/protease), have been found to be 100 fold less virulent than wild type cells (Schneider and Parker, 1978). This may be because these mutants were GSP mutants, however, as they were also defective in the secretion of other exoproteins including neuraminidase. It has been recently speculated that HA/protease actually plays a role in the detachment of V. cholerae from the epithelium rather than in its colonization (Finkelstein et al. , 1992). 20 The secretion of heat-labile enterotoxin of E. coli (LT) was used as a model in order to study the secretion of proteins from V.cholerae. The structure, immunological properties and biochemical activity of LT are similar to those of cholera toxin (CT) (Svennerholm and Holmgren, 197 8). LT is a multimeric protein consisting of one A subunit (EtxA) of 28 kDa and five identical B subunits (Eth) of 12 kDa each (Gill et al., 1981; Sixma et al., 1993). The B subunits are necessary for binding to GMl, the ganglioside receptor on target epithelial cells. After this binding, the A subunit is proteolytically cleaved and the A1 portion is taken up into the target cell. Once inside the target cell, the toxin stimulates the production of cyclic AMP (cAMP) by cleaving NAD+ and linking the resulting ADP-ribose moiety to the accessory G protein of the adenlyate cyclase system. The now activated G protein stimulates adenylate cyclase to produce large amounts of cAMP. This increase in cAMP leads to a release of ions and fluids causing watery diarrhea in the host (Sixma et al., 1993). Both the A and B peptides of LT are synthesized with signal peptides that are cleaved off during export across the cytoplasmic membrane via the general export pathway (Dallas et al., 1979; Palva et al., 1981; Hofstra and Witholt, 1984). The A and B subunits (or the B subunits alone if the A is not present) then assemble in the periplasm to form the mature protein (Hirst et al., 1983; Hofstra and Witholt, 1984; Hofstra and Witholt, 1985). In wild type V. cholerae, the folded LT is then secreted across the outer membrane by the GSP (Figure 1.5), just as CT is (Sandkvist et al., 1993); Chapter 2). When either protein is expressed in E. coli though, it remains in the periplasm (Pearson and Mekalanos, 1982; Neill et al., 1983; Hirst et al., 1984). Thus the GSP secretion machinery present in V. cholerae is likely absent in E. coli. 21 During the course of this project, epsC-N, which are the V. cholerae homologues of pulC-N, have been cloned and sequenced (Sandkvist et al., 1993; Chapters 2 and 3). In addition to enterotoxin, the GSP gene products of V. cholerae are required for secretion of other pathogenicity and colonization factors including protease and chitinase (Chapter 2). They do not, however, appear to be required for the secretion of toxin-coregulated pili (TCP), type IV pili which assist in colonization of the small intestine of the host (Kaufman et al., 1993). A cluster of genes required for the secretion of TCP has been cloned and sequenced (Ogierman and Manning, 1992; Kaufman et al., 1993; Ogierman et al., 1993). Included in the top gene cluster is tcpJ, which encodes a peptidase required for the correct processing of the pilin subunit TcpA (Kaufman et al., 1991). In P. aeruginosa, the TcpJ homologue, chA/PilD, is required for the processing of both type IV pilin and the pilin- like proteins of the GSP of this organism (Tommassen et al., 1992). This does not appear to be the case in V. cholerae however, since TcpJ is unable to process EpsG (M. Sandkvist, personal communication). Although two of the proteins required for secretion of TCP, TcpT and E, show similarity to EpsE and F, this cluster of genes seems to be different from the GSP. The TCP are regulated by ToxR, a global regulatory protein that regulates as many as seventeen virulence genes in V. cholerae (Peterson and Mekalanos, 1988). While the synthesis of cholera toxin is also regulated by ToxR, its secretion is not and the eps genes seem to be expressed constituitively (M. Sandkvist, personal communication). 22 Pro-A Pro-B Cytoplasm iiiiiiiiiiiii iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii ,. Milli)!!! Mlllllllll MtlllllllllllMMHMWMMMMMM CM Periplasm Assembly Rfillllfilfiififififillfil Figure 1.5 Biogenesis of E. coli heat-labile enterotoxin. Signal peptides of the A and B precursors are removed as they cross the cytoplasmic membrane (CM). The subunits then assemble in the periplasm to form the holotoxin which is transported across the outer membrane (OM) by the general secretion pathway in Vibrio cholerae. 23 Since little is known about the mechanism of secretion through the outer membrane, it is important to try and understand how this process works. Increased knowledge of secretory processes will help to increase our understanding of the invasiveness and pathogenicity of Gram‘ bacteria, since many secrete tissue degrading enzymes and toxins harmful to both animals and plants. This knowledge also has industrial importance, since proteins are often easier to isolate and purify when they are secreted as there are fewer proteins present in the external medium than in the periplasm or cytoplasm of bacteria. Although there are limitations to the amount of protein that can be secreted, production may also increase. 24 References Albano, M., Breitling, R. and Dubnau, D. (1989) Nucleotide sequence and genetic organization of the Bacillus subtilis comG operon. J Bacteriol 171: 5386-5404. Bortoil-German, I., Brun, E., Py, B., Chippaux, M. and Barras, F. (1994) Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi. Molec Microbial 11: 545-553. Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, SA. and Palmer, L.M. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Bio/ Technol 3: 817-820. 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J Biol Chem 264: 9083-9089. Delepelaire, P. and Wandersman, C. (1990) Protein secretion in Gram negative bacteria: the extracellular metaloprotease B from Erwinia chrysanthemi contains a C terminal secretion signal analogous to that of Escherichia coli a - hemolysin. J Biol Chem 265: 17118-17125. Dums, F., Dow, J .M. and Daniels, M.J. (1991) Structural characterization of protein export genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to export systems of other Gram-negative bacteria. Mol Gen Genet 229: 357-364. 25 Faast, R., Ogierman, M.A., Stroeher, U.H. and Manning, RA. (1989) Nucleotide sequence of the structural gene, tcpA, for a major pilin subunit of Vibrio cholerae. Gene 85: 227-231. Fath, M.J. and Kolter, R. (1993) ABC transporters: bacterial exporters. Microbiol Rev 57: 995- 1017. Findlay, G., Yu, J. and Hirst, TR. 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(1993) Isolation and analysis of eight exe genes and their involvement in extracellular protein secretion and outer membrane assembly in Aeromonas hydraphila. J Bacterial l7 5: 6695-6703. Hu, N.-T., Hung, M.-N., Chiou, S.-J., Tang, F., Chiang, D.-C., Huang, H.-Y. and Wu, C.-Y. (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. Campestris. J Bacteriol 174: 2679-2687. Jiang, B. and Howard, SP. (1991) Mutagenesis and isolation of Aeromonas hydraphila genes which are required for extracellular secretion. J Bacteriol 173: 1241-1249. Jiang, B. and Howard, SP. (1992) The Aeromonas hydrophila eer gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway. Molec Microbial 6: 1351-1361. Kaufman, M.R., Seyer, J .M. and Taylor, R.K. (1991) Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by Gram-negative bacteria. Gene Develop 5: 1834-1846. Kaufman, M.R., Shaw, C.E., Jones, LB. and Taylor, R.K. (1993) Biogenesis and regulation of the Vibrio cholerae toxin-coregulated pilus: analogies to other virulence factor secretory systems. Gene 126: 43-49. Kellenberger, E. (1990) The "Bayer bridges" confronted with results from improved electron microscopy methods. Molec Microbial 4: 697 -705. 27 Kenny, B., Taylor, S. and Holland, LB. (1992) Identification of individual amino acids required for secretion within the haemolysin (HlyA) C- terminal targeting region. Molec Microbiol 6: 1477-1489. Koronakis, V., Koronakis, E. and Hughes, C. (1989) Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. EMBO J 8: 595-605. Letoffé, S., Delepelaire, P. and Wandersman, C. (1991) Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions. J Bacterial 173: 2160-2166. Lindeberg, M. and Collmer, A. (1992) Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi : sequence comparison with secretion genes from other Gram-negative bacteria. J Bacterial 174: 7385-7397. Michiels, T., Vanooteghem, J .-C., de Rouvroit, C.L., China, B., Gustin, A., Boudry, P. and Cornelis, GR. (1991) Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enteracalitica. J Bacteriol 173: 4994-5009. Miller, J .F., Mekalanos, J .J . and Falkow, S. (1989) Coordinate regulation and sensory transduction in the control of bacterial virulence. Science 243: 916- 922. Nalin, D.R., Daya, V., Reid, R., Levine, M.M. and Cisneros, L. (1979) Adsorption and growth of Vibrio cholerae on chitin. Infect Immun 25: 768- 770. Neill, R.J., Ivins, BE. and Holmes, R.K. (1983) Synthesis and secretion of the plasmid-coded heat-labile enterotoxin of Escherichia coli in Vibrio cholerae. Science 221: 289-291. Nicaud, J.-M., Mackman, N., Gray, L. and Holland, LB. (1986) The C- terminal, 23 kD peptide of E. coli haemolysin 2001 contains all the information necessary for its secretion by the haemolysin (Hly) export machinery. FEBS Letts 204: Nunn, D., Bergman, S. and Lory, S. (1990) Products of three accessory genes, pilB, pilC and pilD are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol 172: 2911-2919. Nunn, D. and Lory, S. (1993) Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins chT, -U, -V, and -W. J Bacterial 175: 4375-4382. 28 Ogierman, MA. and Mamiing, RA. (1992) Homology of TcpN, a putativae regulatory protein of Vibrio cholerae, to the AraC family of transcriptional activators. Gene 116: 93-97. Ogierman, M.A., Zabihi, S., Mourtzios, L. and Manning, RA. (1993) Genetic organization and sequence of the promoter-distal region of the tap gene cluster of Vibrio cholerae. Gene 126: 51-60. Palva, E.T., Hirst, T.R., Hardy, S.J.S., Holmgren, J. and Randall, LL. (1981) Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli. J Bacteriol 146: 325-330. Pasloske, B.L., Finlay, BB. and Paranchych, W. (1985) Cloning and sequencing of the Pseudomonas aeruginosa PAK pilin gene. FEBS Lett 183: 408-412. Pearson, G.D.N. and Mekalanos, J .J . (1982) Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K12. Proc Natl Acad Sci USA 79: 2976-2980. Peterson, KM. and Mekalanos, J .J . (1988) Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization. Infect Immun 56: 2822-2829. Pohlner, J ., Halter, R., Beyreuther, K. and Mayer, T.F. (1987 ) Gene structure and extracellular secretion of Neisseria ganorrhaeae igA protease. Nature (London) 325: 458-462. Possot, O., d'Enfert, C., Reyss, I. and Pugsley, A.P. (1992) Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein. Molec Microbiol 6: 95- 105. Pugsley, A.P. (1992) Translocation of a folded protein across the outer membrane in Escherichia coli. Proc Natl Acad Sci USA 89: 12058-12062. Pugsley, A.P. (1993) The complete general secretory pathway in Gram- negative bacteria. Microbiol Rev 57: 50-108. Pugsley, A.P., d'Enfert, C., Reyss, I. and Komacker, M.G. (1990) Genetics of extracellular protein secretion by Gram-negative bacteria. Annu Rev Genet 24: 67-90. Pugsley, A.P. and Dupuy, B. (1992) An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca. Molec Microbiol 6: 751-760. 29 Pugsley, A.P., Kornacker, M.G. and Poquet, I. (1991) The general protein- export pathway is directly required for extracellular pullulanase secretion in Escherichia coli K12. Molec M icrobiol 5: 343-352. Pugsley, A.P. and Possot, O. (1993) The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex. Molec M icrobiol 10: 665-674. Pugsley, A.P. and Reyss, I. (1990) Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion. Molec Microbiol 4: 365-379. Reeves, P.J., Whitcombe, D., Wharam, 8., Gibson, M., Allison, G., Bunce, N., Barallon, R., Douglas, P., Mulholland, V., Stevens, 8., Walker, D. and Salmond, G.P.C. (1993) Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in Gram- negative bacteria. Molec Microbiol 8: 443-456. Reyss, I. and Pugsley, A.P. (1990) Five additional genes in the pulC-0 operon of the Gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion. Mol Gen Genet 222: 176-184. Russel, M. (1991) Filamentous phage assembly. Molec Microbial 5: 1607- 1613. Salmond, G.P.C. and Reeves, P.J. (1993) Membrane traffic wardens and protein secretion in Gram-negative bacteria. Trends Biochem Sci 18: 7- 12. Sandkvist, M., Morales, V. and Bagdasarian, M. (1993) A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae. Gene 123: 81-86. Schatz, P.J. and Beckwith, J. (1990) Genetic analysis of protein export in Escherichia coli. Annu Rev Genet 24: 215-248. Schneider, DR. and Parker, CD. (1978) Isolation and characterization of protease-deficient mutants of Vibrio cholerae. J Infect Dis 138: 143-151. Sixma, T.K., Kalk, K.H., van Zanten, B.A.M., Dauter, Z., Kingma, J ., Witholt, B. and H01, W.G.J. (1993) Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin. J Mol Biol 230: 890-918. Svennerholm, A.-M. and Holmgren, J. (197 8) Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GMl-ELISA) procedure. Curr Microbiol 1: 19-23. 30 Tamplin, M.L., Gauzens, A.L., Huq, A., Sack, DA. and Colwell, RR. (1990) Attachment of Vibrio cholerae serogroup 01 to zooplankton and phytoplankton of Bangladesh waters. Appl Environ Microbiol 56: 1977- 1980. Tomb, J .-F., El-Hajj, H. and Smith, HQ. (1991) Nucleotide sequence of a cluster of genes involved in the transformation of Haemaphilus influenzae Rd. Gene 104:1-10. Tommassen, J ., Filloux, A., Bally, M., Murgier, M. and Lazdunski, A. (1992) Protein secretion in Pseudomonas aeruginosa. FEMS Microbiol Rev 103: 73-90. Turner, L.R., Lara, J.C., Nunn, D.N. and Lory, S. (1993) Mutations in the consensus ATP-binding sites of chR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J Bacterial 175: 4962-4969. Walker, J .E., Saraste, M., Runswick, M.J. and Gay, NJ (1982) Distantly related sequences in the a- and B-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1: 945-951. Wei, Z.-M. and Beer, S.V. (1993) HrpI of Erwinia amylavora functions in secretion of harpin and is a member of a new protein family. J Bacterial 17: 7958-7967. Woestyn, S., Allaoui, A., Wattiau, P. and Cornelis, GR. (1994) YscN, the putative energizer of the Yersinia yop secretion machinery. J Bacterial 176: 1561-1569. CHAPTER2 GENES REQUIRED FOR EXTRACELLULAR SECRETION OF ENTEROTOXIN ARE CLUSTERED IN VIBRIO CH OLERAE 31 32 Pleiotropic transposon insertion mutants of Vibrio cholerae that are unable to secrete enterotoxin, protease and chitinase through the outer membrane have been isolated. The gene, epsM, responsible for complementation of two of the Tn5 insertion mutations was sequenced. It encodes a putative cytOplasmic membrane protein of 18.5 kDa that exhibits similarity to proteins required for extracellular secretion of pullulanase, pectate lyase or elastase in other Gram' bacteria. It is present on a 15 kb DNA fragment from the V. cholerae genome, containing the epsE gene that was previously shown to be required for secretion of cholera toxin [Sandkvist et al., Gene 123 (1993) 81-86]. Partial reading frames flanking epsM also demonstrated similarity to genes required for extracellular secretion of pullulanase in Klebsiella oxytoca. 33 Introduction The intestinal pathogen Vibrio cholerae that is the causative agent of cholera in humans (Finkelstein, 1973), is also found in estuarine and marine waters (Colwell et al., 1985). V. cholerae secretes a number of proteins through the outer membrane including cholera toxin, protease, chitinase, DNase and pilin. The production and secretion of various exoproteins may aid in the adaptation of this organism to these diverse ecological niches. In the aquatic environment, vibrios are often found attached to chitinous particles (Tamplin et al., 1990). Secreted chitinase may aid in attachment and nutrient acquisition. In the gut, secreted proteins such as mucinase or pili assist the colonization process whereas secretion of HA/protease, and perhaps cholera toxin, may be responsible for the detachment of V. cholerae from epithelial cells (Finkelstein et al., 1992). Although diverse types of proteins are secreted by V. cholerae and other Gram’ bacteria, it is believed that there are two main, highly conserved, pathways of secretion. In the signal peptide independent pathway, which is the one-step mechanism of secretion of Escherichia coli a-hemolysin (Holland et al., 1990) and Erwinia chrysanthemi proteases A, B, and C (Ghigo and Wandersman, 1992), proteins cross the cell envelope without a transient residence in the periplasm. In the general secretion pathway, proteins such as Klebsiella oxytoca pullulanase cross the cytoplasmic and outer membranes in two distinct steps (Pugsley et al., 1990). Cholera toxin (CT) as well as the structurally similar E. coli heat- labile enterotoxin (LT) are secreted through the outer membrane of V. cholerae by the general secretion mechanism (Hirst and Holmgren, 1987; Sandkvist et al., 1993a; Sandkvist et al., 1993b). The toxins each consist of 34 one A subunit and five identical B subunits (Gill et al., 1981; Sixma et al., 1991). The A and B precursor polypeptides contain signal peptides that are cleaved off during export across the cytoplasmic membrane (Dallas et al., 1979; Palva et al., 1981; Hofstra and Witholt, 1984). Assembly of the subunits (or of the B subunits alone if the A is not present) occurs in the periplasm (Hirst et al., 1983; Hofstra and Witholt, 1984; Hofstra and Witholt, 1985). When assembled, both CT and LT are secreted from V. cholerae, but in E. coli, they remain in the periplasm (Pearson and Mekalanos, 1982; Neill et al., 1983; Hirst et al., 1984). Although protein secretion is most likely of primary importance for the pathogenicity as well as for the survival of V. cholerae in diverse environments, only one gene, epsE, essential for the extracellular secretion of CT and protease has been identified to date (Sandkvist et al., 1993a). The aim of this study was to isolate transposon insertion mutants of V. cholerae that were unable to secrete proteins through the outer membrane in order to characterize other genes involved in this process. 35 Materials and Methods Bacterial strains, plasmids, and growth conditions. V. cholerae TRH7000, a thy derivative (Hirst et al., 1984) of the El Tor strain J BK70 (Kaper et al., 1984) was the wild type strain used in this study. It was grown at 37°C in M9 medium (Miller, 1972) supplemented with 20 amino acids (50ug/ml) or LB, both containing 100ug/ml thymine. E. coli strain M01061 (F', ara139 ara lea-7697 lacX74 galU galK hst52 rspl) (Casadaban and Cohen, 1980) carrying pGP1-2, the plasmid encoding T7 RNA polymerase (Tabor and Richardson, 1985) was the host for T7 expression experiments and was grown in LB or M9 medium supplemented with 50ug/ml of 19 aa (no met). The broad host range vector pMMB67EH (Fiirste et al., 1986) was used in the construction of pMMB347, pMMB524, and pMMB528. The inserts from these three plasmids were also cloned as EcoRI-PstI fragments (from the polylinker sites of the appropriate plasmid) and introduced into pT7-5 and pT7-6 (Tabor and Richardson, 1985) which contain the T7 promoter. pWD615 (Dallas, 1983), which carries the eth gene, was introduced into TRH7000 and its mutant derivatives in order to assay the amount of LT B subunit pentamers secreted. Media used for the detection of extracellular enzyme secretion were LB containing 1% skim milk, M9 minimal agar plates containing 0.4% colloidal chitin prepared by the method of Hsu and Lockwood (Hsu and Lockwood, 1975), and DNase test agar (flooded with 1N HCl after growth). Antibiotics used were ampicillin (100 ug/ml), kanamycin (50ug/ml) and tetracycline (2.5ug/ml). 36 Transposon Tn5 mutagenesis. Tn5 was introduced onto pHSGl, a tetracycline-resistant, temperature-sensitive derivative of pSClOl (Hashimoto and Sekiguchi, 1976). This plasmid was used to deliver Tn5 to the chromosome of V. cholerae TRH7000. The KmR transposon insertion mutants were selected at 42°C on LB agar containing skim milk, Km and thymine. Determination of LT B subunit pentamer secretion. The percentage of LT B subunit pentamers present in the growth medium and sonicated cells of wild type TRH7000 and secretion mutants was determined by GM1 - ELISA (Svennerholm and Holmgren, 1978). All strains contained plasmid pWD615. Cells were grown with shaking at 37°C to A650nm = 0.7 in M9 medium (Miller, 1972) containing all twenty aa, thymine and, for cells carrying pMMB plasmids, Ap. One ml of the culture was harvested by centrifugation and cells suspended in 1.0 ml 10 mM phosphate buffer pH 7.3 /150 mM NaCl (PBS) were broken by sonication with two ten second pulses. Expression of genes under phage T7 promoter control. pT7-5/6 and derivatives of these plasmids constructed during this study were introduced into E. coli MC1061[pGP1-2]. Cells were grown at 30°C in LB containing 50 11ng of Ap and Km until A59Onm = 0.5. 0.2 m1 of cells were then pelleted, washed with 5 ml of M9 medium (Miller, 1972) containing 50 ug/ml of 19 aa (no Met) and 10 ug/ml of thiamine, resuspended in 1.0 ml of this medium and incubated at 30°C for 2 h. The temperature was then raised to 42°C to induce production of T7 RNA polymerase and 300 ug/ml rifampicin was added. The temperature was 37 lowered to 30°C and proteins were labeled with [35S]L-methionine (5 uCi/ml) for 5 min. The cells were pelleted, resuspended in sample buffer and heated at 95°C for 5 min. Proteins were separated on a 0.1% SDS-13.5% polyacrylamide gel which was then dried and autoradiographed (Laemmli, 1970). Some derivatives were also introduced into V. cholerae TRH7000. For these, the cells were grown overnight in M9 media supplemented with 19 aa and 100 ug/ml thymine. 200 pl of this culture was then pelleted and resuspended in 1 ml of the same media and incubated for 2 hours at 30°C. Following this, the cells were incubated at 42°C for 15 minutes, after which 200 ug/ml rifampicin was added. After 10 minutes the temperature was lowered to 30°C for an additional ten minutes. The cells were then labeled with [35S]L-methionine (10 uCi/ml) for 5 min. The cells were pelleted, resuspended in sample buffer and electrophoresed as described above. Localization of the EpsM protein in E. coli and V. cholerae. To determine the cellular location of the EpsM protein, E. coli MC 1061 and V. cholerae TRH7000 carrying a pT7-5 derivative which contained the epsM gene were grown as described in the previous section. One ml of labeled E. coli cells was then pelleted and resuspended in 250 ill 100 mM P04 buffer pH 7 containing 0.25 M sucrose. 20 ug/ml lysozyme and 1 mM EDTA were then added and the cells were incubated on ice for 15 minutes to release periplasmic proteins. The cells were then pelleted, resuspended in 250 pl PBS and were broken by sonication with two fifteen second pulses. After a low speed centrifugation to remove any whole cells, the lysate was centrifuged to pellet the membrane proteins. This pellet was resuspended in 250 ul PBS and 0.1% Triton X-100 was added. This was then centrifuged for 30 minutes to pellet the outer membrane fraction. The 38 periplasmic, cytoplasmic, and inner and outer membrane fractions were then mixed with sample buffer and heated at 95°C for 5 min. Proteins were separated on a 0.1% SDS-13.5% polyacrylamide gel which was then dried and autoradiographed (Laemmli, 1970). For V. cholerae fractionation, 1.0 ml of labeled cells were pelleted and resuspended in 250 ill PBS containing 4000 U/ml polymixin B sulfate (Sigma) and incubated on ice for 25 minutes to release periplasmic proteins. The cells were again pelleted, resuspended in 250 pl PBS, sonicated and centrifuged as described for E. coli. The pellet was then resuspended in 250 pl PBS containing 1.0% Triton X-100 and incubated at room temperature for 30 minutes. The lysate was then centrifuged, resuspended in sample buffer and electrophoresed as described for the E. coli cells. DNA sequence analyses. DNA sequence analysis of the insert present in pMMB524 was performed by using Sequenase 2 (US. Biochemical Corp) and the dideoxynucleotide chain termination method (Sanger et al., 1977). A series of unidirectional deletions were prepared in order to sequence the non- coding strand. The coding strand was sequenced by primer walking utilizing oligonucleotide primers synthesized by the MSU Macromolecular Structure Facility. The insertion points of the transposon in the mutants PU3 and PU5 were determined by sequencing chromosomal fragments containing the transposon. DNA and deduced amino acid sequences were analyzed with the GCG sequence analysis software package (Devereux et al., 1984). The sequence presented in this report appears in the GenBank database under the accession number L13660. 39 Results and Discussion Isolation of Tn5 insertion mutants defective in the secretion of exoproteins The temperature-sensitive vector pHSGl::Tn5 was used to deliver Tn5, which encodes kanamycin resistance, into the chromosome of V. cholerae TRH7000. A total of 10,000 transposition mutants were screened on LB agar containing skim milk, thymine and kanamycin in order to screen for mutants unable to secrete protease. Eight colonies with protease zones smaller than those of the wild type TRH7000 were picked. Since the ctxA and cth genes of TRH7000 have been deleted (Kaper et al., 1984), the plasmid pWD615 (Dallas, 1983) that contains the eth gene encoding the B subunit of E. coli LT was introduced into these mutants and the presence of B subunit pentamers in the extracellular media and in sonicated cells from liquid cultures was determined as described. Four mutants, (PU3, PU4, PU5, and PU6) exhibited decreased secretion of the B subunit (Table 2.1), which was found to accumulate in the periplasm of the mutant cells. The mutants were also tested for secretion of chitinase by plating on chitin agar. PU3 and PU5 were found to be defective in the secretion of chitinase (Figure 2.1b). Secretion of another exoprotein, DNase, was not affected (results not shown). The mutants were also found to exhibit an unusual morphology, as they grew as long filaments rather than short, curved rods (Figure 2.2). 40 Table 2.1 Complementation of the LT secretion defect in mutants PU3 and PU5 by chromosomal fragments of V. cholerae TRH7000. LT B subunit pentamers (%):‘il Strain Medium Periplasm Cells TRH7000[pWD615] 79 N.D.b 21 PU3[pWD615] 6 7O 24 PU4[pWD615] 5 7O % PU5[pWD615] 5 74 Z) PU6[pWD615] 3 70 27 PU3[pWD615; pMMB347] 73 N .D. 27 PU5[pWD615; pMMB347] 71 ND. E PU3[pWD615; pMMB524] 58 N .D. 42 PU5[pWD615; pMMB524] 29 ND. 71 PU3[pWD615; pMMB528] 3 ND. 97 PU5[pWD615; pMMB528] 1 ND. 99 a1LT B subunit pentamers present in the growth medium and sonicated cells were determined by the GM1 - ELISA (Svennerholm and Holmgren, 1978). bNot determined 41 Figure 2.1 Plate assays for protease and chitinase secretion in V. cholerae TRH7000 derivatives. A. Skim milk agar to assay secretion of protease. B. Colloidal chitin agar to assay secretion of chitinase. 1, TRH7000 , PU3 3, PU3[pMMB347] 4, PU3[pMMB524] 5, PU3[pMMB528] Figure 2.2 Secretion defective mutants of V. cholerae exhibit a change in morphology which disappears when secretion is restored. (Photos by Dr. Frank Dazzo, Dept. of Microbiology, Mich. State Univ.) 43 Complementation of secretion defect in mutants PU3 and PU5 Plasmid pMMB339, which contains a 15-kb insertion of genomic DNA from the strain TRH7000 complementing the secretion defect in V. cholerae mutant M14 (Sandkvist et al., 1993b), was introduced into the four transposon insertion mutants. Protease and chitinase secretion were restored in PU3 and PU5 as was secretion of LT B subunit. Subcloning localized this complementation function to an approximately 4.5-kb KpnI fragment that was inserted into pMMB67EH to create pMMB347. BAL-31 nuclease digestion of the KpnI fragment further located the gene responsible for complementation on a 1.1-kb fragment present in pMMB524 (Figs. 2.1 and 2.3, Table 2.1). The secretion defect of PU4 and PU6 could not be rescued by pMMB339. Polypeptides encoded by the KpnI fragment In vivo synthesis of the proteins encoded by the KpnI fragment was performed using the T7 polymerase/promoter system of Tabor and Richardson (Tabor and Richardson, 1985). The KpnI fragment of pMMB347 as well as the insert from pMMB524 were introduced into pT7-5 and pT7-6 in order to determine the direction of transcription. As shown in Figure 2.4, pT7-5 containing the insert from pMMB524 directed the synthesis of one protein of approximately 18.5 kDa. pT7-5 containing the KpnI insert from pMMB347 directed the synthesis of two proteins of 18.5 and 45 kDa. No labeled proteins were detected when the fragments were inserted in the opposite orientation in respect to the T7 promoter, or when a deletion derivative of the pMMB524 insert present in pMMB528 (Figure 2.4) not complementing the secretion mutations was present in pT7-5. Complementation of: Production of: lkb PU3 and PU5 18.5 kDa 45 kDa protein protein K B BSK pMMB347 , l I H t + + pMMB524 E 5 + + ' pMMB528 E ’ ' " e sM p -> Figure 2.3 DNA fragments carrying the epsM gene of V. cholerae strain TRH7000. Horizontal lines indicate the insert present in the plasmids listed. Plasmids pMMB524 and pMMB528 contain BAL-31 deletion derivatives of the KpnI fragment. Heavy arrow indicates the location of the epsM gene, dashed lines show the position of the gene in the KpnI fragment. K, KpnI; B, BamHI; S, SacI. Complementation has been determined by assaying secretion of LT B subunit pentamers by GM 1- ELISA. Production of 18.5 and 45 kDa proteins was determined by expressing the inserts of the plasmids under the control of the T7 promoter. 45 kDa 123456 97.4 ~------- 662 m 45.0 "—- 31.0““- 215...... 14.4 ---- Figure 2.4 Expression of eps genes under the control of bacteriophage T7 promoter. DNA inserts from plasmids pMMB347, pMMB524, or pMMB528 (Figure 2) were introduced into either pT7-5 or pT7-6 (Tabor and Richardson, 1985). This resulted in plasmids containing the 10 promoter of T7 at either end of the insert. Molecular size standards (kDa) are indicated. Lanes represent the extracts from M01061[pGP1-2] containing, in addition, the following plasmids: 1, none; 2, pT7-5::insert from pMMB347; 3, pT7- 6::insert from pMMB347; 4, pT7-5::insert from pMMB524; 5, pT7-6zzinsert from pMMB524; 6, pT7-5::insert from pMMB528. The 18.5-kDa protein is EpsM, the 45-kDa protein is likely the putative EpsL protein. 46 Nucleotide sequence of the epsM gene Sequence of the insert of pMMB524 (Figure 2.3) that was complementing mutations in PU3 and PU5 is presented in Figure 2.5. It encodes an ORF of 165 aa that corresponds to a protein with a predicted M r of 18521 Da. This agrees well with the size observed when analyzed by SDS- PAGE. We have proposed the name epsM for this gene since it shows similarity to the pulM gene of K. oxytoca that is required for the secretion of pullulanase (Pugsley and Reyss, 1990). There are two Met residues located downstream from the proposed RBS 5'-AGGAG at nt 193 and 196 in the sequence of the epsM gene. Though it is not known at present which residue is the start codon, it is believed to be Met1 (at nt 196) as it is located five residues away from the putative RBS at nt 186 - 190. There does not appear to be a signal sequence for this protein, as is the case for the EpsM homologues K. oxytoca PulM (Pugsley and Reyss, 1990) and Pseudomonas aeruginosa chZ (Filloux et al., 1990). Purification of the EpsM protein and determination of its N-terminal sequence should reveal where the start codon is and whether this protein possesses a signal peptide. Localization of the protein by fractionation conducted in E. coli and V. cholerae showed that the majority of the EpsM protein was present in the cytoplasmic membrane fraction (results not shown). Both PU3 and PU5 had Tn5 inserted into the epsM gene and the insertion points are indicated in Figure 4. The rescue of mutants PU3 and PU5 by the recombinant plasmids pMMB347 and pMMB524 (Table 2.1) is therefore due to complementation rather than suppression. Sequence determination of DNA flanking the epsM gene on the insert in pMMB524 (Figure 2.3) has identified two partial reading frames showing 47 similarity to the pulL and pulN genes of K. oxytoca (Pugsley and Reyss, 1990). These have therefore been designated epsL and epsN. The translation products of these partial reading frames are shown in Figure 2.5. Comparison of EpsM to proteins required for secretion in other Gram‘ bacteria The deduced aa sequence of the EpsM protein, exhibits identities of 29, 27, and 26% with E. chrysanthemi OutM (Lindeberg and Collmer, 1992), P. aeruginosa chZ (Filloux et al., 1990), and K. oxytoca PulM (Pugsley and Reyss, 1990) respectively. All of these proteins are essential for extracellular secretion. Figure 2.6 shows the alignment of EpsM with these three proteins. Each of the proteins possesses a hydrophobic segment that is a putative transmembrane spanning region near the N-terminus of the protein (boxed in Figure 2.6). Translation of the sequences flanking the epsM gene revealed peptides that showed similarity to other proteins required for secretion. The putative EpsL peptide had 27, 23, and 21% identity when compared to OutL (Lindeberg and Collmer, 1992), chY (Filloux et al., 1990), and PulL (Pugsley and Reyss, 1990). It is likely that the 45 kDa protein shown in Figure 2.4 is the putative EpsL protein as this protein is approximately the same size as the 44 kDa PulL. The putative EpsN peptide exhibited 31% identity to PulN (Pugsley and Reyss, 1990). 48 Figure 2.5 Nucleotide sequence of the insert from pMMB524 (Gene Bank accession number L13660) and the deduced aa sequences. No aa numbers are given for the partial EpsL peptide because the start of this peptide is at present uncertain. The putative RBSs are underlined. Asterisks (*) indicates a stop codon. Tn5 insertion sites in mutants PU3 and PU5 are indicated by arrows (ll). Sequences were analyzed with the GCG sequence analysis software package (Devereux et al., 1984). 49 CTACAACTGCAAAGCATCAAATTTGACAGTAACCGCAGTGAGATTCGCCTAGAAGCGACC L Q L Q S I K F D S N R S E I R L E A T epsL' -> AGTCGTGATTTCCAAAGTTTTGAACAAGCTCGCACTCAGCTTGAGCAGTATTTTGCTGTT S R D F Q S F E Q A R T Q L E Q Y F A V GAACAGGGGCAGCTCAATAAAAATGGCGAGCAAGTGTTTGGCGTGTTTGTGGTGAAGCCC E Q G Q L N K N G E Q V F G V F V V K P AAGTAAQQAQAAATGATGAAAGAATTATTGGCTCCTGTGCAGGCTTGGTGGCGAAGTGTC K * M K E L L A P V Q A W W R S V epsM'-> ACCCCTCGTGAGCAAAAGATGGTAATGGGCATGGGCGCGCTGACGGTACTCGCTATCGCT T P R E Q K M V M G M G A L T V L A I A TATTGGGGAATATGGCAGCCTTTGAGTGAGCGTACCGCCCAAGCTCAAGCACGATTACAA Y W G I W Q P L S E R T A Q A Q A R L Q ACCGAAAAACAGCTACTGAGTTGGGTTAGTGAAAACGCCAACGACATCGTAACGCTCCGT T E K Q L L s w v s E N A N D I v T L R . U Tn5 in PU3 . . GCGCAAGGGGGCAGTGATGCGCCAAGCGATCAACCACTCAATCAGGTGATCACTAACTCG A Q G G s D A P s D Q P L N Q V I T N s 60 120 180 240 15 300 35 360 55 420 75 480 95 . U Tn5 in PU5 ACGCGTCAGTTCAATATTGAGCTGATCCGCGTGCAGCCGCGCGGCGAAATGATGCAGGTC T R Q F N I E L I R V Q P R G E M M Q V TGGATCCAACCGCTACCGTTTTCGCAATTGGTCTCATGGATTGCGTATTTGCAAGAGCGC W I Q P L P F S Q L V S W I A Y L Q E R CAAGGGGTGAGCGTGGATGCGATTGATATTGACCGTGGTAAAGTGAACGGCGTTGTGGAA Q G V S V D A I D I D R G K V N G V V E GTCAAACGTCTGCAACTGAAGCGTGGAQQCTGATATGAAGCGTGCTGTTGGCTATGGTCT V K R L Q L K R G G * M K R A V G Y G L epsN’ -> GTTATTTTCCACAGTGTTAATGACCAGCGTGGTCGTGCATTTGCCTGCCCAAGTGGCGCT L F S T V L M T S V V V H L P A Q V A L TAGCCCGCTACCGCTGCCTGAAGGTTTAGAGCTCACTGGTATAGAGGGTACTCTGTGGCA S P L P L P E G L E L T G I E G T L W Q AGGTCAAGCCGCGCAAGTTCGTTGGCAAGGCATGAGCCTAGGCGATCTCAACTGGGATCT G Q A A Q V R W Q G M S L G D L N W D L CCACCTCTCGGCGTTACTGTTGGGGCAGTTGGAGGCGGATATCCGCTTTGGCCGCGGTAG H L S A L L L G Q L E A D I R F G R G S CAGCACACAACTAAGAGGGAAAGGTGTCGTGGGGGTCGGTTTGAGTGGTCCCTATGCCGA S T Q L R G K G V V G V G L S G P Y A D TGATTTTTTACTCTCCTTACCGGCTGCGCAAGCCATTACTTGGCTACCGCTACCGGTAC D F L L S L P A A Q A I T W L P L P V 540 115 600 135 660 155 720 780 29 840 49 900 69 960 89 1020 109 1079 128 50 EpsM .......... ..MKELLAPV OutM .. .............. MNEL PulM .. ..... ... ......MHNL chZ LRAQAETSQL 68 EpsM OutM PulM chz EpsM 108 OutM PulM chz EpsM 145 OutM Qas. stgTL PulM ogs. RLSLTV ' fi.GQ. A chz EGEGAVQVAL QPAPRAKLLP nLEQthch ........................ ............... ......................... .................................... EpsM OutM PulM chz *.. 166 *0. EK* Figure 2.6 Amino acid alignments of EpsM from V. cholerae, OutM from E. chrysanthemi, PulM from K. oxytoca, and chZ from P. aeruginosa. Conserved residues are shaded and the hydrophobic region is boxed in. Dots represent gaps created to achieve optimal alignment. Asterisks (*) represent stop codons. Alignment performed using the GCG sequence analysis software package (Devereux et al., 1984). 51 Concluding Remarks The function of the epsM gene is required for secretion of LT, protease, and chitinase by V. cholerae. This gene is located approximately 5 kb from epsE, the other gene known to be required for extracellular protein secretion in V. cholerae (Figure 2.7). Two partial ORFs that exhibit similarity to genes required for extracellular secretion of K. oxytoca pullulanase have been identified adjacent to epsM. It seems likely that other secretion genes may also be located in proximity. Protease, chitinase and heat-labile enterotoxin seem to be transported through the outer membrane of V. cholerae by the same general secretion pathway. Acknowledgments This work was supported by grants from US Department of Agriculture (MICLO 6874), NSF Center for Microbial Ecology (BIR 9120006), Biotechnology Research Center at Michigan State University and Research Excellence Fund from the State of Michigan. 52 E SSS K B BSK B B E pMM3339 L l l l l l I l( L/ I eps L MN Figure 2.7 Physical and genetic map of the EcoRI fragment from the V. cholerae TRH7000 chromosome containing the eps genes present in pMMB339. E, EcoRI; S, SacI; K, KpnI; B, BamHI. Completely sequenced genes are indicated by darkened arrows, partially sequenced reading frames are indicated by open arrows. 53 References Casadaban, MC. and Cohen, SN. (1980) Analysis of gene control signals by DNA fusion in Escherichia coli. J Mol Biol 138: 179-207. Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, SA. and Palmer, L.M. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Bia/ Technol 3: 817-820. Dallas, W.S. (1983) Conformity between heat-labile toxin genes from human and porcine enterotoxigenic Escherichia coli. Infect Immun 40: 647-652. Dallas, W.S., Gill, D.M. and Falkow, S. (1979) Cistrons encoding Escherichia coli heat-labile toxin. J Bacteriol 139: 850-858. Devereux, J ., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12: 387-395. Filloux, A., Bally, M., Ball, G., Akrim, M., Tommassen, J. and Lazdunski, A. (1990) Protein secretion in Gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria. EMBOJ 9: 4323-4329. Finkelstein, RA. (1973) Cholera. CRC Crit Rev Microbial 2: 553-623. Finkelstein, R.A., Boesman-Finkelstein, M., Chang, Y. and Hase, CC. (1992) Vibrio cholerae hemagglutinin/protease, colonial variation, virulence, and detachment. Infect Immun 60: 472-478. Fiirste, J.P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M. and Lanka, E. (1986) Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48: 119-131. Ghigo, J .-M. and Wandersman, C. (1992) Cloning, nucleotide sequence and characterization of the gene encoding the Erwinia chrysanthemi B374 PrtA metalloprotease: a third metalloprotease secreted via a C-terminal secretion signal. Mal Gen Genet 236: 135-144. Gill, D.M., Clements, J.D., Robertson, DC. and Finkelstein, RA. (1981) Subunit number and arrangement in Escherichia coli heat-labile enterotoxin. Infect Immun 33: 677-682. Hashimoto, T. and Sekiguchi, M. (1976) Isolation of temperature-sensitive mutants of R plasmid by in vitro mutagenesis with hydroxylamine. J Bacteriol 127: 1561-1563. 54 Hirst, T.R., Hardy, S.J.S. and Randall, LL. (1983) Assembly in viva of enterotoxin from Escherichia coli: formation of the B subunit oligomer. J Bacterial 153:21-26. Hirst, TR. and Holmgren, J. (1987) Transient entry of enterotoxin subunits into the periplasm occurs during their secretion from Vibrio cholerae. J Bacteriol 169: 1037-1045. Hirst, T.R., Sanchez, J ., Kaper, J .B., Hardy, S.J.S. and Holmgren, J. (1984) Mechanism of toxin secretion by Vibrio cholerae investigated in strains harboring plasmids that encode heat-labile enterotoxins of Escherichia coli. Proc Natl Acad Sci USA 81: 7 752-7756. Hofstra, H. and Witholt, B. (1984) Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli. J Biol Chem 259: 15182-15187. Hofstra, H. and Witholt, B. (1985) Heat-labile enterotoxin in Escherichia coli. Kinetics of association of subunits into periplasmic holotoxin. J Biol Chem 260: 16037-16044. Holland, I.B., Blight, M.A. and Kenny, B. (1990) The mechanism of secretion of hemolysin and other polypeptides from Gram-negative bacteria. J Bioenerg Biomemb 22: 473-491. Hsu, SC. and Lockwood, J .L. (1975) Powdered chitin agar as a selective medium for enumeration of Actinomycetes in water and soil. Appl Microbiol 29:422-426. Kaper, J.B., Lockman, H., Baldini, M.M. and Levine, M.M. (1984) Recombinant nontoxinogenic Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature (London) 308: 655-658. Laemmli, UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685. Lindeberg, M. and Collmer, A. (1992) Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi : sequence comparison with secretion genes from other Gram-negative bacteria. J Bacteriol 174: 7385-7397. Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Neill, R.J., Ivins, BE. and Holmes, R.K. (1983) Synthesis and secretion of the plasmid-coded heat-labile enterotoxin of Escherichia coli in Vibrio cholerae. Science 221: 289-291. 55 Palva, E.T., Hirst, T.R., Hardy, S.J.S., Holmgren, J. and Randall, LL. (1981) Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli. J Bacteriol 146: 325-330. Pearson, G.D.N. and Mekalanos, J .J. (1982) Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K12. Proc Natl Acad Sci USA 79: 2976-2980. Pugsley, A.P., d'Enfert, C., Reyss, I. and Kornacker, M.G. (1990) Genetics of extracellular protein secretion by Gram-negative bacteria. Annu Rev Genet 24:67-90. Pugsley, A.P. and Reyss, I. (1990) Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion. Molec Microbiol 4: 365-379. Sandkvist, M., Morales, V. and Bagdasarian, M. (1993a) A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae. Gene 123: 81-86. Sandkvist, M., Overbye, L.J., Sixma, T.K., Hol, W.G.J. and Bagdasarian, M. (1993b) Assembly of Escherichia coli heat-labile enterotoxin and its secretion from Vibrio cholerae. In Molecular Mechanisms of Bacterial Virulence. C. Kado, J. Crossa and L. Sequeira (eds). Dordrecht, The Netherlands: Academic Publishers, pp. 293-309. Sanger, F., Nicklen, S. and Coulson, AR. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467. Sixma, T.K., Prank, S.E., Kalk, K.H., Wartna, E.S., Van Zanten, B.A.M., Witholt, B. and Hal, W.G.J. (1991) Crystal structure of a cholera toxin- related heat-labile enterotoxin from E. coli. Nature (London) 353: 371-377. Svennerholm, A.-M. and Holmgren, J. ( 1978) Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GMl-ELISA) procedure. Curr Microbial 1: 19-23. Tabor, S. and Richardson, CC. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82: 1074-1078. Tamplin, M.L., Gauzens, A.L., Huq, A., Sack, DA. and Colwell, RR. (1990) Attachment of Vibrio cholerae serogroup 01 to zooplankton and phytOplankton of Bangladesh waters. Appl Environ Microbial 56: 1977- 1980. CHAPTER 3 ORGANIZATION OF THE GENERAL SECRETION PATHWAY GENE CLUSTER OF VIBRIO CH OLERAE. 56 57 Abstract The general secretion pathway (GSP) of Vibrio cholerae is required for the secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report on the cloning and sequencing of twelve ORFs, epsC-N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, and Pseudomonas and are therefore believed to be required for protein secretion. Several of the Eps proteins have been identified by use of the T7 polymerase/promoter system. In addition to the two genes, epsE and epsM, which have been described previously (Sandkvist, et al. 1993. Gene. 123:81-86; Chapter 2), it has been shown that epsC also encodes a gene required for protein secretion. Strains with mutations in the epsC and epsM genes exhibit aberrant outer membrane protein profiles, demonstrating that the GSP is not only required for protein secretion but also for proper outer membrane assembly. Results showing that several ORFs of the eps cluster overlap, that the secretion defect in an epsC mutant is not complemented by the epsC gene alone but is complemented by a cosmid containing the entire eps gene cluster and that the insertion of Tn5 into the epsC gene reduces the expression of epsE indicate that the epsC-N genes may be arranged into an operon-like structure. 58 Introduction The general secretion pathway (GSP) is required for the extracellular secretion of several proteins including chitinase, cholera toxin, protease, and cloned E. coli heat-labile enterotoxin (LT) from V. cholerae (Sandkvist et al. 1993; Chapter 2). This pathway likely plays a significant role in the survival and pathogenesis of this bacteria. For instance, secreted chitinase may assist in the attachment of V. cholerae to chitinous waterborne particles (Tamplin et al. 1990) and secreted protease in the detachment of the bacteria from host epithelial cells and dissemination (Finkelstein et al. 1992). Secretion of some proteins such as LT, a multimeric protein which consists of a single A subunit and a pentamer of B subunits (Gill et al. 1981; Sixma et al. 1991; Sixma et al. 1993), from V. cholerae is a two step process. The first step is translocation of the proteins across the cytoplasmic membrane. This is mediated by the general export pathway (GEP), which is presumably similar to the sec system of E. coli (Schatz and Beckwith 1990). During this step, the signal peptides of the A and B precursor polypeptides are cleaved off (Dallas et al. 1979; Palva et al. 1981; Hofstra and Witholt 1984) and the polypeptides are transported to the periplasm. They undergo folding here and assemble to form the holotoxin (Hirst and Holmgren 1987; Findlay et al. 1993). The second step is secretion of the holotoxin through the outer membrane. This requires the function of the GSP and, as shown in this work, occurs with the assistance of more than twelve gene products. This process seems to be highly conserved: GSP genes have been identified in a variety of bacteria including Aeromonas (Jiang and Howard 1992; Howard et al. 1993), Erwinia (He et al. 1991; Condemine et al. 1992; Lindeberg and Collmer 1992; Reeves et al. 1993), Klebsiella (d'Enfert et al. 1987), 59 Pseudomonas (Tommassen et al. 1992), and Xanthomonas (Dums et al. 1991; Hu et al. 1992). In this study, we report on the cloning and sequencing of a cluster of genes which constitute part of the GSP of V. cholerae. These genes seem to be organized into an operon and appear to play a role not only in secretion or proteins through the outer membrane, but also in the assembly of the outer membrane itself. 60 Materials and Methods Bacterial strains and plasmids The bacterial strains and plasmids used in this study are shown in Table 3.1 and Figure 3.3. DNA sequence analyses The insert present in pMSlO as well as subclones of pMMB339 were inserted into M13 mp18 and/or mp19 (Yanish-Perron et al. 1985) and introduced into E. coli DH5aF' (Gibco-BRL). Part of the DNA sequence analysis of these inserts was performed by the dideoxy chain termination procedure (Sanger et al. 1977) and a Sequenase 2,7-deaza-GTP kit (U.S. Biochemical Corp.). Part was also determined by automated fluorescent sequencing performed by the MSU-DOE-PRL Plant Biochemistry Facility using the ABI Catalyst 800 for Taq cycle sequencing and the ABI 373A Sequencer for the analysis of products. Primers utilized in the sequencing included the M13 forward and reverse primers and oligonucleotides synthesized by the MSU Macromolecular Structure Facility. The insertion point of the transposon in the mutant PU6 was determined by sequencing a PCR amplified fragments containing the IS sequence of Tn5 and the adjacent chromosomal DNA using the method of Thien (1989). DNA and deduced amino acid sequences were analyzed with the GCG sequence analysis software package (Devereux et al. 1984). The sequence presented in this report appears in the GenBank database under the accession number L33796. PCR amplification of mutant DNA containing Tn5 In order to determine the location of Tn5 in mutants PU4 and PU6, Table 3.1 Strains and plasmids used in this study. 61 Strains/Plasmids Relevant characteristics Reference or source Wm TRH7000 PU3 PU6 E l' DH5aF' MC 1061 E26 pMMB67EH pMMB68 pMSlO pWD615 pGP1-2 pT7-5/6 thy Hg-r A(ctxA-cth) TRH7000 epsM::Tn5 TRH7000 epsszTn5 F' ¢80dlacZAM15 A(lacZYA- argF) U169 endAI recAI hst17 (rK'mK+) deoR thi-I supE44 A' gyrA96 relAI F' araD139 A(ara-leu)7697 A(lac) X74 rpsL hst2 mcrA mchI pLAFR5::epsC-N ApR, Ptac, mob+ ApR, eth ApR, epsC-D’ TcR, eth KnR, oripP15A, c1857, pL-T7 gene 5 ApR, T7 ¢10 promoter ori pMBl Hirst et al. 1984 Chapter 2 Chapter 2 Gibco-BRL (Life Technologies) Casadaban and Cohen 1980 Keen et al. 1988 This study Fiirste et al. 1986 Sandkvist et al. 1987 This study Dallas 1983 Tabor and Richardson 1985 Tabor and Richardson 1985 62 PCR was performed using converging primers. Primer MMB41 (5'- CGCACGATGAAGAGCAGA-S') was located in the IS of Tn5 and primer MMB73 (5'-TGC'I"I‘GGC'I"I‘CCATCTG-3') was located in the epsD gene. PCR reactions (100ul final volume) contained 10mM Tris-HCl, pH 8.3 (at 25°C), 50mM KCl, 4mM MgClz, 0.001% (w/v) gelatin (Sigma), 0.5 uM of each oligonucleotide primer, 200uM of each dNTP, and 1 ug genomic DNA. The reaction mixture was overlayed with 100 ul mineral oil (Sigma) and then heated to 95°C for five minutes at which point 2.5 U AmpliTaq DNA polymerase (Perkin Elmer Cetus) was added. The DNA was then amplified in a Perkin Elmer Cetus thermal cycler for 30 cycles consisting of the following steps: 1) 30 sec. at 94°C; 2) 30 sec. at 64°C and; 3) 30 sec. at 72°C with the last cycle having the 72°C incubation extended to seven min. Expression of genes under phage T7 ¢10 promoter control pT7-5/6 (Tabor and Richardson 1985) and derivatives of these plasmids constructed during this study (Figure 3.3) were introduced into E. coli MC1061[pGP1-2]. The expression of proteins encoded by the recombinant plasmids was performed as described in Chapter 2. Isolation of outer membrane fractions from Vibrio cholerae V. cholerae TRH7000 and eps mutant strains were grown overnight in LB with or without 0.4% maltose at 37°C. 1.0 ml of cells were pelleted and resuspended in 250 pl 10 mM phosphate buffer pH 7.3 /150 mM NaCl (PBS) containing 4000 U/ml polymixin B sulfate (Sigma) and incubated on ice for 25 minutes to release periplasmic proteins. The cells were then centrifuged at 12,000 rpm in an Eppendorf microcentrifuge at 4°C for five min., resuspended in 250 pl PBS, placed on ice and broken by sonication with two fifteen sec. 63 pulses. After a low-speed centrifugation (5,000 rpm for two minutes) to remove any whole cells, the lysate was centrifuged at 12,000 rpm at 4°C for thirty mintues to pellet the membrane fraction. This pellet was resuspended in 250 ul PBS containing 1.0% Triton X-100 and incubated at room temperature for 30 minutes. The lysate was centrifuged at room temperature for 30 minutes at 12,000 rpm in an Eppendorf microcentrifuge, and the pellet resuspended in 601.1] of sample buffer. The outer membrane proteins were separated on a 0.1% SDS-15.0% polyacrylamide gel. Immunablotting techniques Western analysis was performed basically as described by Towbin et al. (1979). The proteins from a culture grown in LB medium were mixed with sample buffer and heated at 95°C for five mintues. They were then separated on a 0.1% SDS- 13.5% polyacrylamide gel. The samples were transferred to a nitrocellulose membrane and soaked in 3% bovine serum albumin (BSA) in PBS for one hour. After a one hour incubation with a 1:1000 dilution of EpsE antiserum in 2% skim milk the blot was washed in 3 x 10 minutes PBS- containing 0.05% Tween 20 and incubated with a 1:3000 dilution of anti-IgG coupled with alkaline phosphatase. The wash step was repeated and the blot was developed with BCIP/NBT color development substrate (Promega). Quantitative determination of LT B subunit pentamer secretion eth, the gene which encodes the LT B subunit was introduced into V. cholerae on plasmids pMMB68 (Sandkvist et al. 1987) or pWD615 (Dallas 1983). LT B subunit pentamers present in the growth medium and sonicated cells were determined by GM1 - ELISA (Svennerholm and Holmgren 1978) as described in Chapter 2. 64 Results and Discussion Isolation and analysis of the epsC - N gene cluster We have isolated and sequenced a fragment from a V. cholerae TRH7000 gene library that encoded twelve ORFs, epsC-N which are contiguous and have the same orientation (Figure 3.1; Figure 3.3). The location of these ORFs and the characterisitics of their deduced polypeptide sequences are presented in Table 3.2. Two of the genes, epsE and epsM, have been described previously (Sandkvist et al. 1993; Chapter 2) and are required for the secretion of proteins through the outer membrane of V. cholerae. The epsC gene is preceded by the sequence GTGGAA, a possible ribosome binding site (RBS). No obvious promoter sequence, however, was detected upstream of epsC. This could indicate that there is an additional ORF in the gene cluster even though no such ORF was identified. Further DNA sequencing will have to be performed on the region upstream of epsC to identify any additional genes. It seems likely that additional genes required for protein secretion since expression of the epsC-N genes in E. coli [pMMB68] does not lead to the secretion of LT B subunit pentamers (results not shown). There is an inverted repeat (WOW which follows the epsN gene and could be a transcriptional terminator. DNA sequence downstream of epsN did not reveal any ORFs which showed homology to GSP genes but did identify a partial ORF in the opposite orientation of epsN that showed similarity to the cysQ gene of E. coli (Neuwald et al. 1992). 65 Figure 3.1 Nucleotide sequence of the eps genes. The sequence of the mRNA-like strand is given along with its translation. Potential RBS are underlined. Hydrophobic regions of Eps polypeptides are doubly underlined. The insertion points of Tn5 in the epsC mutant PU6 and the epsM mutant PU3 are indicated by 11. A potential signal peptidase cleavage site following the hydrophobic sequence at the N terminus of EpsD is indicated by A. The inverted repeat of a sequence predicted to be a rho independent terminator is underlined with arrows. The sequence is entered in GenBank database under accession number L337 96. 61 121 181 241 301 361 421 481 541 601 661 721 781 841 941 961 1021 1081 1141 66 AAGCTTATTACATTTGACTATTATTCATGCTTAACAATGGTGTTTGCAGGGCTGTTTTTC AAGCAAGTCGACAATTTAACGTTTGAGACACTTCGCTCCACTATGCTTGTGTTATATTGC GATGCCTGCATAAAGTATTGAAAAACATCTGTACAAACAGATTTGCGCAATATGAAGCGT AAAAAGTACAGAAAGGAATAACQIQGAAATTTATGGAATTTAAACAACTTCCTCCGTTGG M E F K Q L P P L A epsC —) CAGCGTGGCCACGTTTATTGAGCCAGAATACGCTGCGGTGGCAAAAGCCGATCAGCGAAG A W P R L L S Q N T L R W Q K P I S E .9 GATTAACGCTACTGTTATTAGTTGCTTCGGCGTGGACGCTGGCCAAGATGGTGTGGGTCG L T L L L L V A S A W T L A K M V W V V TCTCGGCTGAACAAACACCAGTGCCGACTTGGAGCCCCACGTTATCAGGGCTTAAGGCCG S A E Q T P V P T W S P T L S G L K A E AACGTCAGCCACTCGATATCAGCGTATTGCAAAAAGGTGAGCTGTTTGGTGTGTTTACTG R Q P L D I S V L Q K G E L F G V F T E AGCCGAAAGAAGCTCCGGTTGTGGAGCAACCTGTGGTGGTGGATGCTCCGAAAACGCGCT P K E A P V V E Q P V V V D A P K T R L TGAGCCTTGTGCTGTCTGGTGTGGTGGCGAGTAATGATGCGCAAAAAAGTTTGGCTGTTA S L V L S G V V A S N D A Q K S L A V I TCGCCAATCGTGGTGTGCAAGCGACGTATGGCATTAATGAAGTGATTGAAGGGACTCAGG A N R G V Q A T Y G I N E V I E G T Q A CCAAGCTAAAAGCCGTGATGCCGGATCGGGTCATCATCAGCAACTCCGGGCGTGATGAAA K L K A V M P D R V I I S N S G R D E T UTn5 in PU6 CCTTGATGCTTGAAGGGTTAGACTACACCGCGCCTGCGACGGCTTCGGTATCCAACCCTC L M L E G L D Y T A P A T A S V S N P P CGCGTCCACGACCCAATCAACCTAATGCTGTGCCGCAGTTTGAGGATAAAGTGGATGCAA R P R P N Q P N A V P Q F E D K V D A I TTCGTGAAGCAATCGCACGAAATCCGCAGGAAATTTTTCAATATGTGCGCCTATCTCAGG R E A I A R N P Q E I F Q Y V R L S Q V TAAAACGCGATGACAAAGTACTCGGCTATCGCGTCAGTCCCGGTAAAGATCCGGTACTGT K R D D K V L G Y R V S P G K D P V L F TTGAATCAATAGGTTTACAAGATGGCGATATGGCAGTGGCACTGAATGGCCTAGATCTGA E S I G L Q D G D M A V A L N G L D L T CCGATCCTAATGTAATGAACACGCTATTTCAGTCGATGAATGAGATGACTGAAATGAGTC D P N V M N T L F Q S M N E M T E M S L TGACCGTTGAGCGTGATGGTCAACAACATGATGTATATATTCAATTTTAACGCTAAGGCG T V E R D G Q Q H D V Y I Q F * AGTAACGACGCCTTAGTGAGGCAAQQQAGTTCCCAGTGAAATATTGGCTGAAAAAAAGTT V K Y W L K K S S epsD —) 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 67 CATGGCTGTTGGCGGGCAGTTTGCTGTCTACCCCTCTAGCGATGGCAAACGAGTTTAGCG W L L A G S L L S T P L A M A N E F S S A CCAGCTTTAAAGGCACCGACATTCAAGAATTTATCAATATTGTGGGACGCAATCTTGAGA S F K G T D I Q E F I N I V G R N L E K AAACCATCATTGTCGACCCTTCGGTACGCGGCAAAGTCGATGTGCGCAGTTTTGATACCT T I I V D P S V R G K V D V R S F D T L TAAACGAAGAGCAATATTACAGTTTCTTCCTCAGTGTGCTCGAAGTGTACGGTTTCGCGG N E E Q Y Y S F F L S V L E V Y G F A A CCGTTGAAATGGATAATGGCGTACTGAAAGTCATCAAATCGAAAGATGCAAAGACCTCAG V E M D N G V L K V I K S K D A K T S A CAATTCCGGTGTTGAGTGGTGAAGAGCGCGCCAATGGCGATGAAGTCATTACCCAAGTGG I P V L S G E E R A N G D E V I T Q V V TCGCGGTGAAAAACGTTTCCGTACGCGAGTTGTCCCCTTTGCTGCCCCAACTGATTGATA A V K N V S V R E L S P L L P Q L I D N ACGCAGGGGCGGGGAACGTAGTGCACTACGATCCGGCGAACATCATTTTGATCACGGGGC A G A G N V V H Y D P A N I I L I T G R GAGCTGCGGTAGTGAACCGTTTAGCGGAAATTATTCGTCGCGTTGACCAAGCCGGTGACA A A V V N R L A E I I R R V D Q A G D K AAGAGATTGAAGTGGTTGAGCTTAATAATGCATCGGCAGCTGAAATGGTGCGGATTGTTG E I E V V E L N N A S A A E M V R I V E AAGCGCTCAACAAAACGACAGACGCGCAAAACACCCCTGAATTCTTAAAGCCCAAGTTTG A L N K T T D A Q N T P E F L K P K F V TGGCAGACGAGCGTACCAACTCGATTTTGATTTCTGGCGATCCTAAAGTGCGCGAGCGCC A D E R T N S I L I S G D P K V R E R L TCAAGCGTCTGATCAAGCAGTTGGATGTTGAGATGGCGGCCAAAGGCAATAACCGCGTGG K R L I K Q L D V E M A A K G N N R V V TGTATTTGAAATACGCCAAAGCTGAAGATCTGGTCGAAGTACTGAAAGGGGTGTCTGAGA Y L K Y A K A E D L V E V L K G V S E N ACCTGCAAGCGGAAAAAGGCACCGGACAGCCGACCACTTCAAAACGTAATGAAGTGATGA L Q A E K G T G Q P T T S K R N E V M I TTGCCGCGCACGCTGACACCAACTCGTTAGTGCTTACTGCGCCGCAAGACATTATGAATG A A H A D T N S L V L T A P Q D I M N A CGATGCTGGAAGTGATTGGACAGCTAGATATTCGCCGTGCGCAAGTGTTGATTGAAGCGC M L E V I G Q L D I R R A Q V L I E A L TGATTGTCGAAATGGCAGAGGGCGATGGGATCAACCTTGGTGTGCAGTGGGGCTCGCTGG I V E M A E G D G I N L G V Q W G S L E AAAGTGGTTCAGTTATCCAATATGGCAACACTGGCGCGTCGATTGGCAATGTGATGATTG S G S V I Q Y G N T G A S I G N V M I G 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 68 GTCTTGAAGAAGCCAAAGACACAACCCAAACCAAAGCGGTTTATGATACTAATAACAACT L E E A K D T T Q T K A V Y D T N N N F TCTTGAGAAATGAAACGACGACCACCAAAGGGGATTACACCAAGTTAGCCTCTGCATTGT L R N E T T T T K G D Y T K L A S A L S CGAGCATTCAAGGTGCTGCAGTCAGCATCGCGATGGGCGACTGGACGGCCTTAATCAACG S I Q G A A V S I A M G D W T A L I N A CGGTCTCTAATGATTCCAGCTCGAATATCCTGTCATCACCCAGCATTACGGTGATGGATA V S N D S S S N I L S S P S I T V M D N ACGGTGAAGCCTCCTTTATCGTGGGTGAAGAAGTGCCGGTTATCACAGGTTCTACTGCAG G E A S F I V G E E V P V I T G S T A G GCTCTAATAACGACAACCCATTCCAAACCGTGGATCGTAAAGAAGTCGGTATCAAGCTTA S N N D N P F Q T V D R K E V G I K L K AAGTGGTGCCGCAGATCAACGAAGGTAACTCAGTCCAGCTCAATATTGAGCAAGAAGTCT V V P Q I N E G N S V Q L N I E Q E V S CGAACGTGTTGGGCGCCAATGGAGCGGTAGACGTGCGTTTTGCGAAGCGTCAGCTCAACA N V L G A N G A V D V R F A K R Q L N T CTTCTGTGATGGTGCAAGATGGGCAGATGTTGGTGCTCGGCGGTTTGATTGATGAGCGAG S V M V Q D G Q M L V L G G L I D E R A CTCTTGAGAGTGAGTCGAAAGTCCCGCTCTTGGGGGATATTCCTCTGCTGGGTCAACTGT L E S E S K V P L L G D I P L L G Q L F TCCGCTCAACCAGCTCGCAAGTGGAAAAGAAAAACCTGATGGTGTTTATCAAGCCGACCA R S T S S Q V E K K N L M V F I K P T I TTATTCGTGATGGCGTGACGGCCGATGGCATCACCCAACGTAAATACAACTACATCCGCG I R D G V T A D G I T Q R K Y N Y I R A CCGAGCAACTGTTCCGCGCCGAAAAAGGTTTACGTCTGCTGGATGATGCTAGCGTGCCTG E Q L F R A E K G L R L L D D A S V P V TGTTGCCGAAATTTGGCGATGACCGCCGCCATTCACCTGAAATTCAAGCCTTTATTGAGC L P K F G D D R R H S P E I Q A F I E Q AGATGQAAQCCAAGCAATGACCGAAATGGTGATCTCTCCAGCTGAGCGACAGTCGATTCG M E A K Q * M T E M V I S P A E R Q S I R epsE —) TCGTCTGCCCTTTAGCTTCGCCAATCGCTTTAAGTTGGTGCTGGATTGGAATGAGGATTT R L P F S F A N R F K L V L D W N E D F CTCCCAAGCCAGCATTTATTACTTAGCCCCGTTGTCGATGGAGGCGCTCGTCGAAACCAA S Q A S I Y Y L A P L S M E A L V E T K GCGGGTGGTCAAGCACGCTTTCCAACTGATTGAGCTCTCTCAAGCGGAGTTTGAAAGCAA R V V K H A F Q L I E L S Q A E F E S K GCTAACCCAAGTCTATCAGCGTGATTCTTCAGAAGCGCGTCAATTGATGGAAGACATTGG L T Q V Y Q R D S S E A R Q L M E D I G 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 69 TGCCGACAGTGATGACTTCTTCTCACTGGCGGAAGAGCTGCCTCAAAACGAAGATTTGCT A D S D D F F S L A E E L P Q N E D L L AGAAAGTGAAGATGATGCGCCGATCATCAAACTGATCAACGCCATGTTGGGCGAAGCGAT E S E D D A P I I K L I N A M L G E A I CAAAGAAGGCGCGTCAGATATTCACATTGAAACTTTTGAAAAAACACTGTCGATCCGTTT K E G A S D I H I E T F E K T L S I R F TCGAGTCGATGGCGTGCTGCGCGAAGTACTGGCACCCAGCCGCAAACTCTCATCGCTGCT R V D G V L R E V L A P S R K L S S L L CGTTTCACGGGTCAAAGTGATGGCCAAGCTCGACATTGCCGAAAAACGTGTGCCGCAAGA V S R V K V M A K L D I A E K R V P Q D TGGCCGTATTTCGCTGCGTATCGGTGGCCGCGCGGTCGATGTGCGGGTTTCAACCATGCC G R I S L R I G G R A V D V R V S T M P TTCATCACATGGTGAGCGAGTGGTAATGCGTTTGCTGGATAAAAACGCGACGCGCCTTGA S S H G E R V V M R L L D K N A T R L D TCTGCACAGTTTGGGTATGACGGCGCATAACCATGATAATTTCCGCCGTTTGATTAAGCG L H S L G M T A H N H D N F R R L I K R GCCGCATGGGATCATTCTGGTGACTGGCCCAACCGGCTCGGGTAAATCGACCACCTTGTA P H G I I L V T G P T G S G K S T T L Y CGCGGGTTTGCAAGAGCTCAACAGCAGCGAGCGCAACATTTTAACCGTAGAAGATCCGAT A G L Q E L N S S E R N I L T V E D P I CGAATTTGATATTGATGGTATCGGTCAGACCCAAGTCAACCCAAGGGTAGACATGACCTT E F D I D G I G Q T Q V N P R V D M T F CGCACGGGGGCTACGCGCTATTTTGCGTCAAGACCCCGATGTGGTGATGGTGGGGGAAAT A R G L R A I L R Q D P D V V M V G E I TCGTGATTTGGAAACGGCGCAAATTGCAGTGCAAGCCTCGCTGACTGGTCACTTAGTGAT R D L E T A Q I A V Q A S L T G H L V M GTCGACTCTGCACACCAACACGGCCGTTGGGGCGGTGACTCGGCTGCGCGATATGGGCAT S T L H T N T A V G A V T R L R D M G I CGAGCCTTTTTTGATCTCCTCTTCACTGCTCGGTGTTCTGGCTCAGCGCTTGGTGCGCAC E P F L I S S S L L G V L A Q R L V R T CTTATGCCCAGATTGCAAAGAGCCTTACGAGGCGGACAAAGAGCAGCGCAAACTGTTTGA L C P D C K E P Y E A D K E Q R K L F D TAGCAAGAAAAAAGAACCGCTGATCCTTTATCGTGCAACGGGCTGCCCTAAATGTAACCA S K K K E P L I L Y R A T G C P K C N H CAAAGGTTACCGTGGCCGAACCGGTATCCACGAGCTGCTGCTGGTGGATGACGCGTTGCA K G Y R G R T G I H E L L L V D D A L Q GGAGCTGATCCATAGCGAGGCGGGCGAACAGGCGATGGAGAAACACATTCGCGCGACCAC E L I H S E A G E Q A M E K H I R A T T 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 70 GCCGAGCATTCGTGATGATGGTCTAGATAAGGTGCGCCAAGGCATTACTTCGCTAGAAGA P S I R D D G L D K V R Q G I T S L E E AGTGATGCGGGTGACTAAGQAGTCCTAATGGCCGCGTTTGAATACAAAGCGCTGGATGCC M A A F E Y K A L D A V M R V T K E S * epsF —9 AAGGGACGCCATAAAAAAGGCGTGATTGAGGGCGATAATGCACGTCAGGTGCGTCAGCGC K G R H K K G V I E G D N A R Q V R Q R CTGAAAGAGCAAAGCCTAGTGCCGATGGAGGTGGTGGAGACTCAAGTCAAAGCCGCGCGC L K E Q S L V P M E V V E T Q V K A A R AGTCGCAGCCAAGGTTTTGCGTTTAAGCGTGGGATCAGTACGCCGGATCTGGCGCTGATC S R S Q G F A F K R G I S T P D L A L I ACTCGCCAATTGGCGACTTTAGTTCAATCCGGTATGCCACTGGAAGAGTGTTTACGCGCG T R Q L A T L V Q S G M P L E E C L R A GTTGCCGAGCAGTCGGAAAAACCGCGGATTCGCACCATGTTGGTGGCGGTGCGCGCTAAA V A E Q S E K P R I R T M L V A V R A K GTGACCGAAGGTTACACCCTCTCTGATAGTCTTGGCGATTATCCGCACGTGTTTGATGAG V T E G Y T L S D S L G D Y P H V F D E CTGTTTCGTTCTATGGTTGCGGCGGGCGAAAAATCTGGCCACCTCGATTCCGTTCTCGAA L F R S M V A A G E K S G H L D S V L E CGTTTAGCTGACTACGCCGAAAACCGCCAGAAAATGCGCTCTAAACTGCAACAGGCCATG R L A D Y A E N R Q K M R S K L Q Q A M ATTTACCCTGTGGTGCTGGTGGTGTTTGCGGTCGGTATCGTGGCGTTTTTGTTGGCAGCG I Y P V V L V V F A V G I V A F L L A A GTAGTGCCGAAAATCGTGGGTCAGTTTGTGCAAATGGGGCAAGCCTTGCCGGCATCGACT V V P K I V G Q F V Q M G Q A L P A S T CAGTTTCTGCTTGATGCCAGCGATTTCCTGCAACATTGGGGGATTTCGCTGCTGGTCGGT Q F L L D A S D F L Q H W G I S L L V G CTCTTGATGCTGATTTATCTGGTGCGCTGGTTGCTGACCAAGCCCGATATTCGTTTGCGT L L M L I Y L V R W L L T K P D I R L R TGGGATCGCCGAGTGATTTCCTTGCCTGTGATTGGCAAGATTGCACGCGGTCTGAATACT W D R R V I S L P V I G K I A R G L N T GCGCGCTTTGCGCGTACGCTGTCGATCTGTACCTCAAGTGCAATCCCGATTCTGGATGGT A R F A R T L S I C T S S A I P I L D G ATGCGCGTTGCGGTGGATGTGATGACCAATCAGTTTGTGAAACAGCAAGTGTTGGCTGCG M R V A V D V M T N Q F V K Q Q V L A A GCCGAAAACGTACGCGAAGGCTCTAGCCTGCGCAAAGCGCTAGAGCAGACCAAGCTCTTT A E N V R E G S S L R K A L E Q T K L F CCTCCCATGATGCTGCACATGATTGCCAGTGGTGAGCAGAGTGGAGAATTGGAAGGCATG P P M M L H M I A S G E Q S G E L E G M 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5761 5821 5881 5941 6001 6061 6121 6181 6241 6301 6361 6421 6481 6541 6601 6661 6721 6781 6841 71 TTGACGCGCGCTGCGGATAACCAAGACAACAGTTTTGAATCAACGGTCAACATCGCGCTT L T R A A D N Q D N S F E S T V N I A L GGCATTTTTACCCCGGCCTTGATTGCCTTGATGGCGGGGATGGTGCTGTTTATTGTGATG G I F T P A L I A L M A G M V L F I V M GCGACCCTGATGCCGATTTTGGAAATGAATAACTTAATGAGTCGTTAAGTCGTCACGCGC A T L M P I L E M N N L M S R * GGCGAAAACGACATAGTGTGGAGTAACTATGAAAAAAATGCGTAAACAAACGGGCTTTAC M K K M R K Q T G F T epsG —) CCTGCTCGAAGTAATGGTGGTTGTGGTGATTTTGGGCATTCTGGCCAGCTTTGTTGTCCC L L E V M V V V V I L G I L A S F V V P CAACCTCTTAGGTAACAAAGAGAAAGCGGATCAACAGAAAGCGGTGACCGATATCGTCGC N L L G N K E K A D Q Q K A V T D I V A GCTGGAAAATGCGTTGGATATGTACAAGCTTGACAACAGCGTTTACCCGACGACTGATCA L E N A L D M Y K L D N S V Y P T T D Q AGGTTTGGAAGCGTTAGTGACTAAGCCAACCAATCCAGAGCCGCGTAACTATCGCGAAGG G L E A L V T K P T N P E P R N Y R E G CGGTTACATCAAGCGTCTGCCTAAAGATCCTTGGGGTAACGACTACCAATACTTGAGCCC G Y I K R L P K D P W G N D Y Q Y L S P AGGCGATAAAGGCACGATTGATGTGTTCACCTTAGGTGCGGACGGTCAAGAAGGTGGTGA G D K G T I D V F T L G A D G Q E G G E AGGTACCGGTGCCGATATCGGTAACTGGAATATCCAAGATTTTCAATAAGCTTGGCTAAT G T G A D I G N W N I Q D F Q * TAGCGGTAACTGACGQAACCTTATGACAGCGACACGCGGTTTTACTTTGTTGGAAATTTT M T A T R G F T L L E I L epsH —) GCTGGTGCTGGTGCTGGTTTCGGCCAGTGCGGTGGCGGTCATCGCCACCTTTCCGGTTTC L V L V L V S A S A V A V I A T F P V S CGTCAAAGATGAAGCCAAAATCAGTGCGCAGAGTTTTTATCAGCGTCTGTTGCTGCTCAA V K D E A K I S A Q S F Y Q R L L L L N TGAGGAAGCGATTCTCAGCGGGCAAGATTTTGGCGTGCGGATCGATGTCGATACGCGCCG E E A I L S G Q D F G V R I D V D T R R TCTCACTTTTTTGCAACTCACCGCCGACAAAGGTTGGCAAAAGTGGCAAAACGACAAGAT L T F L Q L T A D K G W Q K W Q N D K M GACCAACCAAACCACCCTTAAAGAAGGGTTACAGCTCGACTTTGAACTCGGTGGTGGCGC T N Q T T L K E G L Q L D F E L G G G A TTGGCAAAAAGACGATCGTCTGTTTAATCCCGGCTCTCTGTTTGATGAAGAGATGTTTGC W Q K D D R L F N P G S L F D E E M F A CGATGAGAAAAAAGAGCAAAAACAGGAACCGGCTCCGCAACTGTTTGTGCTATCGAGTGG D E K K E Q K Q E P A P Q L F V L S S G 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6901 6961 7021 7081 7141 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861 7921 72 CGAAGTGACCCCATTTACGCTGAGCATTTTCCCTAAAGGGCAGGAGCCCGATGAGCAGTG 6960 E V T P F T L S I F P K G Q E P D E Q W GCGAGTGACCGCGCAAGAAAATGGCACTCTGCGTCTACTGGCTCCQGQAQAAAGTGATGA 7020 R V T A Q E N G T L R L L A P G E S D E M K epsI —9 AGAGTAAACGCGGTTTTACTTTGCTTGAAGTGCTGGTCGCGCTGGCAATTTTTGCTACCG '7080 E * S K R G F T L L E V L V A L A I F A T A CGGCGATCAGTGTGATCCGCTCGGTCAGTCAACACATCAATACGGTCAATTATCTTGAAG '7140 A I S V I R S V S Q H I N T V N Y L E E AGAAGATGTTTGCGGCCATGGTCGTGGATAACCAAATGGCGCAAGTGATGCTCAATCCGC 7200 K M F A A M V V D N Q M A Q V M L N P Q AATCTTTAGCGGCGCGTGAGGGCAGTGAGCAGATGGCCGGACGGACTTGGTACTGGAAGC 7260 S L A A R E G S E Q M A G R T W Y W K L TGAGTCCTGTCAAAACCGCCGACAATCTGCTCAAAGCCTTTGATGTCAGTGTCGCCACAG '7320 S P V K T A D N L L K A F D V S V A T E AAAAAGGCGCGACCCCAGTCGTGACGGIQCGTAGCTATGTGGCGAACTAACCAAGTATCT 7380 K G A T P V V T V R S Y V A N * M W R T N Q V S esz -) TCTCGCCAGAATATGGCGGGCTTTACTTTGATTGAAGTGTTGGTGGCGATTGCGATTTTC 7440 S R Q N M A G F T L I E V L V A I A I F GCGAGCTTGAGTGTGGGCGCCTATCAGGTGCTCAATCAAGTCCAACGCAGCAATGAAATT '7500 A S L S V G A Y Q V L N Q V Q R S N E I TCTGCCGAGCGCACCGCGCGTTTGGCTGAATTGCAACGCGCCATGGTGATCATGGATGCC 7560 S A E R T A R L A E L Q R A M V I M D A GATTTTCGGCAGATGGCCCTGCGCCAATTTCGCACCGATGGCGAAGCGCCGAGTGAGCAA '7620 D F R Q M A L R Q F R T D G E A P S E Q ATCCTACAATGGAAAGAATCGCTGCTCGATTCGGATCAGCACGGTTTGTTGTTTGTACGC 7680 I L Q W K E S L L D S D Q H G L L F V R TTGGGTTGGCATAACCCACAGCAACAATTTCCACGCGGTGAAGTGGCGAAAGTCGGTTAC 7740 L G W H N P Q Q Q F P R G E V A K V G Y CGCCTGTTTGAAAACCGCTTAGAGCGGGTCTGGTGGCGCTACCCAGATACTCCAGCGGGG '7800 R L F E N R L E R V W W R Y P D T P A G CAGCAAGGGCTGATCTCTCCGTTGTTAACTGGGGTGGAAGATTGGGCAGTACAGTTTTAT 7860 Q Q G L I S P L L T G V E D W A V Q F Y TTGCAAGGTGAATGGAGTAAGGAGTGGGTGCCCACTAACGCCTTGCCTGAAGCCGTTGAA 7920 L Q G E W S K E W V P T N A L P E A V E AGTGACTTGCGCCTAAAAGATTACGGTGAGATTGAGCGGATATACCTTACAGGGGGCGGT 7980 S D L R L K D Y G E I E R I Y L T G G G 7981 8041 8101 8161 8221 8281 8341 8401 8461 8521 8581 8641 8701 8761 8821 8881 8941 9001 73 TCACTCAATATGACGCAAQAQAQTGTTGAAAATGCGGGCTAAACAGCGCGGCGTGGCGTT S L N M T Q E S V E N A G * M R A K Q R G V A L epsK —) AATTGTGATTCTGCTCCTGCTGGCGGTGATGGTCTCGATTGCTGCGACCATGGCCGAACG I V I L L L L A V M V S I A A T M A E R TCTGTTTAGTCAGTTTCAGCGTGCCACGCATCAGCTCAACTATCAGCAGGCTTATTGGTA L F S Q F Q R A T H Q L N Y Q Q A Y W Y CAGTCTTGGCGTCGAAGCGCTAGCGAAAAAAGGCATTGAGCAGAGCTACCAAGACAGTGA S L G V E A L A K K G I E Q S Y Q D S E AACCATCAATTTGAGTCAGCCTTGGGCTTTGAAAGAGCAAACTTATCCGCTCGATTACGG T I N L S Q P W A L K E Q T Y P L D Y G ACAGGTGCGCGGCAAAATCCGCGATATGCAAGCTTGCTTTAATCTCAATGCACTCGCGGG Q V R G K I R D M Q A C F N L N A L A G GGTAAAGCTCACGCCAGACAGCGTGAAAAAACCGTATTTGCTGACGGTGCTGCAAGCGCT V K L T P D S V K K P Y L L T V L Q A L GCTTGAAGGGCTGGAAGTGGAGAGTTATCAAGCGGAAGTGATTGCCGATTCGACGTTAGA L E G L E V E S Y Q A E V I A D S T L E GTTTATTGATAAAGATGACTCTGTACGCACCGCTTATGGGGTGGAAGACAGTTACTATGA F I D K D D S V R T A Y G V E D S Y Y E ATCGATGATCCCGGCCTATATGGCGGGGGATACTTGGTTGGCCGATGCGAGCGAGTGGCG S M I P A Y M A G D T W L A D A S E W R TGCGGTACAGCAAGTGGGGGGAGAAACGATGAATAAAGCCTTACCTTATGTGTGTGCGTT A V Q Q V G G E T M N K A L P Y V C A L GCCAACCGATCAATGGCGCTTGAATGTGAACACGTTACCCGCTGAGCAAGCGGCACTGCT P T D Q W R L N V N T L P A E Q A A L L GGCGGCCATGTTTAGCCCAACATTGAGTCCGGAAAGCGCGAAAACCTTGCTCGAAGGGCG A A M F S P T L S P E S A K T L L E G R ACCTTTCGATGGTTGGGCGAGTGTGGATGATTTTCTTGCCCAATCGGCACTCACCGGAGT P F D G W A S V D D F L A Q S A L T G V GGATAACGCGGTGCGTGAGGAAGCGAAGAAATACCTCAGTGTAGATAGCCATTATTTTGA D N A V R E E A K K Y L S V D S H Y F E ATTAGATGCACAGGTGCTGGTCGATACGTCTCGCGTGCGGATCCGCAGCCTGTTTTACAG L D A Q V L V D T S R V R I R S L F Y S TAACGATAAGAAAACTGCGACGGTGATACGCCGCCGCTTTQQAQGGATCAGTGAGCGAGT N D K K T A T V I R R R F G G I S E R V V S E F epsL -9 TTCTGACCGTTCGACTGAGTAGTCAAAAAGAGGCCGATATCCCTTGGCTGGTTTGGTCTG S D R S T E * L T V R L S S Q K E A D I P W L V W S A 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9061 9121 9181 9241 9301 9361 9421 9481 9541 9601 9661 9721 9781 9841 9901 9961 10021 10081 10141 74 CCGAGCAGCAAGAAGTGATTGCCAGCGGCCAAGTGGCTGGTTGGGAAGCCTTGCATGAAA E Q Q E V I A S G Q V A G W E A L H E I TTGAGTCTTATGCTGATCAGCGCAGCGTAGTGGTATTACTGGCGGCGAGTGATTTGATTT E S Y A D Q R S V V V L L A A S D L I L TAACGTCAGTGGAGATCCCGCCCGGCGCTTCTCGTCAGCTTGAAAATATGCTGCCGTATT T S V E I P P G A S R Q L E N M L P Y L TGTTAGAAGATGAAATCGCCCAAGATGTGGAAGATGTGCACTTTTGTGTGCTGAGTAAAG L E D E I A Q D V E D V H F C V L S K G GACGAGAAACGGCGGATGTGGTCGGTGTCGATCGTCTTTGGCTGCGCGCTTGCTTAGATC R E T A D V V G V D R L W L R A C L D H ATCTCAAAGCGTGCGGTTTTGATGTGAAGCGCGTATTGCCTGATGTACTGGCGATCCCTC L K A C G F D V K R V L P D V L A I P R GCCCAGAGCACGGTTTAGCTGCCCTGCAATTGGGTGATGAGTGGTTAGTGCGTAAAAGCA P E H G L A A L Q L G D E W L V R K S T CTACGCAAGGGATGGCAGTGGACGCGCAGTGGTTAAGCTTACTGGCCGCTTCCGATTGGG T Q G M A V D A Q W L S L L A A S D W V TGCAGAATGAGGGTGAGTATTTGCCTTTGCAAGCGCTCACGCCGCTTCCAGAGCTGAGCT Q N E G E Y L P L Q A L T P L P E L S L TAGCCGAAACCCAAGAGTGGCGTTATGAGCCGAGCGGTTTAGTCATGCAACTGCTGACTC A E T Q E W R Y E P S G L V M Q L L T Q AGGAGGCCTTAACCAGCAAGTTCAACTTGCTGACGGGGAGTTTTAAACTCAAGTCTTCTT E A L T S K F N L L T G S F K L K S S W GGCTGCGGTATTGGCAAATATGGCGCAAAGTGGCGATCGCTGCTGGGCTATTTGTCGCGG L R Y W Q I W R K V A I A A G L F V A V TATCCATCAGTTATTCGCTGTTTCAGGCGCATCAATACGAAGCACAAGCGGACGCTTACC S I S Y S L F Q A H Q Y E A Q A D A Y R GCGCGGAAAGTGAGCGGATTTTCCGCAGCATCTTCCCTGATAAACAGAAAATCCCGACCG A E S E R I F R S I F P D K Q K I P T V TGACTTATTTGAAAAGACAGATGAGTGATGAGATGGCGCGTTTATCTGGCGGTGCCAGTG T Y L K R Q M S D E M A R L S G G A S V TGGGCAGCGTTTTGAAGTGGCTAACCCCGTTGCCTGAGGCTTTGAAAGGGGTCAATCTAC G S V L K W L T P L P E A L K G V N L Q AACTGCAAAGCATCAAATTTGACAGTAACCGCAGTGAGATTCGCCTAGAAGCGACCAGTC L Q S I K F D S N R S E I R L E A T S R GTGATTTCCAAAGTTTTGAACAAGCTCGCACTCAGCTTGAGCAGTATTTTGCTGTTGAAC D F Q S F E Q A R T Q L E Q Y F A V E Q AGGGGCAGCTCAATAAAAATGGCGAGCAAGTGTTTGGCGTGTTTGTGGTGAAGCCCAAGT G Q L N K N G E Q V F G V F V V K P K * 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10201 10261 10321 10381 10441 10501 10561 10621 10681 10741 10801 10861 10921 10981 11041 11101 11161 11221 11281 75 AAQQAQAAATGATGAAAGAATTATTGGCTCCTGTGCAGGCTTGGTGGCGAAGTGTCACCC10260 M K E L L A epsM —) CTCGTGAGCAAAAGATGGTAATGGGCATGGGCGCGCTGACGGTACTCGCTATCGCTTATT R E Q K M V M G M G A L T V L A I A Y W P V Q A W W R S V T P GGGGAATATGGCAGCCTTTGAGTGAGCGTACCGCCCAAGCTCAAGCACGATTACAAACCG G I w Q P L s E R T A Q A Q A R L Q T E AAAAACAGCTACTGAGTTGGGTTAGTGAAAACGCCAACGACATCGTAACGCTCCGTGCGC K Q L L s w v s E N A N D I v T L R A Q U Tn5 in PU3 AAGGGGGCAGTGATGCGCCAAGCGATCAACCACTCAATCAGGTGATCACTAACTCGACGC G G s D A P s D Q P L N Q v I T N s T R GTCAGTTCAATATTGAGCTGATCCGCGTGCAGCCGCGCGGCGAAATGATGCAGGTCTGGA Q F N I E L I R V Q P R G E M M Q V W I TCCAACCGCTACCGTTTTCGCAATTGGTCTCATGGATTGCGTATTTGCAAGAGCGCCAAG Q P L P F S Q L V S W I A Y L Q E R Q G GGGTGAGCGTGGATGCGATTGATATTGACCGTGGTAAAGTGAACGGCGTTGTGGAAGTCA V S V D A I D I D R G K V N G V V E V K AACGTCTGCAACTGAAGCGTQQAQGCTGATATGAAGCGTGCTGTTGGCTATGGTCTGTTA R L Q L K R G G * M K R A V G Y G L L epsN —) TTTTCCACAGTGTTAATGACCAGCGTGGTCGTGCATTTGCCTGCCCAAGTGGCGCTTAGC F S T V L M T S V V V H L P A Q V A L S CCGCTACCGCTGCCTGAAGGTTTAGAGCTCACTGGTATAGAGGGTACTCTGTGGCAAGGT P L P L P E G L E L T G I E G T L W Q G CAAGCCGCGCAAGTTCGTTGGCAAGGCATGAGCCTAGGCGATCTCAACTGGGATCTCCAC Q A A Q V R W Q G M S L G D L N W D L H CTCTCGGCGTTACTGTTGGGGCAGTTGGAGGCGGATATCCGCTTTGGCCGCGGTAGCAGC L S A L L L G Q L E A D I R F G R G S S ACACAACTAAGAGGGAAAGGTGTCGTGGGGGTCGGTTTGAGTGGTCCCTATGCCGATGAT T Q L R G K G V V G V G L S G P Y A D D TTTTTACTCTCCTTACCGGCTGCGCAAGCCATTACTTGGCTACCGCTACCGGTACCACTG F L L S L P A A Q A I T W L P L P V P L ATGGCGCAAGGGCAGTTGGAGATGGCCGTCAAACAGTACCGCTTTGGTGAGCCTTACTGC M A Q G Q L E M A V K Q Y R F G E P Y C CAGCAAGCCGAAGAGCTTAGCTTGGTCAGCCGCGCAGTAGAATCGCCGATTGGTGCGCTG Q Q A E E L S L V S R A V E S P I G A L CAGCTTGGTACTGTCGTGTCGGATTTTACCTGCCAAGAGAGCGTTGTGACCCTGAAAGGT Q L G T V V S D F T C Q E S V V T L K G GGCCAAAAAACTGCGCAGGTGAGCAGTGAATTTAACCTCAGTTTACAGCCGGACAATCGC G Q K T A Q V S S E F N L S L Q P D N R 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11341 11401 11461 11521 11581 11641 11701 11761 11821 11881 11941 12001 12061 76 TATCAAGCACAAGCGTGGTTTAAACCAGAAGCTGAATTTCCTGAGAGTTTAAAGGAGCAG Y Q A Q A W F K P E A E F P E S L K E Q TTGAGCTGGCTACCGCAGCCTGATGGGCAAGGTCGCTATCCGTTCAATCAACAAGGTCAG L S W L P Q P D G Q G R Y P F N Q Q G Q CTCTAGGATGTGTTATCCCATTCATTTCACGCCCAACTGGGCGTGAAATTTTATCGGCTA L ‘k < ........... * GTCTTTGATTTTTAAGATCTCATTCCAAGGTAAATCCGCATCACCAAGCACGATGAAGTT D K I K L I E N W P L D A D G L V I F N CGGGTTTTCCAAGGTTTCCCGCTCATTGTACGAGAGGGGCGATAATTGAGTGCTCAGAAT P N E L T E R E N Y S L P S L Q T S L I GCGCCCACCCGCTTCTTCGACAATGCACTGGGTTGCGGCGGTATCCCATTCGCCAGTTGG R G G A E E V I C Q T A A T D W E G T P ACCAAGGCGCAGATAGCAATCTACCGCACCTTCTGCCACTAAGCAGGCTTTCAGTGCTGC G L R L Y C D V A G E A V L C A K L A A AGAGCCCAGTGGCACCAAGTCATAATTCCAAGCACTGCTTAAACGGCGCGTGATTTTGTT S G L P V L D Y N W A S S L R R T I K N GATGTCTTGGCGACGGCTGATGGCAATCGCGATCGAGCTGCTCGGCAGCTCATGTTTGTG I D Q R R S I A I A I S S S P L E H K H AGTCTGAATTTTGAGGCTCTGCGCCATGTCGGGGATCTTCCACGCCCCTTTGCCTGCGTA T Q I K L S Q A M D P I K W A G K G A Y ACCGTAGTAAGTCACACCAGAAACGGGGCCATACACCACCCCCATCACCGGATGGTTATT V Y Y T V G S V P G Y V V G M V P H N N TTCCACGAGCGCAATGATGGTCGCGAAGTCGCCGCTACGTGCGATGAACTCTTGAGTGCC E V L A I I T A F D G S R A I F E Q T G ATCCAGCGGATC 12072 D L P D 6— oer‘ 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 77 Table 3.2 Location and characteristics of deduced Eps gene products of the epsC - N gene cluster. Protein Position (bp) in sequence N0. of residues Predicted MR EpsC 213-1130 305 33592 EpsD 1176-3200 674 73337 EpsE* 3197-4703 503 56358 EpsF* 4703-5923 406 44916 EpsG 5969-6409 146 16063 EpsH 6443-7027 194 21739 EpsI* 7017-7370 117 13493 Esz* 7364-8022 210 23757 EpsK* 8012-9022 336 37599 EpsL* 3991-10202 403 45343 EpsM 10209-10709 166 13521 EpsN* 10711-11466 251 27322 * Initiation codon of ORF overlaps termination codon of preceding ORF or is separated by two nucleotides or less. 78 The deduced aa sequence of the eps ORFs were analyzed by the method of Kyte and Doolittle (1982) to identify possible hydrophobic, membrane spanning regions. These regions were found in each ORF with the exception of EpsE, a cytoplasmic protein loosely associated with the cytoplasmic membrane (Sandkvist et al. 1993). The hydrophobic regions are doubly underlined in Figure 3.1. Stretches of hydrophobic residues typical of those found in signal peptides (van Heijne 1986) were found at the N terminus of EpsD and EpsN. It is possible, therefore, that both of these proteins are translocated through the cytoplasmic membrane. Hydrophobic sequences similar to prepilin peptidase signal sequences found in type IV pilin subunits were found in Eps G - K, although the similarity was not as strong in EpsK as it was in the other proteins (Figure 3.2). These signal sequences consist of a short leader sequence, followed by a conserved region (G-VF-T-L/I-Q) surrounding the prepilin peptidase cleavage site (V), which is followed by a region of hydrophobic aa (Strom et al. 1993). It is likely, therefore, that EpsG-J and possibly EpsK are also recognized and processed by a prepilin peptidase encoded by V. cholerae. EpsC, L, and M each possess one hydrophobic region while EpsF has three. N0 typical signal sequence was detected in any of these proteins, and, since hydrophobic regions often serve as membrane anchors (Davis and Model 1985), these proteins may be associated with the cytoplasmic membrane. We have previously noted that EpsM was localized in the cytoplasmic membrane fraction of fractionated V. cholerae cells (Chapter 2). The hydrophobic regions identified in the Eps proteins are conserved among their homologues in other Gram' bacteria (Table 3.3). 79 TcpA MQLLKQLFKKKFVKEEHDKKTGQEg MTLLEVIIVLGIMGVVSAGVVTLAQ EpsG MKKMRKQTG FTLLEVMVVVVILGILASFVVPNLL EpSH MTATRG FTLLEILLVLVLVSASAVAVIATFP EpSI MALCVYWLREKVMKSKRG FTLLEVLVALAIFATAAISVIRSVS EpSJ MWRTNQVSSRQNMAG FTLIEVLVAIAIFASLSVGAYQVLN EpsK MRAKQRG VALIVILLLLAVMVSIAATMAERLF Figure 3.2 Putative prepilin cleavage sites of TcpA, EpsG, EpsH, EpsI, Esz, and EpsK. Probable cleavage sites are indicated by 11. 80 Comparison of the V. cholerae GSP genes and gene products to those of other bacteria Table 3.3 shows identities and similarities between the putative sequences of Eps proteins of V. cholerae (obtained by translation of the appropriate ORFs) and GSP proteins of other organisms including the Exe proteins of A. hydraphila (Jiang and Howard 1991; Howard et al. 1993), the Out proteins of E. chrysanthemi (He et al. 1991), the Pul proteins of K. oxytoca (d'Enfert and Pugsley 1989; Pugsley and Reyss 1990; Reyss and Pugsley 1990; Possot et al. 1992), and the ch proteins of P. aeruginosa (Bally et al. 1991; Bally et al. 1992). Similarity also exists between the Eps proteins and the Pil proteins of P. aeruginosa (Bally et al. 1991; N unn and Lory 1991) and the Tcp proteins of V. cholerae (Ogierman et al. 1993) which are required for processing and secretion of type IV pilin. One difference between the pul gene cluster of Klebsiella oxytoca (Reyss and Pugsley 1990) and the eps gene cluster of V. cholerae is that the pul gene cluster includes a gene, pulO, located just downstream of the pulN gene that encodes a prepilin peptidase which is involved in the cleavage and methylation of PulG and likely PulH-J (Pugsley and Dupuy 1992). The eps gene cluster of V. cholerae does not encode a similar gene however. Neither does P. aeruginosa. In this organism, chA (PilD), which is involved in the processing of chT-W (Nunn and Lory 1993), is not located in the xcp gene cluster, but rather in the pil gene cluster which encodes gene products required for the assembly and translocation of type IV pilin (Bally et al. 1991; Nunn and Lory 1991). A gene similar to chA (PilD), tcpJ, exists in V. cholerae and is part of the toxin-coregulated pilus (TCP) operon. TcpJ cannot process EpsG however (M. Sandkvist, unpublished) and tcpJ mutants are still able to secrete cholera toxin (Kaufman et al. 1991). This and the fact 81 Ugoommwbm 55 59353! cm 36m 2 635m. Hut—a ab Oo5um1m5 em @3589 Mum 3255.2 39 3855's 4.55 359. Eagle 5374mm 5 “.555 $8.35: 5m Mama MSU WEE wpmw MEG Mum: NEH MEL max Eva—I Mumps $32 was -- -- m m. Q m H m N r a 2 mm 83 am 8: mm 33 3 $8 mm 3.8 mm 315 mm 6% S 68 3 GE 8 38 OS. 0 U w my Q m H m N r g -- GS 38 «a SS 3 68 mm 88 mm G8 mm $3 3 $8 mm 38 mm $8 ac $8 3: O U m m. Q. m H a N m. 2— 2 68 88 am a: am 63 mm 38 a.» $8 an $3 ma 89 mm 63 3 ES .8 $8 mc $8 N8 w 0 w m e C < ¢< N a. N - - 63 69 3 88 .3 38 am 88 mm $8 ma $8 a» $8 an a: me $8 an 33 e8 6 m aw 38 A4 39 w: a w 0 3 39 am Sb 8 $3 9925958 89.6 85pman 55m $5 930 9.8.55 m5. 000.. <55; «.725 m5. 55:5. 3355 «5.9. 8 $5 pmnomsemmm 5539134 59 5253‘ as 65533. MS 5.35% 4.35 «53:83.5 3.3.3523 05.. 5.3551. $65 @2355 oSQmaanSmSR TE 3335 4.55 NFSKEQ 88.35“ New 634.53. 4.35 Wmmzmoaeaam 9955483“ 68 565:5 #65 5.9.3 39338 mu: 56554”. ~35 Tmmumouguom 52.355. wee Sun. m8. Bmmnmbnmm. 82 that EpsG is known to be processed by the Neisseria gonorrhoeae prepilin peptidase PilD (M. Sandkvist, unpublished) indicates that V. cholerae likely specifies a second prepilin peptidase which functions in the processing of EpsG-J . Expression of the eps genes by the T7 promoter / polymerase system In order to identify the proteins produced by the eps genes, we utilized the T7 promoter/polymerase system of Tabor and Richardson (1985). Fragments of DNA containing different eps genes were placed under the control of the the 4:10 promoter of T7 by cloning them into pT7-5 or pT7-6 (which have the polycloning sites in the opposite orientation with respect to the promoter). These recombinant plasmids were transformed into E. coli MClOGHpGPl-Z], which contains the T7 RNA polymerase gene and labeled with [35S]-Met under conditions when only the genes controlled by the $10 promoter were expressed. DNA fragments cloned into pT7-5/6 are shown in Figure 3.3 and the proteins expressed by these fragments are shown in Figure 3.4. The insert in pMMB560, which encodes epsC and the 5' portion of epsD, expresses a 35 kDa protein, likely the EpsC protein, which has a predicted MR of 33.5 kDa. The proteins expressed by pMMB547, which encodes the 3' portion of epsD and epsE-epsN, were identified based on their expression by the subclones in the following lanes and their size in comparison to the predicted MR of the proteins. The 56 kDa protein is EpsE (Sandkvist et al. 1993), which appears as two bands due to two different redox forms (M. Sandkvist, personal communication). The 45 kDa protein has been identified as EpsL, which has an apparent MR of 44.3 kDa. The 37 kDa protein could be EpsK as it is also expressed from pMMB551. It is not 83 expressed by pMMBSBl however, which also encodes epsK. Another possibility is that the 37 kDa protein is EpsF, which could be migrating aberrantly since, as stated earlier, it has several hydrophobic regions and is predicted to be an integral membrane protein. This protein does not appear to be expressed by pMSlQ, however, which also encodes the epsF gene. The 28 kDa protein is likely the 27.3 kDa EpsN protein. The 26 kDa protein is expressed from the insert in pMMB552 which encompasses the region downstream of the eps genes. It may, therefore, be unrelated to the Eps proteins. The strongly expressed 22 kDa protein is likely EpsG. EpsG appears to migrate aberrantly on SDS-polyacrylamide gels since the predicted MR of this protein is 16.0 kDa. The 18.5 kDa protein is EpsM (Chapter 2). Varying levels of eps gene expression were evident in this experiment. For instance, while the pilin-like protein EpsG is expressed strongly by this system, the other pilin-like proteins downstream of it, EpsH-J, do not appear to be expressed at a detectable level. This may be due to some type of differential expression due, for example, to translational regulation. Nunn and Lory (1993) found that in P. aeruginosa the relative amounts of chT, U, V, and W (homologues of EpsG, H, I, and J) produced were 16:1:1:4. If similar differences in expression exist in V. cholerae, it may explain why EpsG is visible while EpsH, I, and J are not. The eps genes are better expressed from pMMB547, which contains epsD'-N, than from smaller subclones. For instance, the 27 kDa EpsN protein is expressed quite well from pMMB547 (Figure 3.4). When expressed from pMMB543, which contains only part of the epsM gene in addition to epsN, it is barely visible after a one day exposure to X-ray film (Figure 3.4). After a longer exposure, however, the protein band is visible (Figure 3.4, last lane), indicating that the 84 1? WET“??? Hnl‘KH if? “it"? S‘s/E W. Plasmid epsC D E F GHIJ K L MN pMMBS6O pMMB547 pMMBSSl pMSlQ pMMBS3 1 pMM13543 __ pMM13552 L———'1kb Figure 3.3 Physical map of the eps genes showing subclones cloned into pT7- 5/6 used for deletion mapping of the genes. The insert present in each of the subclones is indicated as a line underneath the portion of the eps genes that it encodes. B, BamHI; E, EcoRI; H, HindIII; K, KpnI; S, SacI; Sm, SmaI; X, XbaI. 85 9 0 xx -' .... or: o: no a (0 90%) of the B subunit cell associated (Table 4.3). The ability to secrete at least part of the B subunit pentamer through the outer membrane appears to be confined to bacteria which are members of Vibrianaceae and Aeramanadaceae (MacDonell and Colwell, 1985; Colwell et al., 1986). For instance, while most Vibrio species can secrete the B subunit through the outer membrane, Plesiamanas shigellaides, which was proposed to be moved into Enterabacteriaceae, cannot. On the other hand, Shewanella putrefaciens, which was proposed to be moved into Vibrianaceae, can secrete at least part of the total B subunit pentamers to the external millieu. The members of the family Aeramanadaceae are also able to secrete the B subunit, which implies that they may be more closely related to the Vibrianaceae than those bacteria which cannot, such as Alcaligenes and Erwinia. The B subunit pentamer of LT is known to be secreted from V. cholerae via the GSP (Chapter 2). This system is present in many Gram' bacteria including Aeromonas (Jiang and Howard, 1992; Howard et al., 1993), Erwinia (He et al., 1991; Condemine et al., 1992; Lindeberg and Collmer, 1992; Reeves et al., 1993), Klebsiella (d'Enfert et al., 1987), Pseudomonas (Tommassen et al., 1992), Vibrio (Sandkvist et al., 1993; Chapters 2 and 3) and Xanthomonas 109 (Dums et al., 1991; Hu et al., 1992). Although there seems to be little cross- complementation among the systems, the two species of Aeromonas assayed were able to secrete the B subunit pentamer. A marine species of Vibrio is able to secrete aerolysin, a toxin produced by A. hydraphila and secreted via the GSP of that organism (Wong et al., 1990). The GSP of members of Aeromonas, therefore, appears to be able to recognize and secrete Vibrio proteins while the GSP of more distantly related bacteria cannot. When comparisons are made between the GSP proteins of V. cholerae and other bacteria, they are the most similar to those from A. hydraphila (Chapter 3). It is possible that the species of Vibrio and Aeromonas produce proteins similar to each other which could be secreted by both GSP systems. GMl- ELISA (Svennerholm and Holmgren, 1978) was used to assay the bacteria in Table 4.1 for the presence of proteins which cross-reacted with a polyclonal antibody to cholera toxin. No cross-reacting proteins were detected (results not shown) but other researchers have found that Vibrio mimicus (Spira and Fedorka-Cray, 1984) and Aeromonas hydraphila (Chopra et al., 1986; Schultz and McCardell, 1988) produce toxins that are related to cholera toxin. It is not known if additional members of these two families do. Cholera toxin related sequences have also been found in bacteria unable to secrete the B subunit pentamer such as Salmonella (Chopra et al., 1991), enterotoxigenic E. coli (Yamamoto et al., 1987) and Plesiamanas (Proteus) shigellaides (Gardner et al., 1987). It is possible, however, that these particular bacteria do not possess the GSP genes which would be necessary for secretion of these toxins. E. coli, for instance, secretes very few proteins. One protein that is secreted, (at-hemolysin, does not utilize the GSP but rather the signal peptide independent pathway (Chapter 1). E. coli and Vibrio natriegens, the only Vibrio unable to secrete LT, do not produce a protease halos when plated on 110 skim milk agar. Protease is secreted via the GSP in V. cholerae (see Chapter 2); therefore, it seems likely that E. coli and V. natriegens do not have a GSP. If the GSP system of V. cholerae could be reconstituted in these bacteria, perhaps they would be able to secrete LT through their outer membranes. According to the 16S rRNA sequence analysis of Kita-Tsukamoto et al (1993), V. natriegens is part of a large cluster of Vibrio species, however it is on a distinct branch in this cluster. It is possible that this organism lost or modified its GSP somewhere during the course of its evolution. Concluding remarks The results of this study indicate that the GSP, although conserved among the Gram' bacteria, has a high level of specificity for the recognition of secretable proteins by the secretion apparatus. The secretion of LT, for instance, seems to be confined to Vibrianaceae and Aeramanadacae. 1 1 1 References Bagdasarian, M.M., Aman, E., Lurz, R., Riickert, B. and Bagdasarian, M. (1983) Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad host range, controlled- expression vectors. Gene 26: 273-282. Baumann, L., Bang, SS. and Baumann, P. (1980) Study of relationship among species of Vibrio, Phatabacterium, and terrestrial enterobacteria by an immunological comparison of glutamine synthetase and superoxide dismutase. Curr Microbial 4: 133-138. Chopra, A.K., Houston, C.W., Genauz, C.T., Dixon, MD. and Kurosky, A. (1986) Evidence for production of an enterotoxin and cholera toxin cross- reactive factor by Aeromonas hydraphila. J Clin Microbial 24: 661-664. Chopra, A.K., Peterson, J .W., Houston, C.W., Pericas, R. and Prasad, R. (1991) Enterotoxin-associated DNA sequence homology between Salmonella and Escherichia coli. FEMS Microbial Lett 77: 133-138. Colwell, R.R., MacDonell, M.T. and De Ley, J. (1986) Proposal to recognize the family Aeramanadaceae fam. nov. Int J Syst Bacterial 36:473-477. Condemine, G., Dorel, C., Hugovieux-Cotte-Pattat, N. and Robert-Baudouy, J. (1992) Some of the out genes involved in the secretion of pectate lyases in Erwinia chrysanthemi are reulated by kng. Molec Microbial 6: 3199- 3211. d'Enfert, C., Ryter, A. and Pugsley, A.P. (1987) Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase. EMBO J 6: 3531- 3538. Dallas, W.S. (1983) Conformity between heat-labile toxin genes from human and porcine enterotoxigenic Escherichia coli. Infect Immun 40: 647 -652. de Groot, A., Filloux, A. and Tommassen, J. (1991) Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other Gram-negative bacteria. Mol Gen Genet 229: 278-284. Dums, F., Dow, J .M. and Daniels, M.J. (1991) Structural characterization of protein export genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to export systems of other Gram-negative bacteria. Mol Gen Genet 229: 357-364. Figurski, DH. and Helinski, DR. (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76: 1648-1652. 112 Frey, J. and Bagdasarian, M. (1989) The molecular biology of Ian plasmids. In Promiscuous Plasmids of Gram-Negative Bacteria. C. M. Thomas (eds). New York: Academic Press, Inc., pp. 7 9-94. Fiirste, J .P., Pansegrau, W., Frank, R., Blocker, H., Scholz, P., Bagdasarian, M. and Lanka, E. (1986) Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48: 119-131. Gardner, S.E., Fowlston, SE. and George, L.W. (1987) In vitro production of cholera toxin-like activity by Pleisiamanas shigellaides. J Infect Dis 156: 720-722. Gormley, ER and Davies, J. (1991) Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis. J Bacteriol 173: 6705-67 08. He, S.Y., Lindeberg, M., Chatterjee, AK. and Collmer, A. (1991) Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its millieu. Proc Natl Acad Sci USA 88: 1079-1083. Hirst, T.R., Sanchez, J ., Kaper, J .B., Hardy, S.J.S. and Holmgren, J. (1984) Mechanism of toxin secretion by Vibrio cholerae investigated in strains harboring plasmids that encode heat-labile enterotoxins of Escherichia coli. Proc Natl Acad Sci USA 81: 7 752-7 756. Howard, S.P., Critch, J. and Bedi, A. (1993) Isolation and analysis of eight exe genes and their involvement in extracellular protein secretion and outer membrane assembly in Aeromonas hydraphila. J Bacterial 175: 6695-6703. Hu, N .-T., Hung, M.-N., Chiou, S.-J., Tang, F., Chiang, D.-C., Huang, H.-Y. and Wu, C.-Y. (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. Campestris. J Bacterial 174: 2679-2687. Jiang, B. and Howard, SP. (1992) The Aeromonas hydraphila eer gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway. Molec Microbial 6: 1351-1361. Kita-Tsukamoto, K., Oyaizu, H., Nanba, K. and Simidu, U. (1993) Phylogenetic relationships of marine bacteria, mainly members of the family Vibrianaceae, determined on the basis of 16S rRNA sequences. Int J Syst Bacterial 43: 8-19. Krieg, N .R. and Holt, J .G. (1984) Bergey's Manual of Systematic Bacteriology. Baltimore, MD: Williams and Wilkins. 113 Lindeberg, M. and Collmer, A. (1992) Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi : sequence comparison with secretion genes from other Gram-negative bacteria. J Bacterial 174: 7385-7397. MacDonell, M.T. and Colwell, RR. (1985) Phylogeny of the Vibrianaceae and recommendation for two new genera, Listanella and Shewanella. System Appl Microbial 6: 171-182. Miller, J .H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Reeves, P.J., Whitcombe, D., Wharam, 8., Gibson, M., Allison, G., Bunce, N., Barallon, R., Douglas, P., Mulholland, V., Stevens, S., Walker, D. and Salmond, G.P.C. (1993) Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in Gram- negative bacteria. Molec Microbial 8: 443-456. Sancar, A., Hack, AM. and W.D., R. (1979) Simple method for identification of plasmid-coded proteins. J Bacterial 137: 692-693. Sandkvist, M., Hirst, T.R. and Bagdasarian, M. (1987) Alterations at the carboxyl terminus change assembly and secretion properties of the B subunit of Escherichia coli heat-labile enterotoxin. J Bacteriol 169: 4570- 4576. Sandkvist, M., Morales, V. and Bagdasarian, M. (1993) A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae. Gene 123: 81-86. Scholz, P., Haring, V., Wittmann-Liebold, B., Ashman, K., Bagdasarian, M. and Scherzinger, E. (1989) Complete nucleotide sequence and gene organization of the broad host range plasmid RSF1010. Gene 75: 271-288. Schultz, A.J. and McCardell, BA. (1988) DNA homology and immunological cross-reactivity between Aeromonas hydraphila cytotonic toxin and cholera toxin. J Clin Microbial 26: 57-61. Spira, W.M. and Fedorka-Cray, P.J. (1984) Purification of enterotoxins from Vibrio mimicus that appear to be identical to cholera toxin. Infect Immun 45: 679-684. Svennerholm, A.-M. and Holmgren, J. (1978) Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GMl-ELISA) procedure. Curr Microbial 1: 19-23. 114 Tommassen, J ., Filloux, A., Bally, M., Murgier, M. and Lazdunski, A.,(1992) Protein secretion in Pseudomonas aeruginosa. FEMS Microbial Rev 103: 73-90. Wong, K.R., McLean, D.M. and Buckley, J.T. (1990) Cloned aerolysin of Aeromonas hydraphila is exported by a wild-type marine Vibrio strain but remains periplasmic in pleiotropic export mutants. J Bacterial 172: 372- 376. Yamamoto, T., Gojobori, T. and Yokota, T. (1987) Evolutionary origin of pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae 01. J Bacterial 169: 1352-1357. CHAPTER5 CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH 115 116 Little was known about the genes whose products were required for the secretion of proteins through the outer membrane of V. cholerae when this project was started. Several transposon mutants which were defective in the secretion of protease (identified by plating the mutants on skim milk agar plates and selecting those without a protease halo) were isolated and found to be defective in the secretion of enterotoxin and chitinase as well. A cluster of genes (epsC-N) which restored secretion to these mutants were cloned and sequenced. Five of these genes, epsC, E, F, G, and M, were shown to be required for the secretion process since strains carrying mutations in these genes are not able to secrete proteins (Chapters 2 and 3; Sandkvist et al., 1993; M. Sandkvist, personal communication.) The Eps proteins showed similarity to proteins from other Gram' bacteria which are part of the general secretion pathway (GSP) and are involved in secretion of proteins through the outer membrane of these bacteria. Results showing that the insertion of Tn5 into the epsC gene reduces the expression of epsE, that several ORFs of the eps cluster overlap and that the secretion defect in an epsC mutant is not complemented by the epsC gene alone but is by a cosmid containing the entire eps gene cluster indicate that the epsC-N genes may be arranged into an operon-like structure. It was found that although the GSP is conserved amongst the Gram' bacteria, the system is quite specific in recognition and secretion of proteins. The Eps proteins are able to recognize and promote the secretion of proteins such as E. coli heat-labile enterotoxin (which is closely related to cholera toxin), protease and chitinase, but will not recognize proteins such as B-lactamase, which is located in the periplasm. The GSP systems of other bacteria such as Erwinia chrysanthemi or Klebsiella pneumoniae will generally not recognize foreign proteins such as enterotoxin even 117 though they possess genes similar to the eps genes of V. cholerae. Most species of Vibrio and Aeromonas will recognize and secrete enterotoxin however, even if they do not produce a similar protein themselves. The Eps proteins of V. cholerae appear to not only be required for the secretion of proteins but also for the assembly of the outer membrane. eps mutants are defective in the production of several outer membrane components. The Eps proteins may be required for the secretion of these components to the outer membrane or may help anchor them in place. Interactions between the components of the GSP need to be identified. A strategy which is currently being employed to identify what the EpsE protein interacts with is to express it with subclones containing different eps genes. EpsE is soluble when expressed alone. When expressed with certain other Eps proteins however, it becomes associated with the cytoplasmic membrane (M. Sandkvist, personal communication). It thus appears that EpsE is interacting with these other proteins. A second approach which could be used to study interactions among the Eps proteins would be to look for suppressor mutations which rescue primary mutations in various Eps proteins. If, for instance, a primary mutation in epsM, was rescued by a secondary mutation in epsN, it would indicate that these proteins are interacting. Although this approach should work in theory, it may not if the primary mutation causes such a major change in the conformation of the protein that it simply cannot be rescued by a second-site suppressor. It is also of interest to determine how the GSP system recognizes proteins to be secreted. A bacterium may possess two or three different secretion systems responsible for secreting proteins across the outer membrane and it also must retain certain proteins in the periplasm. Since 118 proteins such as enterotoxin (Hirst and Holmgren, 1987) are secreted across the outer membrane in a folded conformation, it is likely that a three-dimensional secretion signal exists in proteins destined to be secreted via the GSP. A possible approach to identifying the secretion signal of enterotoxin is the construction of mutants which can no longer be secreted. One could then isolate suppressor mutants which are able to secrete these proteins. These would presumably possess mutations in a protein which is interacting with enterotoxin as it is secreted. Another approach may be to look for intragenic suppressors which allow the protein to be secreted. Studies of the structures of these suppressor mutants could give an idea of where the secretion signal is located. The process by which proteins are secreted across the outer membrane in V. cholerae and other Gram' bacteria will require extensive study to deduce its mechanism. Knowledge of this mechanism is important however since it seems to be an almost universal system amongst the Gram' bacteria. In the future, it may be possible to selectively control secretion, whether blocking it to decrease the pathogenicity of organisms to enhance it for the production of proteins which could be secreted from the cell and more easily purified. 119 References Hirst, T.R. and Holmgren, J. (1987) Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae. Proc Natl Acad Sci USA 84: 7418-7422. Sandkvist, M., Morales, V. and Bagdasarian, M. (1993) A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae. Gene 123: 81-86. MIC llllllllllll 3 QRIES Jill/m 4'62