:3... a. -F.~{ .3 “mm? . . (Ia—.1: £$£= 2.3.1.: 3:}:‘. .3: L 5.11. 31.55.}...19. 1.: wilt.» -. , , . . ifll 4 t l "Luau . . , . 2....5gii..u,9.nx> , . . . . .. . 3... . x; . . . . .thfiumMPIxmvii . . :33: . . . . Slauflunfiw 4:23): u... 5...: , . . . . Exit-5.11.3.7? «V8.3... 1.. 353.41.: ,. 0:3 91.8‘ n (1.2;? .‘ :3 I...2$.:.£....l h?!..=x§il§f5§!$. 5:5... 1... .1 5.23:1. . 11.. I. 1:: 142.1: «x... 3.:- f .2... ‘75.. i 9 1.....Azlt...):lx. . .IfziLL... Shift!!! 1 5r . \I r): “13111311: 13......115. 1L2? E.“ $2 11):: .. gammwa __ . . . :3: éfi aéigg m IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII This is to certify that the dissertation entitled PROTEINS OF THE GENERAL SECRETION PATHWAY IN VlBR/O CHOLERAE: INDICATIONS FOR A MULTIPROTEIN COMPLEX presented by Lloyd Patrick Hough has been accepted towards fulfillment of the requirements for L degree in W // /‘.S qF‘f/M‘ _ Major éytfessor Date November 29, 1999 LIERARY Michigan State University PROTEINS OF THE GENERAL SECRETION PATHWAY IN VIBRIO CHOLERAE: INDICATIONS FOR A MULTIPROTEIN COMPLEX By Lloyd Patrick Hough A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1999 ABSTRACT PROTEINS OF THE GENERAL SECRETION PATHWAY IN VIBRIO CHOLERAE: INDICATIONS FOR A MULTIPROTEIN COMPLEX By Lloyd Patrick Hough The type II protein secretion pathway, or general secretion pathway (GSP), is responsible for the transport of proteases, cholera toxin and the related heat-labile enterotoxin, and other putative virulence factors, across the outer membrane of Vibrio cholerae. The function of the GSP in Gram-negative bacteria is dependent on the combined function of 14-16 genes and their associated gene products. In this study the production of polypeptides from several of those genes is demonstrated using the T7 promoter/polymerase system in E. coli. The processing of EpsG by a prepilin peptidase has been shown to occur in a V. cholerae tcpJ mutant, which is defective in the peptidase that processes the TcpA pilin precursor. This predicted the existence in V. cholerae of a second prepilin peptidase specific for the Eps prepilin-like proteins. Four proteins, EpsD, EpsG, EpsF, and EpsM have been purified as C-terminal fusion proteins with an oligohistidine-tag. Two of the Eps proteins have been characterized by gel filtration analysis, revealing that both purified EpsM(His)6 and wild-type EpsL present in 1% Triton X-100 extract are dimeric proteins. In addition, the subcellular localization of EpsC, EpsD, EpsG, EpsL, and EpsM is determined by sucrose gradient separation of the inner and outer membranes. The EpsD protein is shown to fractionate with the outer membrane while the bitopic cytoplasmic membrane proteins EpsC, EpsG, EpsL, and EpsM are found to predominantly sediment with the cytoplasmic membrane, but peaks of EpsC, EpsG, and EpsD also sedimented with outer membrane vesicles. The EpsC and EpsD proteins can be coimmunoprecipitated with Anti-EpsD antiserum, and with Anti-EpsC antiserum after in vivo crosslinking with the cleavable, homobifunctional crosslinker dithiobis(succinimidyl propionate) (DSP). Furthermore, a in vivo stabilization of EpsD and a C-terminal truncation of EpsD, is demonstrated in the presence of EpsC, indicating that the interaction of EpsC and EpsD is likely to be direct, and that the interaction occurs through the N— terminal domain. Additionally, the coimmunoprecipitation, and crosslinking and coimmunoprecipitation of the integral CM protein, EpsL, and the OM secretin, EpsD, is reported. This evidence suggests that not only do EpsC and EpsD interact, but EpsL and EpsD are associated in a complex. Finally, gel filtration fractionation of Triton X-100 and Triton X-100/EDTA solubilized proteins shows that 4 integral CM proteins elute together in the same fraction. Since EpsL has been previously shown to interact directly with EpsE, an autophosphorylating peripheral cytoplasmic membrane protein, and with EpsM, another integral CM protein a hypothesis that each of the CM proteins can be found together in a single complex with the OM components of the GSP has been formulated. Copyright by LLOYD PATRICK HOUGH 1999 To my wife Kimberly Hough. Thank you for your love, support, and patience. ACKNOWLEDGMENTS Special thanks go to the following people for their assistance: Dr. Michael Bagdasarian, my advisor, for the many hours of discussion and guidance. Dr. Wendy Champness, Dr. Rawle Hollingsworth, Dr. Martha Mulks, and Dr. Pat Oriel, my guidance committee members, for their advice and time. The Biotechnology Training Program, for the support, educational opportunities, and industrial exposure it provided. The Institute of Genomic Research, for access to the preliminary genomic sequences of Vibrio cholerae. Charles and Elizabeth Hough, my parents, for the support and encouragement they have given me over the years. Finally, special thanks go to Kimberly Hough for your unconditional love, support, and understanding, without whom I would not have made it through this. vi TABLE OF CONTENTS LIST OF TABLES ................................................................................................. ix LIST OF FIGURES ................................................................................................ x LIST OF ABBREVIATIONS .................................................................................. xi CHAPTER 1 INTRODUCTION ....................................................................... 1 Mechanisms of Protein Secretion .............................................. 2 The General Export Pathway ..................................................... 3 Type I Protein Secretion ............................................................ 3 Type III Protein Secretion .......................................................... 4 Autotransporters ........................................................................ 5 Type II Protein Secretion ........................................................... 7 References .............................................................................. 22 CHAPTER 2 PROTEINS INVOLVED IN THE TYPE II SECRETION CHAPTER 3 SYSTEM ENCODED BY THE EPS GENES OF VIBRIO CHOLEFIAE ............................................................................. 29 Abstract ................................................................................... 30 Introduction .............................................................................. 31 Materials and Methods ............................................................ 33 Results ..................................................................................... 38 Discussion ............................................................................... 45 References .............................................................................. 50 PURIFICATION AND CHARACTERIZATION OF EPS PROTEINS .............................................................................. 54 Abstract ................................................................................... 55 Introduction .............................................................................. 56 vii CHAPTER 4 CHAPTER 5 Materials and Methods ............................................................ 58 Results ..................................................................................... 73 Disscussion ............................................................................. 88 References .............................................................................. 94 INTERACTIONS BETWEEN EPS PROTEINS IN THE TYPE ll SECRETION APPARATUS ........................................ 98 Abstract ................................................................................... 99 Introduction ............................................................................ 100 Materials and Methods .......................................................... 103 Results ................................................................................... 111 Discussion ............................................................................. 122 References ............................................................................ 1 30 CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH ........................................................... 134 References ............................................................................ 140 viii Table 2.1 Table 2.2 Table 2.3 Table 2.4 Table 2.5 Table 3.1 Table 3.2 Table 3.3 Table 3.4 Table 3.5 Table 3.6 Table 4.1 LIST OF TABLES Strains and plasmids used in this study. .................................. 34 DNase and protease activities in the culture medium of eps mutants of Vibrio cholerae TRH7000. ............................... 41 Location and characteristics of predicted Eps reading frames in the sequence of the epsC to epsN gene cluster. ..................................................................................... 45 Percentage of all condons for selected amino acids in V. cholerae eps genes that are low-usage codons" in Escherichia coli. ....................................................................... 46 Putative prepilin cleavage sites of the V. cholerae prepilin-like GSP proteins, EpsG, EpsH, Epsl, Esz, and EpsK, compared with the cleavage sites of two V. cholerae type IV prepilin subunits. ........................................... 49 Bacterial strains used in this study ........................................... 59 Plasmids used in this study. .................................................... 60 Construction of C-terminal oligohistidine tagged fusion proteins with individual Eps proteins. ....................................... 62 Sequences of oligonucleotides used in the construction of oligohistidine fusion proteins with various Eps proteins. ...... 63 Detergent solubility of EpsC, EpsD, EpsG, EpsL, and EpsM from the membranes of V. cholerae TRH7000. ............. 83 Status of different Eps proteins ................................................ 92 Strains and Plasmids used in this study. ............................... 104 Figure 1.1 Figure 2.1 Figure 2.2 Figure 2.3 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 5.1 LIST OF FIGURES Hypothetical model of the type II secretion apparatus in Gram-negative bacteria. .......................................................... 12 Physical and genetic map of the eps gene cluster. .................. 35 Expression of eps genes under the control of the bacteriophage T7 (1)10 promoter ............................................... 4O Processing of the EpsG protein in V. cholerae. ....................... 43 Purification of EpsM(His)6. ....................................................... 74 Purification of EpsG(His)5. ....................................................... 76 Comparison of precusor EpsG(His)5 purified from E. coli DH1OB [pMMBG74] and processed EpsG(His)5 purified from V. cholerae V837 [pMMBB74]. ........................................ 77 Purification of EpsD(His)5 ........................................................ 78 Purification of EpsF(His)6 ......................................................... 79 Size determination of EpsM(His)5 and EpsL. ........................... 81 Sucrose density gradient separation of cytoplasmic and outer membrane proteins of V. cholerae .................................. 86 Coimmunoprecipitation of EpsC, EpsD, or EpsL proteins with EpsC, EpsD, EpsG, EpsL, or EpsM. .............................. 112 In vivo crosslinking of V. cholerae detected with Anti- EpsC antibodies. .................................................................. 114 Coimmunoprecipitation of EpsD with EpsC and EpsL with EpsD after in vivo crosslinking of V. cholerae. ....................... 115 In vivo stabilization of EpsD and EpsD* by EpsC in E. cofl ........................................................................................ 118 Gel filtration fractionation of a proteins solubilized from V. cholerae membranes in 1% Triton X-100/ 10 mM EDTA. ...... 121 An updated model for protein secretion through the type II secretion pathway. .............................................................. 139 LIST OF ABBREVIATIONS ApR ............................................................................................................ Ampicillin ApR ........................................................................................ Ampicillin Resistance ATP .................................................................................. Adenosine Triphosphate bp ........................................................................................................ base pair(s) Cm ............................................................................................... Chloramphenicol CmR ............................................................................ Chloramphenicol resistance CM ..................................................................................... Cytoplasmic Membrane CT ..................................................................................................... Cholera Toxin DSP .................................................................... dithiobis(succinimidyl pr0pionate) GEP .................................................................................. General Export Pathway GSP ............................................................................. General Secretion Pathway IPTG .............................................................. Isopropyl-B-D-thiogalactopyranoside kb ......................................................................................... kilobase(s) or 1000 bp kDa ...................................................................................................... kilodalton(s) KmR ..................................................................................... Kanamycin resistance LB ......................................................................................... Luria-Bertani medium LDAO ........................................................................... Lauryldimethylamine Oxide LT ............................................................ Escherichia coli Heat Labile Enterotoxin MCS ....................................................................................... Multiple Cloning Site Ongo ............................................................................. Optical Density at 280 nm OD550 ............................................................................. Optical Density at 650 nm OG ................................................................................ Octyl-B—D-glucopyranoside OM ............................................................................................... Outer Membrane ORF(s) .............................................................................. Open Reading Frame(s) PAGE ............................................................. Polyacrylamide Gel Electrophoresis PBS .............................................. 10 mM Phosphate Buffer pH 7.4/136 mM NaCI PCR ............................................................................ Polymerase Chain Reaction me ......................................................................................... Polymyxin B Sulfate RBS ..................................................................................... Ribosome binding site Rif ........................................................................................................... Rifampicin RT ............................................................................................ Room Temperature Sm ..................................................................................................... Streptomycin SmR ................................................................................. Streptomycin Resistance SDS .................................................................................. Sodium Dodecyl Sulfate Tc ........................................................................................................ Tetracycline TcR .................................................................................... Tetracycline Resistance WT .......................................................................................................... Wild Type [] .............................................................................. denotes plasmid carrier state B-ME .......................................................................................... B-Mercaptoethanol xi CHAPTER 1 INTRODUCTION Mechanisms of Protein Secretion The process of protein secretion across the cell envelope plays a critical role in the phenomenon of life. The process involves the transport of macromolecules of various sizes, shapes, and biochemical features, across a lipid bilayer that separates the cell from the environment. While this lipid bilayer is responsible for separating and protecting a cell from its environment, it poses a significant problem for the cell: how to transport macromolecules and effect change across the cell envelope without destroying the integrity of the membrane. This problem is compounded in Gram-negative bacteria, in that they have not one, but two membranes separating the cytoplasm from the extracellular environment. Saprophytic and pathogenic Gram-negative bacteria secrete a diverse array of proteins across the cell envelope, for an equally diverse number of reasons. The transport of degradative enzymes is crucial to the procurement of nutrients which are often found in insoluble macromolecules that cannot be transported across the cell envelope. The transport of degradative enzymes and toxins plays a significant role in the pathogenesis and dissemination of these organisms. Protein secretion also involves the transport and assembly of some outer membrane (OM) proteins in Gram-negative bacteria, which permit the cell to respond and adapt to changes in the cell’s environment (Jiang and Howard, 1992; Sandkvist et al., 1997). The General Export Pathway (GEP) Protein export has been defined as the transport of proteins that are retained partially or wholly within the cell boundary (Pugsley, 1993a). The proteins required for this function are encoded by the sec genes. Each protein known to be targeted to the periplasm and outer membrane, and in some cases to the extracellular environment, is synthesized in the cytoplasm with an amino- terminal (N-temIinaI) signal sequence (reviewed in Danese and Silhavy, 1998). Bacterial signal peptides are exclusively N-terminal, and consist of a long hydrophobic H-domain preceded by one or more positively charged amino acid residues in a generally hydrophilic N-domain (Pugsley, 1993a). As a group signal peptides have essentially the same structural features, but no two signal peptides from distinct presecretory proteins have the exact same sequence (Pugsley, 1993a). Signal peptides direct precursor polypeptides to the sec translocation apparatus in the cytoplasmic membrane, and are then removed during translocation (Danese and Silhavy, 1998). For this reason, they are believed to have no influence on downstream targeting (Pugsley, 1993a). Type I Protein Secretion Originally described for a group of highly homologous toxins and proteases in Gram-negative bacteria, transport mechanisms with similarities to type I protein secretion are widespread in eukaryotic and prokaryotic organisms ( reviewed in Schneider and Hunke, 1998). Type I secretion refers to a sec- independent pathway that transports proteins lacking an N-terminal signal sequence from the cytoplasm to the extracellular environment in Gram-negative bacteria in one step (Delepelaire and Wandersman, 1989; Gentschev et al., 1990). Secretion is generally dedicated to the secretion of one or more closely related toxins, proteases, or Iipases through an ABC-protein mediated exporter. The exporter consists of an ABC (ATP-Binding Cassette) transporter and two accessory proteins that are located in both membranes. (Binet et al., 1997). The genes are generally clustered with the secreted protein, consistent with their specificity. However, they can be promiscuous in that the secretion systems will often transport heterologous exoproteins from the same family (Binet eta/., 1997). Type III Protein Secretion The type III secretion mechanism is the most recently discovered system for the transport of proteins across the outer membrane of Gram-negative bacteria. It has been described in a variety of plant and animal pathogens, including Pseudomonas, Salmonella, Shigel/a, and Yersinia. Sometimes also referred to as the contact-dependant secretion pathway, the type III protein secretion systems described to date mediate the transport of proteins directly into the cytoplasm of a target cell and has been shown to share a common mechansim with flagellar assembly pathways (for recent reviews, see Hueck, 1998; Galan and Collmer, 1999). The type III pathway appears to be a specialized mechanism for the transport of virulence factors directly into the cytoplasm of potential host cells. Analysis of genetic elements harbored by pathogenic variants of Gram-negative bacteria have revealed the presence of pathogenicity islands, clusters of genes required for successful initiation of infection often with features characteristic of mobile genetic elements. Contained within these pathogenicity islands are a collection of approximately 20 genes which encode the proteins of the type III protein secretion pathway. Many of the virulence factors delivered by the type III pathway resemble proteins involved in signal transduction pathways. This pathway permits the pathogenic bacterium to persuade a plant or animal cell, through these signal transduction pathways, to lower their defenses, or to undergo cytoskeletal rearrangements that result in the colonization or invasion by the pathogen (Hueck, 1998). The type III secretion pathway consists of a variety of proteins, including an OM protein similar to the secretin family of protein transporters (Genin and Boucher, 1994), several lipoproteins, and a group of integral membrane proteins with a high degree of similarity to components of the flagellar export apparatus. This collection of proteins form a supramolecular structure in the cytoplasmic and outer membranes of Gram-negative bacteria that has recently been isolated from Salmonella typhimurium and visualized by electron microscopy (Kubori et al., 1998). The supramolecular structure that was visualized closely resembles the flagellar basal body, and contains a long needle-like structure that extends from the basal body. This needle-like structure is proposed to be the channel through which secreted proteins are transported, and may be directly involved in the delivery of virulence factors to the cytoplasm of a target cell (Kubori et al., 1998). Autotransporters The Type IV protein secretion mechanism is another example of how Gram-negative bacteria have evolved mechanisms for getting macromolecules to the extracellular milieu. Proteins transported by this mechanism, often termed ’autotransporters’, are unique in that the transport machinery is completely encoded within the precursor of the secreted protein itself (reviewed in Henderson et al., 1998). The IgA1 protease of Neisseria gonorrhoeae is the classic example of the growing number of proteins known to be transported through this mechanism. The autotransporters are typically proteases that have a fairly conserved structure. They consist of three domains, an N-terminal leader peptide, the surface localized mature protein (the a- or passenger domain), and a C-terminal B-domain (Henderson etal., 1998). The N-terminal leader peptide, which shares many characteristics of prototypical sec-dependent signal peptides, mediates transport across the CM into the periplasmic space using the GEP. Upon reaching the periplasm, it is believed that the B-domain spontaneously inserts the polypeptide chain into the OM by adopting a B-barrel configuration, and the passenger domain is then translocated across the OM in an unfolded state. Evidence supporting this model is provided by the predicted structure of the B-domain and the transport of a recombinant IgA1 protease passenger domain containing cysteine. The predicted structure of the B-domains of the 31 described autotransporters results in the prediction of an even number, 10 to 18, of anti-parallel amphipathic B-sheets, consistent with a porin-Iike structure (Henderson et al., 1998). The evidence for a periplasmic intermediate and transport of an unfolded domain comes from studies involving the transport of recombinant proteins in which cysteine residues have been introduced. The introduction of cysteine residues results in the formation of disulfide bonds, presumably by the periplasmic enzyme DsbA, and subsequently inhibits passage through the outer membrane (Jose et al., 1996). Type II Protein Secretion The type II protein secretion system, or general secretory pathway (GSP), is a two-step mechanism that offers the Gram-negative bacterium several advantages over the aforementioned secretion systems. A protein secreted to the extracellular milieu through the GSP is first synthesized in the cytoplasm as a precursor with a traditional N-terminal signal sequence. The N-terminal signal sequence directs the protein to the GEP, provided by the Sec proteins in Escherichia coli and presumably by homologs in other Gram-negative bacteria, which translocates the precursor polypeptide to the periplasm. Upon reaching the periplasm the N-terminal signal sequence is cleaved from the precursor by a signal peptidase, and the protein is permitted to fold and assemble before being secreted. The folding and assembly of exoproteins in the periplasm is the greatest advantage that the GSP offers the bacterium. The periplasmic space of the Gram-negative bacterium is one of the compartments which the bacterium may use to contain and segregate certain chemical reactions. The periplasm is an oxidizing environment, compared to the reducing environment of the cytoplasm, which favors the formation of disulfide bonds (Rietsch and Beckwith, 1998). The formation of disulfide bonds is often critical to the tertiary and quaternary structure of a protein (Missiakas and Raina, 1997). In the case of the E. coli heat-labile enterotoxin (LT), a multi-subunit toxin which is secreted through the GSP of Vibrio cholerae (Sandkvist et al., 1993), the protein requires the formation of disulfide bonds in both the A and B subunits for its biogenesis (Yu et al., 1992), and enzymatic activity (Hol et al., 1995; Orlandi, 1997). In addition to an environment favorable to the formation of disulfide bonds, the periplasm offers the cell a way to increase the relative concentration of a protein. This may be critical in the case of LT, since it has been shown that the LT B subunits will not spontaneously assemble in vitro until a high concentration of subunits has been reached (Sandkvist and Bagdasarian, 1993). The first step of protein secretion through the GSP is the translocation of the protein precusors into the periplasm by the GEP. In E. coli the GEP is provided by the combined actions of the Sec proteins. To date only, two type II secretion systems have been reconstituted in E. coli, the pullulanase secretion system of Klebsiel/a oxytoca and the out system of Erwinia chrysanthemi and En/vinia carotovora (d’Enfert etal., 1987; He etal., 1991a; Lindeberg eta/., 1996). In both instances it was demonstrated that secretion of the heterologous exoproteins specific for each system required a functional Sec mediated export pathway (He at al., 1991b; Pugsley et al., 1991). It is generally assumed the GEP is similar to the Sec system of E. colifunctions in V. cholerae and other Gram-negative bacteria. In support of this assumption, an ORF with 72% identity to both the E. coli SecA and SecB proteins can be located by BLAST in the unfinished V. cholerae genome (unpublished observations using preliminary sequence information made available from The Institute for Genomic Research). The Secretion Genes. The second step of protein secretion through the GSP depends on the combined function of at least 12, and possibly up to 16 gene products. In most systems these genes are designated A-0 and S. In V. cholerae, a fragment containing the epsC-N genes has been described and demonstrated to be involved in the translocation of LT across the OM (Overbye et al., 1993; Sandkvist etal., 1993; Sandkvist etal., 1997). Recently, a prepilin peptidase similar in function to the product of the K. oxytoca pulO was predicted (Sandkvist et al., 1997), and subsequently cloned by Marsh and Taylor (1998), as the vch gene, bringing the number of genes involved in type II secretion in V. cholerae to at least 13. Several genes identified by analysis of homologous secretion systems have not yet been described in V. cholerae. The genes exeA and exeB have been identified and demonstrated to be required for secretion of aerolysin in Aeromonas hydrophila (Howard at al., 1996). Homologs of exeB have been described within the cluster of secretion genes in both the K. oxytoca pullulanase and E. chrysanthemi pectinase secretion systems, but mutations in these genes reportedly had no effect on pullulanase secretion, and decreased the secretion of pectinase by only 30%, suggesting that they may be dispensable in some systems (d’Enfert and Pugsley, 1989; Condemine eta/., 1992). Additionally, another gene that encodes an outer membrane Iipoprotein, the S protein, encoded by pulS in K. oxytoca and outs in E. chrysanthemi, has been identified and demonstrated to be essential for the function of the GSP (d’Enfert and Pugsley, 1989; Lindeberg eta/., 1996). The Secretion Substrates Each of the secretion systems is specific for a particular set of exoproteins. In addition to the CT and LT molecules, V. cholerae has been shown to secrete protease(s), lipase, and chitinase through the eps encoded GSP (Overbye et al., 1993; Sandkvist et al., 1997), while the secretion of at least some DNases and amylases occurs through other pathways (Sandkvist et al., 1997, and unpublished observations). Other proteins secreted through type II secretion pathways include aerolysin in Aeromonas (Howard and Buckley, 1985), pullulanase in Klebsiella (d’Enfert etal., 1987), pectinases and cellulases in E. chrysanthemi and E. carotovora (Murata et al., 1990), alkaline phosphatase and elastase in Pseudomonas aeruginosa (Lazdunski et al., 1990), and proteases and pectinases in Xanthomonas campestris (Dums et al., 1991 ). While all of these proteins are secreted through type II secretion systems, they are generally not secreted through heterologous systems. For example, E. chrysanthemi secretes a pectate lyase, but is unable to secrete a similar pectate Iyase from E. carotovora (He etal., 1991a; Py eta/., 1991), and the K. oxytoca Pul system cannot secrete P. aeruginosa or E. chrysanthemi proteins (de Groot et al., 1991; He eta/., 1991a), suggesting that there must be species-specific recognition signal encoded within an exoprotein. However, no common secretion amino acid sequences in proteins secreted from any one organism have been identified, thereby implying that secretion signals must be a discontinuous, or 10 structural signal, contained within the folded protein (Pugsley, 1993a; Connell et al., 1995). The nature of the discontinuous patch signal, or structural signal, is unknown. Indications for a discontinuous secretion signal have been shown in pullulanase, in which two regions consisting of 78 amino acids at the N-terminus and 80 amino acids near the C-terminus, were shown to be necessary for secretion of pullulanase, and sufficient for secretion of B-lactamase (Sauvonnet and Pugsley, 1996). However, a single 60 amino acid region of P. aeruginosa exotoxin A, was found to be sufficient for secretion of a B-lactamase fusion protein through the ch system (Lu and Lory, 1996), but then a region including these 60 amino acids could be deleted from exotoxin A without effecting secretion (McVay and Hamood, 1995). The complete lack of sequence homology between any of the identified regions, and the lack of strucutural information about the folding of truncated or fusion proteins, will make identification of any signal difficult. Furthermore, addition, or even substitution of various reporters to proteins which contain the appropriate signals can also inhibit secretion. Sauvonnet et al. have shown that while most small insertions had no effect on secretion of pullulase, additions of B-Iactamase, alkaline phosphatase, or other domains to the C-terminus usually prevented their secretion (Sauvonnet et al., 1995). Similarly, the insertion of B-lactamase or alkaline phosphatase to the A subunit of CT which properly assembled into a holotoxin like molecules, were not secreted by V. cholerae (Jobling and Holmes, 1992). Since these fusions are to a chain which is not required in the secretion of CT, 11 Figure 1.1 Hypothetical model of the type II secretion apparatus in Gram- negative bacteria. and should not have affected the folding and consequently the signals contained within the B subunit, steric hindrances by a passenger domain will also hamper efforts to identify a signal. The Proteins, the Interactions, and the Model. The current model of Type II protein secretion in Gram-negative bacteria (Figure 1.1) is based upon the subcellular locations and identified interactions of the 12-14 secretion components. Subcellular locations of each of the 14 proteins identified thus far in most secretion systems, were initially predicted from computerized analyses of the amino acid sequences. Most of the proteins were predicted to have a cytoplasmic membrane location, with two proteins localized to the OM, and one localized to the cytoplasm. The subcellular location of many of the proteins have been experimentally evaluated using a variety of techniques including selective detergent solubilization, topology analysis by genetic fusions with reporter domains B-lactamase and alkaline phosphatase, and sucrose density gradient separation. However, many of the initial studies of subcellular localization have suffered from analysis of highly expressed protein(s), production in isolation from other GSP proteins, or production at non- stoichiometric amounts. The membrane location of the C protein was initially determined by detergent solubilization of a highly expressed protein (Bleves et al., 1996). Subsequent analysis by genetic fusions with B-Iactamase (BIaM) has determined the membrane topology of the C protein (Thomas et al., 1997), and more recently, evidence obtained by sucrose gradient fractionation of membranes showed that the C protein is actually distributed between both the 13 cytoplasmic and outer membrane (Possot et al., 1999). Antibodies have been used to demonstrate that the D protein separates with the outer membrane by sucrose gradient fractionation (Hardie et al., 1996a). The E protein is the only GSP protein located exclusively in the cytoplasm, it is however associated with the membrane through other GSP proteins (Possot and Pugsley, 1994; Sandkvist et al., 1995). The F protein is one of two proteins predicted to be a polytopic cytoplasmic membrane protein, and its membrane topology has been determined through fusions to BlaM (Thomas et al., 1997). Nunn and Lory performed analyses of both precursor forms and processed and N-methylated forms of the prepilin-like proteins G, H, I, and J from the ch system of P. aeruginosa. Their analysis of the localization of the G, H, I, and J, proteins revealed that the proteins are solubilized in detergents that selectively solubilize CM proteins. Sucrose gradient separation of the CM and OM fractions demonstrated an association with both membranes, with each protein predominantly in the CM fractions. Their analysis also revealed that processing resulted in little change in the overall distribution of the prepilin-like proteins (Nunn and Lory, 1993) . However analysis of the G protein from K. oxytoca suggested that both the precursor and mature forms of the protein were predominantly located in the OM (Pugsley and Dupuy, 1992; Pugsley, 1993b; Pugsley and Possot, 1993). This observation was supported by both detergent solubilization and sucrose gradient fractionations. Localization of the K, L, M, N, and 0 proteins to the CM was done exclusively by genetic fusions to BlaM or alkaline phosphatase (Reeves eta/., 1994; Thomas etal., 1997). Subcellular 14 location of the last of the 14 proteins included in this model, the 8 protein, was determined by palmitate labeling and sucrose gradient fractionation (d’Enfert and Pugsley, 1989). The model presented in (Figure 1.1) presents a hypothetical organization of the type II secretion appratatus, or secreton, using only the 14 proteins known to be involved in the secretion of pullulanase and pectinase from reconsititution of the appropriate secretion pathways in E. coli. This model does not include the A or B proteins of Aeromonas, since these may not be required for the secretion of all proteins (Jahagirdar and Howard, 1994). The N protein is included in this model even though no N homolog has been identified in the reconstituted E. chrysanthemi Out secretion system. The model presented in Figure 1.1 demonstrates some of the interactions that have been identified, and some that have been proposed, between proteins of the type II secretion pathway. Interactions between the E protein and L protein have been identified through studies with the V. cholerae EpsE and EpsL proteins (Sandkvist et al., 1995). Interactions between the 8 protein and the D protein have been demonstrated through studies with the K. oxytoca PulD and PulS (Hardie etal., 1996a; Hardie et al., 1996b), and with the E. chrysanthemi OutD and OutS (Shevchik and Condemine, 1998). Interactions between the E protein and the G proteins have been suggested by isolation of suppressors with the P. aeruginosa chR and chT proteins (Kagami et al., 1998). Indications of interactions between the prepilin-like proteins have also been reported (Lu et al., 1 997). 15 While the molecular architecture of the type II secretion pathway is slowly being worked out through biochemical and genetic analyses, precious little is currently known about the functions played by each of these proteins. Roles have been identified for the S proteins, the D proteins, and the O proteins. Parital roles have been identified for the E proteins, and the L proteins. The only component for which a certain enzymatic function has been assigned is the C protein, a prepilin peptidase. The 0 proteins encoded by pilD in P. aeruginosa, by tapD in A. hyrdophila, and vch in V. cholerae, have been shown to be functional in both pilus biogenesis and in protein secretion (Strom et al., 1993; Pepe et al., 1996; Marsh and Taylor, 1998). These proteins are responsible for the cleavage and N-methylation of the prepilin-like G, H, I, J, and possibly K proteins of the type II secretion pathway at a cleavage site that resembles the type IV-A prepilin subunit processing site (Nunn and Lory, 1992; Nunn and Lory, 1993; Bleves et al., 1998; Fullner and Mekalanos, 1999). This function is also performed by the PuIO and OutO genes of K. oxytoca and E. chrysanthemi, respectively (Nunn and Lory, 1991; Pugsley and Dupuy, 1992; Pugsley, 1993b). A second component for which a function has been identified is the 8 protein. The S protein has a piloting and chaperone-like function in its interaction with the D protein (Hardie et al., 1996a). Expression of the K. oxytoca D protein, PulD, in E. coli resulted in multimeric PulD that was not efficiently inserted into the outer membrane (Hardie et al., 1996a). However, when PuID was expressed together with the 8 protein, the D became protected from proteolysis and efficiently inserted into the OM. Initially, it was suspected that the S protein was 16 exclusively a piloting protein responsible for transporting assembled secretin to the OM, however, when Hardie et al. replaced the Iipoprotein-type signal peptide of PulS with the signal peptide of maltose binding protein, the resulting recombinant protein was able to protect the D protein from proteolysis , but no longer properly localized the D protein in the OM (Hardie et al., 1996b). Daefler et al. then identified an S protein binding domain in the D protein by constructing hybrid proteins between N-terminal fragments from the filamentous bacteriophage pr protein and C-terminal fragments of PuID (Daefler et al., 1997). However, the 8 protein has only been identified in the Klebsiella Pul and the Erwinia Out secretion systems. It remains to be determined whether S homologs or analogs exist in other Gram-negative bacteria. The D proteins have been putatively assigned the function as the pore though which secreted proteins cross the OM and as such are referred to as secretins. The function of the pore was initially proposed because the D protein is the only integral OM component of the type II secretion pathway, and because it shared homology with OM proteins involved in filamentous phage biogenesis and type III secretion systems. It was initially demonstrated that the M13 pr protein could be found in E. coli as a homomultimers of approximately 10-12 subunits (Kazmierczak et al., 1994), that were resistant to denaturation by SDS (Linderoth et al., 1996). It has since been determined that D protein multimers of pr, PulD, PilQ, or chQ consist of approximately 14 monomeric subunits by visualization of purified multimers under an electron microscope (Linderoth et al., 1997; Bitter etal., 1998; Nouwen etal., 1999). The dimensions of the putative 17 pore formed by various secretins has also been estimated from the EM visualization of the mutlimeric structures (Linderoth et al., 1997; Bitter et al., 1998; Nouwen et al., 1999). Images of the bacteriophage M13 pr protein suggested that the central pore was approximately 8 nm, large enough to permit extrusion of an M13 phage (Linderoth et al., 1997). The existence of an open pore with a diameter of 8 nm in the OM of any Gram-negative bacterium would more than likely have deleterious effects. Thus, such a pore must be gated. Evidence in support of this hypothesis has been reported by Shevchick et al. The authors demonstrated that the E. chrysanthemi OutD expressed in E. coli in the presence of an appropriate pectinase resulted in the lysis of the host (Shevchik et al., 1997), that was not observed when each gene was expressed in the same host separately. Additionally, the toxicity of the E. chrystanthemi OutD expressed in E. coli, was not caused by the pectinase from E. carotovora, which cannot be secreted by E. chrysanthemi (Shevchik et al., 1997). This finding implies that the gate may normally be closed, and is opened upon substrate binding. This finding also suggests that the species specificity of the secretion apparatus may be limited to the N-terminal, or periplasmic, portion of the D protein. The last protein for which a function is beginning to be understood is the E protein. Analysis of the amino acids sequences of several E homologs revealed a common Walker A motif, a motif commonly associated with nucleotide binding proteins. A functional Walker A motif has been demonstrated to be required for secretion through the GSP in the E homologs of K. oxytoca (Possot and Pugsley, 18 1994), V. cholerae (Sandkvist et al., 1995), P. aeruginosa (Turner et al., 1993), and A. hydrophilia (Howard at al., 1996). The presence of the Walker A motif suggested that these proteins were likely to be ATP-binding proteins, and potentially ATPases. Purified protein of the V. cholerae E protein, EpsE, was demonstrated to be an autophosphorylase (Sandkvist et al., 1995). However, the authors were unable to detect any catalytic ATPase activity of the purified EpsE protein, even when incubated in the presence of V. cholerae membranes (Sandkvist etal., 1995). Additionally, it was demonstrated that the EpsE protein became membrane associated in the presence of EpsL, and integral bitopic membrane protein, an interaction that requires the N-terminus of EpsE to occur. It has been proposed that the role of the E protein is to energize the process of secretion through the hydrolysis of ATP. However, there are indications that the process of secretion may only require ATP hydrolysis for certain secretion systems or possibly substrates. Letellier et al. found that both ATP and proton motive force (PMF) were required for the secretion of aerolysin from A. hydrophila (Letellier et al., 1997). On the other hand, Possot et al. determined that ATP, when reduced to less than 10% of cellular pools, had no effect on secretion of mature proteins already in the periplasm (Possot et al., 1997). Additionally, it was also noticed by Possot et al. that the presence of the E protein is required while the remaining 13 or so proteins are being produced, and presumably assembled, and cannot be added to the system later (Possot et al., 1992). This would suggest that the role of the E protein is not in the energetics of secretion, but perhaps in the assembly of the secretion apparatus. 19 No function has yet been determined for the remaining proteins, however it is likely that many may play purely structural roles, while still others may be involved in assembly of the secretion apparatus. The prepilin-like proteins G, H, l, and J are hypothesized to form a pilus-like structure in the periplasm, but no evidence for such a structure has been reported. Limited evidence for a higher order structure formed by the prepilin-like proteins stems from in vivo crosslinking experiments which permitted the detection of a homodimer of the G protein (Pugsley, 1996), and heterodimers of the H, l, and J proteins copurifying with the G protein (Lu et al., 1997). The same experiments identified a crosslinking product between the G (chT) protein of the type II secretion pathway, and PiIA, the type IV pilus subunit of P. aeruginosa, suggesting that there may be some cross-talk or an intimate relationship between piliation and exoprotein transport systems in Pseudomonas. No function has been reported for the C, F, M, and N proteins in the functioning of the type II secretion pathway. Although, two recent reports on the C protein have provided indications that the C protein may interact with the D protein in the OM. Bleves et. al. reported a proteolytic protection conferred upon the C protein by the presence of D (Bleves et al., 1999). Additional, indications for function of the E protein come from Possot et al. in which they determined that the C protein fractionates with both the CM and OM fractions in sucrose gradient separations. However, they also report that the fractionation pattern of the C protein is not dependent on the presence of the D or any other protein from the GSP. In vivo crosslinking results also suggest that the C protein can form 20 high-order oligomers, that may depend on the presence of the D protein (Possot etal,1999) Thus, the dissection of the interactions between proteins in the secretion pathway has not yet been completed. The implications of membrane association of the autophosphorylating E protein with the integral cytoplasmic membrane protein have yet to be defined. Is the E protein responsible for energizing the transport of secreted proteins across the outer membrane? Does the C protein interact with proteins in the OM? Do any of the other CM proteins with extended periplasmic domains interact with proteins in the OM? The present study addressed primary the question of protein-protein interactions in the type II secretion pathway of V. cholerae. The indication of multiple interactions suggests that a multiprotein complex involving all Eps proteins may be the functional unit of the type II secretion pathway. 21 References Binet, R., S. Létoffé, J. M. Ghigo, P. Delepelaire and C. Wandersman (1997). Protein secretion by Gram-negative bacterial ABC exporters--a review. Gene 192: 7-11. Bitter, W., M. Koster, M. Latijnhouwers, H. de Cock and J. Tommassen (1998). Formation of oligomeric rings by chQ and PiIQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mal. Microbial. 27: 209-219. Bleves, S., M. Gerard-Vincent, A. Lazdunski and A. Filloux (1999). Structure- function analysis of chP, a component involved in general secretory pathway- dependent protein secretion in Pseudomonas aeruginosa. J. Bacterial. 181: 4012-4019. Bleves, S., A. Lazdunski and A. Filloux (1996). Membrane topology of three ch proteins involved in exoprotein transport by Pseudomonas aeruginosa. J. Bacterial. 178: 4297-4300. Bleves, S., R. Voulhoux, G. Michel, A. Lazdunski, J. Tommassen and A. Filloux (1998). The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, chX (GspK family). Mal. Microbial. 27: 31-40. Condemine, G., C. Dorel, N. Hugovieux-Cotte-Pattat and J. Robert-Baudouy (1992). Some of the out genes involved in the secretion of pectate lyases in Eminia chrysanthemi are reulated by kng. Mal. Microbial. 6: 3199-3211. ' Connell, T. D., D. J. Metzger, M. Wang, M. G. Jobling and R. K. Holmes (1995). Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae. Infect. Immun. 63: 4091-4098. Daefler, S., l. Guilvout, K. R. Hardie, A. P. Pugsley and M. Russel (1997). The C- terminal domain of the secretin PulD contains the binding site for its cognate chaperone, PuIS, and confers PulS dependence on prf1 function. Mal. Microbial. 24: 465-475. Danese, P. N. and T. J. Silhavy (1998). Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli. Annu Fiev Genet 32: 59-94. de Groot, A., A. Filloux and J. Tommassen (1991). Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other Gram-negative bacteria. Mal. Gen. Genet. 229: 278-284. Delepelaire, P. and C. Wandersman (1989). Protease secretion by Erwinia chrysanthemi. Proteases B and C are synthesized and secreted as zymogens without a signal peptide. J. Biol. Chem. 264: 9083-9089. 22 d’Enfert, C. and A. P. Pugsley (1989). Klebsiella pneumoniae pulS gene encodes an outer membrane lipoprotein required for pullulanase secretion. J. Bacterial. 171 : 3673-3679. d’Enfert, C., A. Ryter and A. P. Pugsley (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. Dums, F., G. M. Dow and M. J. Daniels (1991). Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other Gram-negative bacteria. Mal. Gen. Genet. 229: 357-364. Fullner, K. J. and J. J. Mekalanos (1999). Genetic characterization of a new type lV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect. Immun. 67: 1393-1404. Galan, J. E. and A. Collmer (1999). Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284: 1322-1328. Genin, S. and C. A. Boucher (1994). A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain. Mal. Gen. Genet. 243: 112-118. Gentschev, I., J. Hess and W. Goebel (1990). Change in the cellular localization of alkaline phosphatase by alteration of its carboxy-terminal sequence. Mol. Gen. Genet. 222: 211-216. Hardie, K. R., S. Lory and A. P. Pugsley (1996a). Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein. EMBO J. 15: 978- 988. Hardie, K. R., A. Seydel, I. Guilvout and A. P. Pugsley (1996b). The secretin- specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions. Mal. Microbial. 22: 967-976. He, S. Y., M. Lindeberg, A. K. Chatterjee and A. Collmer (1991a). Cloned En/vinia chrysanthemi aut genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu. Prac. Natl. Acad. Sci. USA 88: 1079- 1083. He, 8. Y., C. Schoedel, A. K. Chatterjee and A. Collmer (1991b). Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane. J. Bacterial. 173: 4310- 4317. 23 Henderson, I. R., F. Navarro-Garcia and J. P. Nataro (1998). The great escape: structure and function of the autotransporter proteins. Trends Microbial 6: 370- 378. Hol, W. G. J., T. K. Sixma and E. A. Merritt (1995). Structure and function of E. coli heat-labile enterotoxin and cholera toxin B pentamer. Bacterial Toxins and Virulence Factors in Disease. J. Moss, B. lglewski, M. Vaughan and A. T. Tu. New York, Marcel Dekker, Inc. 8: 185-223. Howard, S. P. and J. T. Buckley (1985). Protein export by a Gram-negative bacterium: production of aerolysin by Aeromonas hydrophila. J. Bacterial. 161: 1118-1124. Howard, S. P., H. G. Meiklejohn, D. Shivak and R. Jahagirdar (1996). A TonB- like protein and a novel membrane protein containing an ATP-binding cassette function together in exotoxin secretion. Mal. Microbial. 22: 595- 604. Hueck, C. J. (1998). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbial Mal Biol Rev 62: 379-433. Jahagirdar, R. and S. P. Howard (1994). Isolation and characterization of a second exe operon required for extracellular protein secretion in Aeromonas hydrophila. J. Bacterial. 176: 6819-6826. Jiang, B. and S. P. Howard (1992). The Aeromonas hydrophila eer gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway. Mal. Microbial. 6: 1351-1361. Jobling, M. G. and R. K. Holmes (1992). Fusion proteins containing the A2 domain of cholera toxin assemble with B polypeptides of cholera toxin to form immunoreactive and functional holotoxin-like chimeras. Infect. Immun. 60: 4915- 4924. Jose, J., J. Kramer, T. Klauser, J. Pohlner and T. F. Meyer (1996). Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the lgA B autotransporter pathway. Gene 178: 107-110. Kagami, Y., M. Ratliff, M. Surber, A. Martinez and D. N. Nunn (1998). Type II protein secretion by Pseudomonas aeruginosa: genetic suppression of a conditional mutation in the pilin-like component chT by the cytoplasmic component chR. Mal. Microbial. 27: 221 -233. Kazmierczak, B. I., D. L. Mielke, M. Russel and P. Model (1994). pr, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer. J. Mal. Biol. 238: 187-198. 24 Kubori, T., Y. Matsushima, D. Nakamura, J. Uralil, M. Lara-Tejero, A. Sukhan, J. E. Galan and S. I. Aizawa (1998). Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280: 602-605. Lazdunski, A., J. Guzzo, A. Filloux, M. Bally and M. Murgier (1990). Secretion of extracellular protein by Pseudomonas aeruginosa . Biachimie 72: 147-156. Letellier, L., S. P. Howard and J. T. Buckley (1997). Studies on the energetics of proaerolysin secretion across the outer membrane of Aeromonas species - Evidence for a requirement for both the protonmotive force and ATP. J. Biol. Chem. 272: 11109- 11113. Lindeberg, M., G. P. C. Salmond and A. Collmer (1996). Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway. Mal. Microbial. 20: 175-190. Linderoth, N. A., P. Model and M. Russel (1996). Essential role of a sodium dodecyl sulfate-resistant protein IV multimer in assembly-export of filamentous phage. J. Bacterial. 178: 1962-1970. Linderoth, N. A., M. N. Simon and M. Russel (1997). The filamentous phage pr multimer visualized by scanning transmission electron microscopy. Science 278: 1635-1638. Lu, H. M. and S. Lory (1996). A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa. EMBO J. 15: 429-436. Lu, H. M., S. T. Motley and S. Lory (1997). Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion. Mal. Microbial. 25: 247-259. Marsh, J. W. and R. K. Taylor (1998). Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation. Mal. Microbial. 29: 1481 -1492. McVay, C. S. and A. N. Hamood (1995). Toxin A secretion in Pseudomonas aeruginosa: the role of the first 30 amino acids of the mature toxin. Mal. Gen. Genet. 249: 515-525. Missiakas, D. and S. Raina (1997). Protein folding in the bacterial periplasm. J. Bacterial. 179: 2465-2471. 25 Murata, H., M. Fons, A. Chatterjee, A. Collmer and A. K. Chatterjee (1990). Characterization of transposon insertion Out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements autmutations in E. carotovora subsp. carotovora, E. carotovora subsp. atraseptica, and Erwinia chrysanthemi. J. Bacterial. 172: 2970-2978. Nouwen, N., N. Ranson, H. Saibil, B. Wolpensinger, A. Engel, A. Ghazi and A. P. Pugsley (1999). Secretin PulD: Association with pilot PulS, structure, and ion- conducting channel formation. Prac. Natl. Acad. Sci. USA 96: 8173-8177. Nunn, D. and S. Lory (1991). Product of the Pseudomonas aeruginosa gene pi/D is a prepilin leader peptidase. Prac. Natl. Acad. Sci. USA 88: 3281-3285. Nunn, D. and S. Lory (1993). Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins chT, -U, -V, and -W. J. Bacterial. 175: 4375-4382. Nunn, D. N. and S. Lory (1992). Components of the protein-excretion apparatus of Pseudomonas aeruginosa are processed by the type IV prepilin peptidase. Prac. Natl. Acad. Sci. USA 89: 47-51. Orlandi, P. A. (1997). Protein-disulfide isomerase-mediated reduction of the A subunit of cholera toxin in a human intestinal cell line. J. Biol. Chem. 272: 4591- 4599. Overbye, L. J., M. Sandkvist and M. Bagdasarian (1993). Genes required for extracellular secretion of enterotoxin are clustered in Vibrio cholerae. Gene 132: 101-106. Pepe, C. M., M. W. Eklund and M. S. Strom (1996). Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N- methyltransferase required for extracellular protein secretion. Mal. Microbial. 19: 857-869. Possot, O., C. d’Enfert, I. Reyss and A. P. Pugsley (1992). Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein. Mal. Microbial. 6: 95-105. Possot, O. and A. P. Pugsley (1994). Molecular characterization of PuIE, a protein required for pullulanase secretion. Mal. Microbial. 12: 287-299. Possot, O. M., M. Gerard-Vincent and A. P. Pugsley (1999). Membrane association and multimerization of secreton component PulC. J. Bacterial. 181: 4004-4011. 26 Possot, O. M., L. Letellier and A. P. Pugsley (1997). Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway. Mal. Microbial. 24: 457-464. Pugsley, A. P. (1993a). The complete general secretory pathway in Gram- negative bacteria. Microbial. Rev. 57: 50-108. Pugsley, A. P. (1993b). Processing and methylation of PulG, pilin-like component of the general secretory pathway of Klebsiella oxytoca. Mal. Microbial. 9: 295-308. Pugsley, A. P. (1996). Multimers of the precusor of a type IV pilin-like component of the general secretory pathway are unrelated to pili. Mal. Microbial. 20: 1235- 1245. Pugsley, A. P. and B. Dupuy (1992). An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca. Mal. Microbial. 6: 751 -760. Pugsley, A. P., M. G. Kornacker and l. Poquet (1991). The general protein-export pathway is directly required for extracellular pullulanase secretion in Escherichia coli K12. Mal. Microbial. 5: 343-352. Pugsley, A. P. and O. Possot (1993). The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into multiprotein complex. Mal. Microbial. 10: 665-674. Py, B., G. P. C. Salmond, M. Chippaux and F. Barras (1991). Secretion of cellulases in Erwinia chrysanthemi and E. carotovora is species-specific. FEMS Microbial. Lett. 79: 315-322. Reeves, P. J., P. Douglas and G. P. C. Salmond (1994). Beta-Iactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus. Mal. Microbial. 12: 445-457. Rietsch, A. and J. Beckwith (1998). The genetics of disulfide bond metabolism. Annu Rev Genet 32: 163-184. Sandkvist, M. and M. Bagdasarian (1993). Suppression of temperature-sensitive assembly mutants of heat-labile enterotoxin B subunits. Mal. Microbial. 10: 635- 645. Sandkvist, M., M. Bagdasarian, S. P. Howard and V. J. DiRita (1995). Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. EMBO J. 14: 1664-1673. 27 Sandkvist, M., L. 0. Michel, L. P. Hough, V. M. Morales, M. Bagdasarian, M. Koomey, V. J. DiRita and M. Bagdasarian (1997). General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae. J. Bacterial. 179: 6994-7003. Sandkvist, M., V. Morales and M. Bagdasarian (1993). A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae. Gene 123: 81 -86. Sauvonnet, N., l. Poquet and A. P. Pugsley (1995). Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end. J. Bacterial. 177: 5238-5246. Sauvonnet, N. and A. P. Pugsley (1996). Identification of two regions of Klebsiella oxytoca pullulanase that together are capable of promoting [5- lactamase secretion by the general secretory pathway. Mal. Microbial. 22: 1-7. Schneider, E. and S. Hunke (1998). ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbial. Rev. 22: 1-20. Shevchik, V. E. and G. Condemine (1998). Functional characterization of the Erwinia chrysanthemi OutS protein, an element of a type II secretion system. Microbiology 144: 3219-3228. Shevchik, V. E., J. Robert-Baudouy and G. Condemine (1997). Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins. EMBO J. 16: 3007-3016. Strom, M. 8., D. N. Nunn and S. Lory (1993). A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Prac. Natl. Acad. Sci. USA 90: 2404-2408. Thomas, J. D., P. J. Reeves and G. P. C. Salmond (1997). The general secretion pathway of Erwinia carotovora subsp carotovora: Analysis of the membrane topology of OutC and OutF. Microbiology-UK 143: 713- 720. Turner, L. R., J. C. Lara, D. N. Nunn and S. Lory (1993). Mutations in the consensus ATP-binding sites of chR and PIIB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J. Bacterial. 175: 4962-4969. Yu, J., H. Webb and T. R. Hirst (1992). A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae. Mal. Microbial. 6: 1949-1958. 28 CHAPTER 2 PROTEINS INVOLVED IN THE TYPE || SECRETION SYSTEM ENCODED BY THE EPS GENES OF VIBRIO CHOLEFIAE The results presented in this chapter appeared as part of the publication: Sandkvist et. al. 1997. General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in V. cholerae. J. Bacterial. 179:6994-7003. 29 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, while not required for the secretion of DNase or amylase. Proteins produced from several of the previously identified open reading frames in the eps gene cluster are identified by the use of the T7 polymerase-promoter system in Escherichia coli. One of the proteins, EpsG, was also analyzed in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that EpsG might be processed or otherwise modified by prepilin peptidase not found in E. coli. It is also shown that the TcpJ prepilin peptidase, required for processing of the TcpA subunit of the toxin-coregulated pilus (TCP), is not involved in this modification. It is thus concluded that a second prepilin peptidase is encoded by the genome of V. cholerae. 3O Introduction In Vibrio cholerae the type II secretion system, or general secretory pathway (GSP) is required for the extracellular secretion of several proteins, including cholera toxin (CT), chitinase, and protease (Overbye et al., 1993; Sandkvist et al., 1993). This pathway is likely to play a significant role in the survival of V. cholerae in different environments, as well as in its pathogenicity . Chitinase and protease may allow Vibrio to detach from waterborne chitinaceous particles or allow the procurement of nutrients from macromolecular sources in the environment. CT, the major determinant of the diarrheal disease cholera, may also be involved in the dissemination of Vibrio. Human isolates of enterotoxigenic Escherichia coli produce a heat labile enterotoxin (LT) that is 81% and 78% identical with the cholera toxin A and B subunits, respectively. These toxins are multimeric proteins consisting of a single A subunit and a pentamer of B subunit polypeptides (Spangler, 1992), which also have a similar biological effect and the ability to be secreted by V. cholerae (Neill etal., 1983). The secretion of LT from V. cholerae proceeds through a two step mechanism, similar to CT, in which the toxin is first translocated to the periplasm, presumably by a pathway similar to the sec system of E. coli (Danese and Silhavy, 1998). The signal peptides of the precursor A and B polypeptides are removed and the mature subunits are released into the periplasm where they undergo folding and assembly(Hirst et al., 1984; Hofstra and Witholt, 1984; Hirst and Holmgren, 1987a; Hirst and Holmgren, 1987b; Yu et al., 1992; Sandkvist et al., 1994). The second step of translocation requires the 31 function of the GSP that is believed to occur by the combined action of 12 or more proteins. Several of the proteins in V. cholerae have been identified through complementation of spontaneous secretion defect mutants and through transposon mutagenesis. These techniques resulted in the identification of the epsC, epsE, and epsM genes, and subsequently resulted in the cloning and sequencing of a 12 kb fragment of V. cholerae chromosomal DNA (Overbye et al., 1993; Sandkvist etal., 1993; Sandkvist etal., 1997). Analysis of the sequenced region identified 12 open reading frames (ORFs) that share homology with genes identified in homologous secretion systems in other Gram—negative bacteria (Sandkvist et al., 1997). In this work, the visualization of polypeptides produced from the putative ORFs is demonstrated. The in viva processing of one of the polypeptides, EpsG, and the requirement for a prepilin peptidase other than the previously described TcpJ prepilin peptidase is demonstrated. Finally, it is shown that mutations in the eps genes result in a specific secretion defect and is not a general secretion defect, through the analysis of secreted DNase and protease. 32 Materials and Methods Bacterial Strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 2.1. Strains of Escherichia coli and Vibrio cholerae were grown at 30°C or 37°C as indicated. LB medium, liquid or solidified with 1.5% Bacto-agar (Difco), supplemented with 100 ug/ml thymine was used for subculturing bacteria. M9 medium (Miller, 1972) supplemented with 0.4% glucose, 100 ug/ml thymine, 10 ug/ml thiamine, and 20 ug/ml each of 19 amino acids (all except methionine) was used for the radioactive labeling of proteins. For selection of plasmids, antibiotics were added to the media at the following concentrations: Ap and Km 10 ug/ml, Cm 25 ug/ml. For selection of resistance genes in the chromosome, Km was used at 50 ug/ml. Expression of genes under bacteriophage T7 (D10 promoter control. Derivatives of pT7-5 or pT7-6 containing different fragments of the eps gene cluster (Figure 2.1) were introduced into E. coli MC1061 carrying plasmid pGP1-2. Cells were grown at 30°C in M9 medium for 2 hr. The temperature was then raised to 42°C to induce production of T7 RNA polymerase and 300 ug/ml rifampicin was added 30 min. later. Twenty minutes after adding rifampicin, the temperature was lowered to 37°C and proteins were labeled with 10 uCi/ml L- [358]-methionine (1,000 Ci/mmol) (NEN Life Science, Boston, MA) for 10 min. The cells were pelleted, resuspended in sample buffer and heated to 95°C for 5 min. Proteins were separated on a 0.1% SDS-15% polyacrylamide gel, which 33 Table 2.1 Strains and plasmids used in this study. Strain or Plasmid Relevant Characteristic(s) Source or Reference Strains V. cholerae TRH7000 J71 K-1 E. coli MC1061 Plasmids pGP1-2 pT7-5 pT7-6 pMMBSSO pMMB547 pMMB551 pMMB564 pMM8586 pMMB608 pMS19 pMS37 pMMB531 pTKK4 pMS38 pTKK5 pM821 El Tor thy HgR A(ctxA-cth) O395 tcpJ::Tn5 F— araD139 A(ara-leu)7697 A(Iac)X74 rpsL hst2 mcrA mch1 KmR T7 gene 1 under PAL control, cl857 ApR T7 ()1 0 promoter ApR T7 (p10 promoter, MCS in opposite orientation of pT7-5 pT7-6::epsC pT7-5::epsE-N pT7-5::epsE-J pT7-5::epsG pGP1-2 with KmR replaced with CmR pT7-5::epsE pT7-5::epsF-J pT7-5::epsH-K pT7-5::epsH-M pT7-5::epsKL pT7-5::epsL pT7-6::epsLM pMMB207::tcpJ 34 (Hirst etal., 1984) (Kaufman etal., 1991) (Casadaban, 1980) (Tabor and Richardson, 1985) (Tabor and Richardson, 1985) (Tabor and Richardson, 1985) (Sandkvist etal., 1997) (Sandkvist etal., 1997) (Sandkvist eta/., 1997) (Sandkvist etal., 1997) (Sandkvist etal., 1997) (Sandkvist etal., 1997) (Sandkvist eta/., 1997) (Sandkvist etal., 1997) (Sandkvist etal., 1997) (Sandkvist eta/., 1997) (Sandkvist etal., 1997) (Sandkvist eta/., 1997) (Sandkvist etal., 1997) )|(T EH X HKH HBH BKB SE .‘r ........................................................................................................................................................ ..' epsC D E FGHIJK LMN pMMB547 pMMB551 pMMB608 pMS19 pM837 pMMB531 pTKK4 pMS38 m pTKK5 Figure 2.1 Physical and genetic map of the eps gene cluster. The extent of DNA fragments inserted into the pT7-5 or pT7-6 vectors used for deletion mapping and visualization of gene products is indicated by horizontal lines. Restriction site abbreviations: B, BamHl; E, EcaRl; K, Kpnl; S, Smal; X, Xbal. was then stained with Coomassie Brilliant Blue R-250, dried, and subjected to autoradiography. EpsG was expressed in V. cholerae by introducing pMM8586 carrying the gene for T7 RNA polymerase, and pMMB564, carrying the epsG gene cloned behind the T7 ()1 0 promoter, into strains TRH7000 and J71K-1. Cells were grown at 30°C overnight in M9 medium. Two hundred microliters of this culture was then pelleted and resuspended in 1 ml of fresh medium, and incubated at 30°C for 2 hr, followed by 30 min. of incubation at 42°C. Rifampicin was added to a final concentration of 300 ug/ml, and the incubation continued for another 20 min., when the temperature was lowered to 37°C for 10 min. The cells were 35 labeled for 10 min. with 10 uCi/ml (1,000 Ci/mmol) L-[3SS]-methionine, pelleted and resuspended in sample buffer, and heated to 95°C for 5 min. Proteins were separated on a 0.1% SOS-15% polyacrylamide gel, which was stained with Coomassie Brilliant Blue R250, dried, and subjected to autoradiography. For pulse-chase labeling of V. cholerae J71K-1 [pMMB586 pMMB564], cells from 1 ml of an overnight culture in M9 medium were pelleted and resuspended in 11 ml of fresh medium and grown at 30°C, induced at 42°C, and treated with rifampicin as described above. It was pulsed with 10 uCi/ml L-[358]-methionine for 30 s and chased with 0.6 mM non-radioactive L-methionine. Two-milliliter samples were withdrawn at 0.5, 1, 2, 5, and 10 min. into 2 ml of ice-cold 10% trichloroacetic acid (TCA). After 15 min. on ice, the samples were centrifuged, washed with acetone, suspended in sample buffer, and heated at 95°C for 10 min. Proteins were separated on a 0.1% SDS-17% polyacrylamide gel, stained with Coomassie Brilliant Blue R-250, dried and subjected to autoradiography. Enzyme Assays. Extracellular DNase was determined by combining In a 1.5 ml microfuge tube: Sephadex G-25 filtered culture medium (3.0 to 12.0 ug of protein/ml), 400 ug/ml salmon sperm DNA, and buffer (25 mM HEPES, 4 mM MgCl2, 4 mM CaClg, pH 7.4). Assay mixtures were incubated for 0, 15, and 30 min. at 37°C, before an equal volume of 6% HCIO4 was added to the samples to stop the reaction and precipitate the high molecular weight DNA. After 15 min. on ice, the 36 samples were centrifuged at 20,000 x g and the OD250 was determined. One OD unit was considered equivalent to 50 ug of DNA. Extracellular protease was determined in the same samples of culture medium by a modification of the method of Julius et. al. (Julius et al., 1984). The mixture contained, in 10 ul, 100 mM HEPES adjusted to pH 7.0 with 100 mM Tris base, and 0.1 mM N-tert-butoxy-carbonyl-GIn-Ala-Arg-7-amido-4-methylcoumarin (Sigma Chemical Co., St. Louis, MO). The mixture was placed in the microcuvette of the DynaQuant 2000 fluorometer (Amersham Pharmacia- Biotech, Piscataway, NJ) and the signal was recorded every minute for 10 min. at RT. 37 Resufls Expression of the eps genes by the T7 (2)10 promoter-polymerase system. DNA fragments inserted into pT7-5 or pT7-6 are shown in Figure 2.1, and the proteins specified by these fragments are shown in Figure 2.2. Only those plasmids in which the DNA fragments were inserted in the orientation presented in Figure 2.1 produced detectable amounts of labeled proteins. Although different genes were expressed at different levels and no polypeptide band could be assigned to some of the ORFs, six, possibly seven, bands were identified as corresponding to certain ORFs on the basis of their MS and the expression by the appropriate DNA fragment (Figure 2.1). Thus, the insert in plasmid pMMB560, which contains epsC and the 5’ portion of epsD, produces two bands, a 32-kDa band, likely the EpsC protein, which has a predicted molecular mass of 33.5 kDa, and a 23-kDa band, presumably the truncated EpsD. The bands of approximately 33 kDa, specified by plasmids pMMBS47 (epsE to -N), pMMB551 (epsE to -J), and pMS19 (epsF to -J) (lanes 3, 4, and 5 in ), which should not encode epsC, may represent an aberrantly migrating EpsF protein which has an expected molecular mass of 44 kDa. The insert in plasmid pMMB547, which contains the 3’ portion of epsD and the entire epsE through epsN genes, produced several bands. The band of 52 kDa corresponds to the EpsE protein (Sandkvist et al., 1993). The 37.2-kDa protein is presumably EpsL. The predicted molecular mass of this protein is 45.3 kDa. The question of whether this discrepancy is due to the abnormal migration on the SDS gel or to a possible processing of the EpsL protein has not been resolved yet. The strongly 38 expressed 17.9-kDa protein is likely EpsG, whose predicted molecular mass is 16.0 kDa. The 16-kDa protein, produced by the cells carrying plasmids pMMB547, pMMB531 (epsH to -M), and pTKK5 (epsL to -M) (lanes 3, 8, and 11 in Figure 2.2), is most likely EpsM (Overbye et al., 1993). The band running at 13 kDa in lane 10 of Figure 2.2 is presumably a truncated EpsM. In these experiments, EpsG protein was synthesized in large amounts, whereas ORFs downstream of the EpsG ORF, encoding pilin-like proteins EpsH to -J, were not expressed at similar levels. The band of 14 kDa visible in lanes 3, 4, 5, and 8 of Figure 2.2 may be the Epsl protein, whereas the band of 25 kDa in lanes 3, 4, 5, and 8 could be Esz. Different intensities of the different pilin-like protein bands may indicate differences in expression due, for example, to translational regulation or weaker ribosome binding sites . Nunn and Lory (Nunn and Lory, 1993) found that in Pseudomonas aeruginosa the ratio of amounts of chT, -U, -V, and -W (homologs of EpsG, -H, -l, and -J) produced was 16:1 :1 :4, respectively. If similar differences in expression exist for the eps genes, it may explain why EpsG is visible on our gels while, for instance, EpsH is not. The EpsN protein could not be detected in these experiments. However, in another experiment a band with the mobility expected of EpsN was visible. Requirement of eps functions for secretion. It has been reported that, in addition to being unable to secrete LT, eps mutants are also defective in secretion of protease(s) and chitinase(s), based on 39 Control 2 m “.I x. E. "I E o LI'J u'.I u'. Lu I I :2 _I _'I 203.0 — 105.0 — 70.8 -— — E 43.6 — ,. ~ m w W— I. _ C 28.3 — 17.9 — — G 15.1 — __ I? 1234567891011 Figure 2.2 Expression of eps genes under the control of the bacteriophage T7 (>10 promoter. Cells of E. coli MC1061 containing recombinant plasmids indicated in Figure 2.1 were labeled, separated by SDS-PAGE, and subjected to autoradiography as described in Materials and Methods. The bands which correspond to the molecular masses expected for the Eps proteins (Table 2.3) encoded by the plasmids are indicated by the appropriate letters. Positions of molecular mass markers (in kDa) are indicated on the left. Lanes: 1, pT7-5 vector (without insert); 2, pMMB560; 3, pMMB547; 4, pMMB551; 5, pMS19; 6, pMMB608; 7, pM837; 8, pMMB531; 9, pTKK4; 10, pMS38; 11, pTKK5. 40 Table 2.2 DNase and protease activities in the culture medium of eps mutants of Vibrio cholerae TRH7000. Genotype DNase Activity Protease Activity (m of protein/mini (pmol/mg of protein/min.) WT 230 26.8 epsC 202 1.4 epsE 311 3.9 epsF 408 4.7 epsG 424 8.2 epsL 317 1.6 epsM 317 8.0 non-quantitative analysis of agar plates (Overbye et al., 1993; Sandkvist et al., 1993) and by immunoblotting experiments with antisera specific to the V. cholerae ChiA endochitinase (Connell et al., 1998). To determine whether these proteins belong to a group that is specifically affected by the functions of eps genes, we have screened culture media of V. cholerae for the presence of other soluble proteins. We have found that whereas the activity of extracellular protease(s) was markedly reduced in culture medium of eps mutants, the specific activity of extracellular DNase was not affected (Table 2.2) This indicates that extracellular DNase may be secreted by a pathway different from the GSP. Processing of epsG in V. cholerae As stated earlier, the predicted amino acid sequences of EpsG, EpsH, Epsl, and Esz proteins contain hydrophobic regions which resemble the signal peptides of type IV pilin subunits. It was also shown by Nunn and Lory (Bally et al., 1992; Nunn and Lory, 1992; Nunn and Lory, 1993) that the G, H, I, and J homologs in P. aeruginosa are both cleaved and methylated by the PilD/chA prepilin peptidase. However, no coding region with homology to any known 41 prepilin peptidase has been found immediately upstream or downstream of the eps gene cluster. In order to demonstrate that the prepilin-like Eps proteins are also processed in V. cholerae, the epsG gene was inserted downstream of the T7 ¢10 promoter in pT7-5 to create pMMB564. This plasmid was introduced into E. coli MC1061 and V. cholerae TRH7000, both carrying the T7 RNA polymerase gene on pMMB586. The EpsG protein produced by this plasmid was labeled with [3SS]-Met and visualized by SDS-PAGE and autoradiography (Figure 2.3A). It was found that in E. coli, the EpsG protein was seen as a single band, whereas in V. cholerae, an additional band of lower Mr was produced. This suggested that the second band was a processed form of EpsG. The inefficient processing of radiolabeled EpsG in V. cholerae was most likely due to the continuous labeling conditions, since in experiments in which proteins were pulse-labeled for only 30 s and then chased with unlabeled methionine, most of the EpsG was present in the processed form at the end of the chase (Figure 2.3B). It could be imagined that the prepilin peptidase, encoded by the tcpJ gene of V. cholerae (Kaufman eta/., 1991) for processing of the TCP, might be involved in the processing of the EpsG, EpsH, Epsl, and Esz proteins. This would be analogous to the dual function of the PiID prepilin peptidase in Pseudomonas, in which PilD is responsible for the processing of both the type IV prepilin subunits and the pilin-like proteins chT to chW required for extracellular secretion (Nunn and Lory, 1991; Bally et al., 1992; Nunn and Lory, 1992; Nunn and Lory, 1993). However, the expression of epsG in a tcpJ mutant 42 1 2 3 kDa — 18.4 — 14.3 Figure 2.3 Processing of the EpsG protein In V. cholerae. (A) Cells of E. coli and V. cholerae strains containing plasmid pMMB586, expressing the T7 RNA polymerase, and plasmid pMMBS64, expressing the epsG gene from the T7 ¢10 promoter, were labeled as indicated in the Materials and Methods. Total cell proteins were subjected to SDS-PAGE and autoradiography. Lanes: 1, V. cholerae TRH7000 [pMMB564 pMMB586]; 2, E. coli MC1061 [pMMB564 pMMB586]; 3, V. cholerae J71K-1 [pMMB564 pMMB586]. (B) Cells of the tcpJ :: Tn5 mutant, V. cholerae J71K-1, containing plasmids pMMB564 and pMMB586 were pulse-labeled and chased as described in the Materials and Methods. Lanes: 1, 0.5 min.; 2, 1 min.; 3, 2 min.; 4, 5 min.; 5, 10 min. after chase. 43 of V. cholerae resulted in a two-band pattern of EpsG similar to that in the WT V. cholerae strain (Figure 2.3). We have to conclude, therefore, that the V. cholerae genome encodes at least two prepilin peptidases. One, encoded by the tcpJ gene, functions in the processing of the prepilin TcpA protein, the main component of the TCP type IV pilus. The other, functions in the processing of the pilin-like Eps proteins required, presumably, for the assembly of the type II secretion apparatus. 44 Discussion CT is secreted via a two-step pathway that requires a specific set of genes, the eps genes, for translocation across the OM of V. cholerae. In this study, proteins produced in viva were correlated to putative ORFs identified by sequencing and analysis of a region of the V. cholerae chromosome done by Overbye (Overbye, 1994) and shown in Table 2.3. As mentioned previously, there are significant differences in expression levels of several of the proteins, possibly owing to transcriptional and/or translational regulation. An analysis of the coding sequences of the eps genes, demonstrated differences in the codon usage. Table 2.4 presents an analysis of the percentage of E. coli low-usage codons used in the V. cholerae eps genes. One striking observation made from this analysis was that the coding sequence Table 2.3 Location and characteristics of predicted Eps reading frames in the sequence of the epsC to epsN gene cluster. Protein 0an No. of Amino Predicted MW Acid Residues (kDa) EpsC 213-1 130 305 33,592 EpsD 1 173-3200 374 73,337 EpsEa 3197-4703 503 53,353 EpsFa 4703-5923 403 44,913 EpsG 5939-3409 143 13,033 EpsH 3443-7027 194 21,739 EpsIa 7017-7370 1 17 13,493 1:sza 7334-3022 210 23,757 EpsKa 3012-9022 333 37,599 EpsLa 3991-10202 403 45,343 EpsM 10209-10709 133 13,521 EpsNa 10711-11433 251 27,322 alnitiation codon of ORF overlaps termination codon of preceeding ORF or is separated by less than 2 nucleotides. IDNucleotide numbers given indicate the first nucleotide of the initiation codon and the last nucleotide of the termination codon of the ORF as supplied in record L33796 of the GenBank database. 45 <00 .50 .000 33: ”05 .5033: ”<94 EC ”00: .00: .:0< <8 59 ”000 .30 .00< .<0< 3:: ”000 .<00 39 ”835-22 85228 35; as 23 5553 58 3.. 22803 $8 5 82:32 >0 00:000. 009.: 0B 09003-32 0B “05 020000 36 090E028 0E 9:55.900 E 000: :00 .m E 0:008 0900726.? by mm we we mm mm cm mu ow mm nm em can. em 5 me mm 5 om 5 o .3 E or E 30.. om o o m P F m P .3 o o v m m E... be ow om mm mm mu mu 0 mm we no om 00m 2 t em 5 em om mm o 3 mm 2 E 9< om om em 8 mm mm 5 0 mm 3 0m 5 2.0 280 5.00.0 ._0Q0 x80 .00 _0Q0 100.0 600.0 “.80 M30 030 030 0 MMWJMWJ :00 030.:0comm E E 0200 oEEm 0080.00 Lo.— wconcoo :0 do 0903:0200 ed 030... 46 of epsG is the only eps gene that specifies only low-usage codons for 6 of the 8 prolines present in EpsG, and as seen in lanes 3, 4, and 5 of Figure 2.2, EpsG is one of the most abundant Eps proteins. Additional sequence analysis of the predicted amino acid sequence for EpsG, EpsH, Epsl, and Esz contains a hydrophobic region which resembles the signal sequence of Type IV pilin subunits. In at least three other GSP operons, a prepilin peptidase required for processing of the prepilin-like proteins could be located immediately downstream of the last gene in the GSP operon (Pugsley and Reyss, 1990; Lindeberg and Collmer, 1992; Pugsley and Dupuy, 1992; Reeves et al., 1993; Reeves etal., 1994). However, sequence analysis of regions upstream and downstream of the eps gene cluster identified no gene encoding a prepilin peptidase. The lack of an adjacent prepilin peptidase demonstrates the similiarity between the V. cholerae GSP system and those of Aeromonas and Pseudomonas, in which the prepilin peptidase is found elsewhere on the chromosome (Pepe et al., 1996; Filloux et al., 1998). However, in contrast to the GSP’s of Pseudomonas and Aeromonas, in which a single prepilin peptidase is required for processing the EpsG through Esz homologs and the type IV prepilin subunits (Nunn and Lory, 1991; Bally et al., 1992; Nunn and Lory, 1992; Nunn and Lory, 1993; Pepe et al., 1996), it was demonstrated that in V. cholerae a prepilin peptidase different from TcpJ is required to process EpsG. On the other hand the prepilin peptidase of Neisseria ganorrhaeae, PilD, was shown to process EpsG (Sandkvist et al., 1997), allowing the conclusion that EpsG is a substrate for a type IV prepilin peptidase and that it is likely that, 47 similar to the homolog PuIG (Pugsley, 1993), processing of EpsG is required for its function. The signal sequence of EpsG more closely resembles that of other type IV prepilin signal sequences than that of the V. cholerae TcpA prepilin signal sequence. The shorter signal sequence in EpsG and the presence of Phe instead at Met at position +1 at the predicted cleavage site, may be the reason that TcpJ is unable to process EpsG. Since TcpJ is not involved in the secretion of CT or the processing of EpsG, it is probable that another prepilin peptidase, active in the GSP and with a specificity similar to the prepilin peptidase of N. ganorrhaeae, exists in V. cholerae. The likelihood for another type IV prepilin peptidase became even more probable when the presence of a second type IV pilus, the mannose-sensitive haemagglutinin pilus in V. cholerae was realized (Jonson et al., 1994). Table 2.5 shows the differences between the TcpA signal peptide and those of the Eps prepilin-like proteins and MshA. After completion of this work, two groups (Marsh and Taylor, 1998; Fullner and Mekalanos, 1999), have since cloned the predicted second prepilin peptidase and demonstrated processing of another prepilin-like protein in V. cholerae, Epsl, and its requirement in the GSP. Mutants defective in the genes epsC, epsE, epsF, epsG, epsL, and epsM have been analyzed for their ability to secrete extracellular enzymes. Although eps mutants appear to be defective in a number of different functions, the defect in the extracellular secretion of toxin and protease is not simply a secondary result from a general defect in the OM, since the secretion of another protein, DNase, is not affected. In addition, these findings demonstrate that at least two 48 Table 2.5 Putative prepilin cleavage sites of the V. cholerae prepilin-like GSP proteins, EpsG, EpsH, Epsl, Esz, and EpsK, compared with the known cleavage sites of two V. cholerae type IV prepilin subunits, TcpA and MshA. Pilin Prepilin Peptidase Cleavage Site TcpA MQLLKQLFKKKFVKEEHDKKTGQEG MTLLEVI IVLGIMGVVSAGVVTLAQ EpsG MKKMRKQTG FTLLEVMVVVVILGILASFVVPNLL EpsH MTATRG FTLLE ILLVLVLVSASAVAVIATF P Eps I MALCVYWLREKVMKSKRG FTLLEVLVALAI FATAAI SVUR SVS Eps J MWRTNQVS SRQNMAG FTL I EVLVAIAI FASLSV . GAYQVL EpSK MRAKQRG VAL IVILLLLAVMVS IAATMAERLF MShA MVIMKRQGG FTL IELVVVIVILGI LAVTAAPRFL protease(s) use the GSP, the secretion of DNase occurs by a different, as yet unidentified secretion mechanism. V. cholerae is known to secrete two different DNases into the extracellular environment (Newland et al., 1985; Focareta and Manning, 1991). In this assay for DNase activity, it is not possible to distinguish whether one or both DNases are being detected. Nonetheless, these finding indicate that secretion of at least one of the DNases is unaffected by eps mutations in V. cholerae. 49 References Bally, M., A. Filloux, M. Akrim, G. Ball, A. Lazdunski and J. Tommassen (1992). Protein secretion in Pseudomonas aeruginosa: Characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol. Microbiol. 6: 1121-1131. Casadaban, M. C. (1980). Analysis of gene control signals by DNA fusion in Escherichia coli. J. Mol. Biol. 138: 179-207. Connell, T. D., D. J. Metzger, J. Lynch and J. P. Folster (1998). Endochitinase is transported to the extracellular milieu by the eps-encoded general secretory pathway of Vibrio cholerae. J. Bacterial. 180: 5591-5600. Danese, P. N. and T. J. Silhavy (1998). Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli. Annu Rev Genet 32: 59-94. Filloux, A., G. Michel and M. Bally (1998). GSP-dependent protein secretion in Gram-negative bacteria: the ch system of Pseudomonas aeruginosa. FEMS Microbial. Rev. 22: 177-198. Focareta, T. and P. A. Manning (1991). Distinguishing between the extracellular DNases of Vibrio cholerae and development of a transformation system. Mal. Microbial. 5: 2547-2555. Fullner, K. J. and J. J. Mekalanos (1999). Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae. Infect. Immun. 67: 1393-1404. Hirst, T. R. and J. Holmgren (1987a). Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae. Prac. Natl. Acad. Sci. USA 84: 7418-7422. Hirst, T. R. and J. Holmgren (1987b). Transient entry of enterotoxin subunits into the periplasm occurs during their secretion from Vibrio cholerae. J. Bacterial. 169: 1037-1045. Hirst, T. R., J. Sanchez, J. B. Kaper, S. J. S. Hardy and J. Holmgren (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: 7752-7756. Hofstra, H. and B. Witholt (1984). Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli. J. Biol. Chem. 259: 15182-15187. 50 Jonson, G., M. Lebens and J. Holmgren (1994). Cloning and sequencing of Vibrio cholerae mannose-sensitive haemagglutinin pilin gene: localization of mshA within a cluster of type 4 pilin genes. Mal. Microbial. 13: 109-118. Julius, D., A. Brake, L. Blair, R. Kunisawa and J. Thorner (1984). Isolation of the putative structural gene for the Iysine-arginine- cleaving endopeptidase required for processing of yeast prepro-alpha- factor. Cell 37: 1075-1089. Kaufman, M. R., J. M. Seyer and R. K. Taylor (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. Genes Dev 5: 1834- 1846. Lindeberg, M. and A. Collmer (1992). Analysis of eight out genes in a cluster required for pectic enzyme secretion by Envinia chrysanthemi: sequence comparison with secretion genes from other Gram-negative bacteria. J. Bacterial. 174: 7385-7397. Makrides, S. C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbial. Rev. 60: 512-538. Marsh, J. W. and R. K. Taylor (1998). Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation. Mal. Microbial. 29: 1481 -1492. Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press. Neill, R. J., B. E. Ivins and R. K. Holmes (1983). Synthesis and secretion of the plasmid-coded heat-labile enterotoxin of Escherichia coli in Vibrio cholerae. Science 221 : 289-291 . Newland, J. W., B. A. Green, J. Foulds and R. K. Holmes (1985). Cloning of extracellular DNase and construction of a DNase-negative strain of Vibrio cholerae. Infect. Immun. 47: 691 -696. Nunn, D. and S. Lory (1991). Product of the Pseudomonas aeruginosa gene piID is a prepilin leader peptidase. Prac. Natl. Acad. Sci. USA 88: 3281-3285. Nunn, D. and S. Lory (1993). Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins chT, -U, -V, and -W. J. Bacterial. 175: 4375-4382. Nunn, D. N. and S. Lory (1992). Components of the protein-excretion apparatus of Pseudomonas aeruginosa are processed by the type IV prepilin peptidase. Prac. Natl. Acad. Sci. USA 89: 47-51. 51 Overbye, L. J. (1994). Identification and characterization of the general secretion pathway genes in Vibrio cholerae. Ph.D. thesis. Michigan State University, East Lansing. Overbye, L. J., M. Sandkvist and M. Bagdasarian (1993). Genes required for extracellular secretion of enterotoxin are clustered in Vibrio cholerae. Gene 132: 101-106. Pepe, C. M., M. W. Eklund and M. S. Strom (1996). Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N- methyltransferase required for extracellular protein secretion. Mal. Microbial. 19: 857-869. Pugsley, A. P. (1993). Processing and methylation of PulG, pilin-like component of the general secretory pathway of Klebsiella oxytoca. Mal. Microbial. 9: 295- 308. Pugsley, A. P. and B. Dupuy (1992). An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca. Mal. Microbial. 6: 751-760. Pugsley, A. P. and I. Reyss (1990). Five genes at the 3’ end of the Klebsiella pneumoniae puIC operon are required for pullulanase secretion. Mal. Microbial. 4: 365-379. Reeves, P. J., P. Douglas and G. P. C. Salmond (1994). Beta-lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Enivinia carotovora general secretion pathway apparatus. Mal. Microbial. 12: 445-457. Reeves, P. J., D. Whitcombe, S. Wharam, M. Gibson, G. Allison, N. Bunce, R. Barallon, P. Douglas, V. Mulholland, S. Stevens, D. Walker and G. P. C. Salmond (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. Mal. Microbial. 8: 443-456. Sandkvist, M., L. 0. Michel, L. P. Hough, V. M. Morales, M. Bagdasarian, M. Koomey, V. J. DiRita and M. Bagdasarian (1997). General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae. J. Bacterial. 179: 6994-7003. Sandkvist, M., V. Morales and M. Bagdasarian (1993). A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae. Gene 123: 81 -86. 52 Sandkvist, M., L. J. Overbye, T. K. Sixma, W. G. J. Hol and M. Bagdasarian (1994). Assembly of Escherichia coli heat-labile enterotoxin and its secretion from Vibrio cholerae. Molecular Mechanisms of Bacterial Virulence. C. I. Kado and J. H. Crosa. Netherlands, Kluwer Academic Publishers: 293-309. Spangler, B. D. (1992). Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol. Rev. 56: 622-647. Tabor, S. and C. C. Richardson (1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Prac. Natl. Acad. Sci. USA 82: 1074-1078. Yu, J., H. Webb and T. R. Hirst (1992). A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae. Mal. Microbial. 6: 1949-1958. 53 CHAPTER 3 PURIFICATION AND CHARACTERIZATION OF EPS PROTEINS The results an the purification of EpsM(His)6, preparation of palyclanal antisera against EpsM(His)5, and the gel filtration analysis of EpsL and EpsM(His)6, presented in this chapter appeared as part of the publication: Sandkvist, M., Hough, L. P., M. M. Bagdasarian, and M. Bagdasarian. 1999. Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae. J. Bacterial. 181:3129-3135. The results from the sucrose gradient separation of cytoplasmic and outer membranes of Vibrio cholerae will be submitted to the Journal of Bacteriology as part of a publication with coauthors Maria Sandkvist and Michael Bagdasarian. 54 Abstract The general secretion pathway of Gram-negative bacteria is responsible for the extracellular secretion of several proteins including proteases and the cholera toxin. Proteins secreted through this pathway are secreted through a two step mechanism that is mediated with the assistance of at least 13 proteins. Four of these proteins have been purified and characterized in this study. Most of the 13 proteins are predicted to be membrane proteins, and the majority of those are predicted to be localized to the cytoplasmic membrane. Analysis of subcellular localization through selective detergent solubilization of these proteins and homologous proteins of the GSP in other Gram-negative bacteria corroborates this prediction. However, in this study it is demonstrated that several proteins predicted and determined to be localized to the cytoplasmic membrane exhibit a distribution to both membranes after sucrose gradient separation of the membranes. Additionally, it is shown that two proteins, EpsL and EpsM, are present as homodimers in Triton X-100 extracts of Escherichia coli membranes. 55 Introduction Gram-negative bacteria have evolved several independent mechanisms for the extracellular secretion of proteins (Binet etal., 1997; Hueck, 1998; Russel, 1998). One of these pathways, the type II secretion system or General Secretary Pathway (GSP), supports secretion of proteins across the cytoplasmic membrane (CM) and outer membrane (OM) of Gram-negative bacteria in two steps (Hirst and Holmgren, 1987b). Proteins are translocated across the CM via the sec machinery (Pugsley et al., 1991; Danese and Silhavy, 1998). Then after folding, and sometimes assembly into multimeric protein complexes, they cross the OM by a mechanism that requires the products of 14-16 genes depending on the species (Hirst etal., 1983; Hirst and Holmgren, 1987a; Pugsley, 1992; Russel, 1998). The genes encoding these 14-16 proteins have been designated by the letters A through O and S in most systems (for review, see Russel, 1998). Previous work with the type II secretion system in Vibrio cholerae identified and sequenced 12 ORFs from the chromosome that had homology with some of the 14 genes shown to be required for type II protein secretion in other Gram-negative bacteria. Genetic evidence confirmed the requirement of at least 6 of these ORFs for secretion of toxin and protease (Sandkvist et al., 1999). Eleven of the thirteen genes identified thus far in V. cholerae are predicted by various computer algorithms to contain at least one transmembrane helix, and to localize to the CM in Gram-negative bacteria (von Heijne, 1992; Rost, 1996; Sandkvist eta/., 1997; Marsh and Taylor, 1998; Nakai and Horton, 1999). However, only selective detergent solubilization of a few of these proteins has 56 been used for localization in V. cholerae (Sandkvist et al., 1995; Sandkvist eta/., 1999). In order to permit the biochemical dissection of the type II secretion apparatus it was necessary to clone, express, purity, and develop immunoreagents for the detection of some of the Eps proteins. In this study, the cloning, expression, purification, and the preparation of antisera, of EpsD, EpsF, EpsG, and EpsM is reported. Also reported is the selective solubilization of these proteins from the membranes of WT V. cholerae, the gel filtration determination of the apparent molecular mass of solubilized EpsL and EpsM, and the sucrose density gradient separation of the CM and OM fractions of V. cholerae. 57 Materials and Methods Bacterial Strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 3.1 and Table 3.2. Strains of Escherichia coli and V. cholerae were grown at 30°C or 37°C as indicated. LB medium, liquid or solidified with 1.5% Bacto-agar (Difco), supplemented with 100 ug/ml thymine was used for subculturing bacteria. M9 medium supplemented with 0.4% glucose, 100 ug/ml thymine, 10 ug/ml thiamine, and 20 ug/ml of 19 amino acids (all except methionine) was used for the radioactive labeling of proteins. When required the antibiotics ampicillin (Ap) and kanamycin (Km) were supplemented into both types of media at 100 ug/ml for the selection of plasmid-encoded resistance genes and at 50 ug/ml for chromosomal-encoded resistance genes. Recombinant DNA techniques and generation of constructs. Plasmid pMMB603 was constructed by from pMMB67EH. The Ptac was removed by BaI31 nuclease digestion and replaced with an Xhol linker. The resulting intermediate was digested with Xhol and Hindlll and the MCS of pQE70 was inserted as an Xhol/Hindlll fragment. The resulting broad host-range, Iow- copy number, expression vector allows Iaclq regulated expression from the bacteriophage T5 promoter with tandem lac operators between the promoter and a strong ribosome binding site (RBS). The vector includes a start codon and restriction sites for constructing a fusion with codons for 6 C-terminal His residues, a stop codon, and the rrn transcriptional terminator. 58 Table 3.1 Bacterial strains used in this study. Strain Relevant Characteristic(s) Source or Reference Vibrio cholerae TRH7000 El Tor thy HgR A(ctxA-cth) (Hirst et al., 1934) VB9 TRH7000 epsM::Tn5 PU3 in (Overbye et al., 1993; Sandkvist et al., 1997) VB37 TRH7000 epsG::KmR Mut5 in (Sandkvist et aL,1997) VB109 TRH7000 epstszR Mut6 in (Sandkvist et Escherichia coli DH1 0B MC1061 XL1-Blue MRF’ F' mcrA A(mrr-hstMS-mchC) 080dlacZAM15 AlacX74 endA1 recA1 deaR A(ara-Ieu)7697 araD139 gaIU gaIK nqu rpsL (SmR) i; F' araD139 A(ara-Ieu)7697 A(Iac)X74 rpsL hst2 mcrA mch1 A(mcrA)1 83 A(mchB-hsaSMR- mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F’ praAB IacquAM15 Tn10 (TcR) 59 al,1997) Life Technologies, Inc. (Casadaban, 1980) Stratagene Table 3.2 Plasmids used in this study. Plasmid Relevant Characteristic(s) Source or Reference PGP1-2 KmR T7 gene 1 under PM control, (Tabor and CF57 Richardson, 1985) pMMB67EH ApR Ptac, Iaclq, mab+ (Ftirste eta/., 1986) pMMB587 epsM(His)5 in pQE60 This study pMMB603 ApR, PTSIac/act Iaclq, Codons for 6 C- This study terminal His residues pMMB606 epsM(His)5 in pMMB603 This study pMMB672 epsG(His)6 in pQE70 This study pMMB674 epsG(His)6 in pMMB603 This study pMMB688 epsF(His)6 in pQE60 This study pMMB690 epsF(His)6 in pMMB603 This study pMMB706 ApR T7 010 promoter Codons for 6 This study C-terminal His residues pMMB710 epsD(His)5 in pMMB706 This study pMS44 epsL in pMMB67EH (Sandkvist et al., 1997) pQE60 ApR, FEW/ac, Codons for 6 C- Qiagen, Inc. terminal His residues pQE70 ApR, PTsIadac, Codons for 6 C- Qiagen, Inc. terminal His residues PT7-5 ApR T7 010 promoter (Tabor and 60 Richardson, 1985) Plasmid pMMB706 is a derivative of pT7-5 with the multiple cloning site (MCS) of pQE70 (Qiagen, |nc., Chatsworth, CA) cloned as an EcaRI/Hindlll fragment. This vector provides a RBS, a start codon, codons for 6 His residues, and stop codons in all three reading frames under T7 010 promoter control. Oligohistidine fusion proteins to the C-terminus of EpsD, EpsF, EpsG and EpsM were constructed by the PCR amplification of fragments of each gene with either Pwa DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IA), Pfu DNA polymerase (Stratagene, La Jolla, CA) or Taq DNA polymerase (Gibco, Rockville, MD) and oligonucleotide primers (Genosys Biotechnologies, The Woodlands, TX) as indicated in Table 3.3. Taq-amplified fragments were polished with T4 DNA polymerase. PCR amplified fragments were cloned into plasmids pQE60, pQE70, or pMMB706 as Sphl, Ncal, or blunt-ended Sphl, and Bglll to create C-terrninal oligohistidine fusions. Purification of EpsM. E. caIiTG1 carrying plasmid pMMB587 was grown in 2 L of LB supplemented with 100 ug/ml Ap at 37°C to an OD550 of 0.3. IPTG was added to a final concentration of 0.05 mM, and the culture was allowed to grow overnight at 37°C. Cells were harvested by centrifugation, suspended in 44 ml Lysis Buffer (50 mM sodium phosphate buffer [pH 8.0], 300 mM NaCI), and lysed by sonication in the presence of 1 mg/ml lysozyme. DNase I and MgCI2 were added to a final concentration of 10 U/ml and 10 mM, respectively, and incubated at RT for 10 min. The lysate was centrifuged for 45 min. at 30,000 rpm and 4°C in a 61 00003300 E0205 ._.>> 05. 00 03EEL0..-0 05 9 00000 0060 oEEm 0200_0E 0.00 E 003200. 200 0EE<0 0090:2300 <20 E. 2E2, 000003033 0202, 00:0 <20 005309 05 005 0200_0E 0005:0200 E 00E>Nc0 000030000 0.0 0300. E 0023 000 0.0:.00 00000632096 00 00000300m< 3030220 3230030 2003009 0000220 200 0E 02000 0 22000 2000 500.220 330000 200383 00000 200 9:0 :02: .0 02 2022,. 2000 000220 33.30002 2002200 0030220 200200 0E 02000 0 5.000 200.... 200220 3230009. 2003009 020220 200 003 0.000 0 2.000 2.80 0:03.33 3230022. 2003009 020220 200 0.2 0.000 0 :000 2000 2002.20 e3.30000 2003009 0000.220 200 0E 01000 0 £000 2000 0300220 3330000 2003009 0000 200 0E 0.0000 0 20000 0000 0000220 3230002 2002052 00000 2002.82 0E 00000 0 £000 0000 0 £02220 250000. 2003009 0030.220 .150 :20 00000 0 5000 0000 0:20 - . "£05 “30 29.020 cthfiEthmwu. 23m..__w_h0> ..200> 63020 000W“.%_00 00.55 00%.“.842 95.300: . . con. 6 0206.0 00m. _030_>_0E £3) 0E0§a 0202 00309. 0E000Eom=0 _0EEL000 00 0000320000 0.0 030... 62 Table 3.4 Sequences of oligonucleotides used in the construction of oligohisitidine fusion proteins with various Eps proteins. Oligonucleotide Oligonucleotide Sequence (5’-3’) Name EPSD1 AGTGAAATATTGGCTGAA EPSD2 CGCGGATCCTTGCTTGGGTTCCATCTG EPSF1 TAGCCATGGCCGCGT‘I‘I’GAATACAA EPSF2 CTAGATCTACTCA‘ITAAGTTATTCATTT EPSG1 TATGAAAAAAATGCGTAAACAAACGGG EPSG2 CTAGATC‘ITI'GAAAATC'I‘I'GGATATTCCAG'I'I'AC EPSHI TATGACAGCGACACGCGG'IT'I'I'AC EPS H2 CTAGATCTCTCTTCATCAC'I'I'TCTCCCGGAGC EPSI1 GATGAAGAGTAAACGCGGT‘ITI' EPSI2 CTAGATCTGTTCGCCACATAGCTACGCACC EPSJI TATGTGGCGAACTAACCAAGT EPSJ2 CTAGATCTGCCCGCATTTTCAACACTCT EPSK1 CAGCATGCGGGCTAAACAGCGCGG EPSK2 CTAGATCTCTCAGTCGAACGGTCAGAAA EPSN1 CATGAAGCGTGCTGTTGGCT EPSN2 GAAGATCTGAGCTGACCTTGTTGA'I'I'G MMB1 1 1 AAGAAGATCTGCCTCCACGCTTCAGTTGCAGACGT‘IT MMB1 12 AAAGAATTATTGGCTCCTGTGCAGGC'ITGG Beckman Type 35 rotor, and the resulting pellet was extracted twice with 50 ml Buffer M1 (50 mM sodium phosphate buffer [pH 8.0], 300 mM NaCI, 60 mM imidazole, 0.5% Triton X-100). Insoluble material was pelleted by centrifugation for 45 min. at 30,000 rpm and 4°C in a Beckman Type 35 rotor after each extraction. Both extractions were combined and centrifuged again for 45 min. at 30,000 rpm and 4°C in a Beckman Type 35 rotor before being applied to a 25 ml POROS 20 MC metal chelate column charged with NiSO4 and equilibrated with Buffer M1. After being washed with Buffer M1, proteins were eluted with a linear gradient of 60 to 700 mM imidazole in Buffer M1. EpsM(His)6 eluted at approximately 300 mM imidazole. Fractions containing EpsM(His)6, identified by Coomasie Brilliant Blue staining of 17% polyacrylamide SDS-PAGE gels, were 63 pooled, and dialyzed against Buffer M2 (Lysis Buffer supplemented with 1% Triton X-100). Purification of EpsG. E. coli DH10B carrying plasmid pMMB672 was inoculated into 1 L of LB supplemented with 100 ug/ml Ap and grown at 37°C to an OD650 of 0.3. IPTG was added to a final concentration of 0.1 mM, and the culture was grown at 37°C to an OD550 of 1.0. Cells were harvested by centrifugation, resuspended in 40 ml Lysis Buffer, and lysed by sonication in presence of 1 mg/ml lysozyme. DNAse I and MgClz were added to a final concentration of 10 U/ml and 10 mM, respectively, and incubated at RT for 15 min. The lysate was centrifuged for 20 min. at 35,000 rpm and 4°C in a Beckman Type 35 rotor, and the resulting pellet was extracted 3 times with 40 ml Buffer G1 (Lysis Buffer supplemented with 10 mM imidazole and 1% Triton X-100). Insoluble material was pelleted by centrifugation for 20 min. at 35,000 rpm and 4°C in a Beckman Type 35 rotor after each extraction. The three 40 ml extractions were combined and centrifuged again for 20 min. at 35,000 rpm and 4°C in a Beckman Type 35 rotor before being applied to a 5 ml POROS 20 MC metal chelate column precharged with NiSO4 and equilibrated with Buffer G1. After being washed with Buffer G1, proteins were eluted from the column with a linear gradient of 10 to 200 mM imidazole in Buffer G1. EpsG(His)6 eluted from the column in two broad peaks at approximately 10 and 100 mM imidazole. Fractions containing EpsG(His)6 were dialyzed against Buffer G2 (Lysis Buffer supplemented with 1% Triton X-100) at 4°C to remove imidazole. 64 A small scale purification of EpsG(His)6 was performed essentially as described above. Cultures of E. caIiXL1-Blue MRF’ [pMMB6974] and V. cholerae VB37 [pMMB674] were grown in LB supplemented with 100 ug/ml thymine and 100 ug/ml Ap at 37°C to an ODeso of 0.3. Expression of epsG(His)6 was induced with 100 uM IPTG, and the cultures were incubated at 37°C until and OD650 of 1.0 was reached. Cells from 50 ml of culture were harvested by centrifugation for 10 min. at 6,000 rpm and 4°C in a Beckman JA-20 rotor, and the cell pellet was resuspened in 1 ml Lysis Buffer. Cells were lysed by sonicatio, using 3 pulses of 10 s at 10 3 intervals in an ice bath. Membranes were pelleted by centrifugation for 15 min. at 35,000 rpm and 4°C in a Beckman 70.1 .Ti rotor. The membrane pellets were each resuspended in 1 ml Buffer G1, and centrifuged again for 15 min. @ 35,000 rpm and 4°C in a Beckman 70.1.Ti rotor. The membrane proteins solubilized in Buffer G1 was collected and 600 III was applied to a Ni-NTA Spin Column (Qiagen, Inc.), washed with 600 III Buffer G1, and eluted with 100 III of 300 mM imidazole in Buffer G1. Purification of EpsD. E. coli XL1-Blue MRF' carrying plasmid pGP1-2 and pMMB710 was grown in 2 L of LB supplemented with 100 ug/ml Ap and 100 ug/ml Km to an OD650 of 1.0 at 30°C. Expression of EpsD(His)5 from the T7 010 promoter was induced by incubating the culture at 42°C for 30 min., and then allowing the culture to continue to grow for 2 hours at 37°C. Cells were harvested by centrifugation, resuspended in 100 ml PBS. The cells were again pelleted and frozen at -20°C. 65 The cell pellet was thawed and resuspended in 40 ml Lysis Buffer (50 mM NaPO4 Buffer [pH 8.0], 300 mM NaCI). Resuspended cells were lyzed by sonication with four 15 s pulses at 50 MHz and 4°C at 15 5 intervals. The crude envelope fraction was pelleted by centrifugation for 30 min. at 35,000 rpm and 4°C in a Beckman Type 35 rotor. The pelleted membranes were resuspended in 40 ml Buffer D1 (Lysis Buffer supplemented with 10 mM imidazole and 1% Triton X-100) and incubated on ice for 30 min. to permit solubilization of CM proteins. Pellet insoluble material by centrifugation for 30 minat 35,000 rpm and 4°C. The supernatant was discarded and the pellet was resuspended in 40 ml Buffer D2 (Lysis Buffer supplemented with 5 mM imidazole and 0.5% SDS). Incubate for 30 min. @ 37°C. SDS insoluble material was removed by centrifugation for 30 min. at 35,000 rpm and 4°C in a Beckman Type 35. The supernatant containing EpsD(His)6 was harvested and loaded onto a 2 ml POROS 20 MC column, charged with NiSO4 and equilibrated with Buffer D2, at 0.5 ml/min. The column was washed extensively with Buffer D2, and EpsD(His)6 was eluted from the column in 1 ml fractions with a 40 ml linear gradient of 5 to 200 mM imidazole at 0.5 ml/min. Elution of EpsD(His)6 was monitored by measuring the Ongo and fractions containing EpsD(His)6 were identified by Coomassie Brilliant Blue R-250 staining of 10% polyacrylamide SDS-PAGE gels. Fractions containing EpsD(His)6 were pooled and dialyzed against Buffer D3 (Lysis Buffer supplemented with 0.5% SDS) to remove imidazole. 66 Purification of EpsF. E. coli XL1-Blue MRF’ carrying plasmid pMMB688 was grown in 4 L of LB supplemented 100 ug/ml of Ap to an ODGSO of 0.9 at 37°C. IPTG was added to a final concentration of 0.1 mM, and the culture was incubated for an additional 2 h at 37°C. Cells were harvested by centrifugation and resuspended in 100 ml PBS. The cells were again pelleted and frozen at -20°C. The cell pellet was thawed and resuspended in 40 ml 50 mM Lysis Buffer (50 mM NaPO4 Buffer [pH 8.0], 300 mM NaCI). Resuspended cells were lyzed by sonication with four 15 s pulses at 15 second intervals at 80 MHz. Membranes and insoluble material were pelleted by centrifugation for 30 min. at 35,000 rpm and 4°C in a Beckman Type 35 rotor. The supernatant was aspirated and the pellet was extracted with 40 ml Buffer F1 (Lysis Buffer + 1% Triton X-100) and incubated for 15 min. on ice. The Triton insoluble material was pelleted by centrifugation for 30 min. at 35,000 rpm and 4°C. The supernatant was aspirated and the pellet was extracted with 40 ml Buffer F2 (Lysis Buffer + 0.5% SDS). The SDS insoluble material was pelleted by centrifugation for 30 min. at 35,000 rpm and 4°C in a Beckman Type 35 rotor. The supernatant was harvested and applied to a 2 ml metal chelate column at 1 ml/min., POROS 20 MC (PerSeptive Biosystems, Framingham, MA), charged with NISO4 and equilibrated with Buffer F2. After being washed with Buffer F2, proteins were eluted with a 50 ml linear gradient of 0 to 50 mM imidazole in Buffer F2 at 1 ml/min.. EpsF(His)6 eluted at approximately 25 mM imidazole. The eluted protein was concentrated by centrifugation for 90 min. at 3,000 rpm and 4°C in a Beckman JA-20 rotor 67 through an Ultrafree-15 centrifugal ultrafiltration device with a Biomax-10 10,000 MWCO membrane (Millipore, Bedford, MA). Purified EpsF(His)5 was blotted onto ProBlott PVDF membrane (PE Applied Biosystems, Foster City, CA), and the N-terrninal amino acid sequence was determined by an automated Edman degradation procedure. Fifteen residues matched exactly the amino acid residues 2 through 16 predicted by the nucleotide sequence of the gene, as determined previously (Sandkvist et al., 1997). Antibody Production Polyclonal antisera was raised against EpsD, EpsF, EpsG, and EpsM in New Zealand White rabbits by Genosys Biotechnologies (The Woodlands, TX). For each protein, preimmune serum was collected from a minimum of 4 rabbits, and tested for detection of antigens in whole cell extracts of E. caIiXL1-Blue MRF’, or V. cholerae TRH7000 by western blotting at a 120,000 dilution of the preimmune serum. Two rabbits exhibiting the lowest responses to E. coli and V. cholerae antigens were selected for immunization with each protein. Both rabbits were immunized subcutaneously with 200 ug of the purified protein in Freund’s complete adjuvant. Booster immunizations with 100 ug of the purified protein in Freund’s incomplete adjuvant were administered subcutaneously at 14, 28, 42, 56, and 70 days after immunization. Production bleeds were collected at 49 and 63 days after immunization. Antibody production projects were ended and a final production bleed was collected at 77 days after immunization by exsanguination. 68 Serum collected 77 days after initial immunization was the source of polyclonal antibodies used in all immunological procedures. Gel Filtration Analysis of EpsL and EpsM Approximately 1.6 mg of EpsM(His)5 in 0.5 ml of 50 mM sodium phosphate buffer, 150 mM NaCl, and 30 mM n-octyI-B-D-glucopyranoside (OG) was loaded onto a Superdex 200 HR column 10 mm wide by 30 cm long at a flowrate of 0.25 ml/min. Fractions of 1 ml were collected. A. 0.5 ml of 1% Triton X-100 (in 50 mM sodium phosophate and 150 mM NaCI) extract of E. coli MC1061 [pMS44] expressing epsL without IPTG induction was applied to a 16 mm by 60 cm Sephacryl S-300 HR column (Amersham Pharmacia Biotech) at a flow rate of 0.5 ml/min. Fractions of 3 ml were collected. Elution of proteins for both separations were monitored by measurement of the Ongo. Fractions in which EpsM eluted were identified by SDS-PAGE separation of proteins on 17% polyacrylamide gels stained with Coomassie Brilliant Blue R-250. Fractions in which EpsL eluted were identified by SDS-PAGE and immunoblotting with anti- EpsL(His)6 antibodies. Reference proteins apoferritin (443 kDa), B-amylase (200 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (20 kDa), and myoglobin (17.6 kDa) were analyzed under the same conditions and used to estimate the molecular masses of EpsM(His)5 and EpsL. Extraction of EpsC, EpsD, EpsG, EpsL, and EpsM by Different Detergents V. cholerae TRH700 from an overnight culture grown in LB supplemented with 100 ug/ml thymine and 100 U/ml polymyxin B sulfate at 37°C was inoculated 69 into 200 ml of fresh LB supplemented with 100 ug/ml thymine and grown at 37°C to an OD650 of 1.0. Cells from 100 ml of this culture were pelleted and washed once with 10 ml PBS. The cell pellet was resuspended in 10 ml PBS and the cells were lysed by 4 pulses of 10 s at 10 s intervals in an ice bath with a Vibracell sonicator on a setting of 50 MHz. Aliquots of 1 ml of the lysate were centrifuged for 15 min. at 35,000 rpm and 4°C in a Beckman 70.1.Ti rotor to pellet the membranes and insoluble material. Each pellet was then mechanically resuspended in 1 ml of PBS containing 1% Triton X-100 and 10 mM MgCl2, 1% Triton X-100 and 10 mM EDTA, 4% octylpolyoxyethylene (BACHEM, King of Prussia, PA), 1% Thesit (Roche Molecular Biochemicals), 1% sucrose monolaurate (Roche Molecular Biochemicals), 1% Iauryldimethylamine oxide (LDAO) (Sigma), 1% Zwittergent 3-12 (Roche Molecular Biochemicals), or 1% SDS. Resuspended membranes were incubated for 15 min. on ice (except 1% SDS which was incubated at RT) to allow proteins to be solubilized. Each sample was then centrifuged for 15 min. at 35,000 rpm and 4°C (24°C for 1% SDS) in a Beckman 70.1.Ti rotor, and the supernatant containing proteins solubilized by the detergents being tested were harvested. The detergent insoluble material was then extracted again with PBS containing 1% SDS by mechanical resuspension of the pellet and incubation at RT for 15 min, followed by another centrifugation for 15 min. at 35,000 rpm and 24°C in a Beckman 70.1.Ti rotor. Proteins were separated by SDS-PAGE after acetone precipitation if necessary, transferred to nitrocellulose by semi-dry electroblotting, and immunodetected with antisera specific for EpsC, EpsD, EpsG, EpsL, or EpsM as 70 the primary antibody and horseradish peroxidase conjugated goat anti-rabbit lgG as the secondary antibody. Peroxidase activity was detected with SuperSignal chemiluminescent peroxidase substrate (Pierce Chemical Co., IL) and exposure to X-ray film. Proteins that were not detected in the first extraction, but detected in the subsequent extraction with SDS were considered not to be solubilized by the detergent used in the first extraction.. Proteins detectable in both extractions were considered to be partially solubilized by the detergent used in the first extraction. Proteins that were only detected in the first, and not in the subsequent extraction with 1% SDS were considered to be completely solubilized by the detergent used in the first extraction. Sucrose Gradient Separation of Cytoplasmic and Outer Membrane Fractions V. cholerae TRH7000 from a 50 ml overnight culture grown in LB supplemented with 100 ug/ml thymine and 100 U/ml polymyxin B sulfate at 37°C was inoculated into a 500 ml of fresh LB supplemented with 100 jig/ml thymine and incubated at 37°C to an OD650 of 1.0. Four hundred milliliters of this culture was pelleted by centrifugation for 10 min. at 6,000 rpm and 4°C in a Beckman JA-20 rotor. The pellet was washed once with 50 ml ice-cold PBS (10 mM NagHPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCI, pH 7.4), and resuspended in 20 ml PBS supplemented with 200 III 10 mg/ml PMSF (dissolved in 95% ethanol). The cells were lysed by two passes through an ice-cold French Pressure Cell at 1,000 psi. DNase I (Roche Molecular Biochemicals) was added to a final concentration of 20 U/ml and incubated for 15 min. at RT. Unbroken cells were pelleted by centrifugation for 10 min. at 6,000 rpm and 4°C in a 71 Beckman JA-20 rotor, and the supernatant was harvested. Envelopes from 10 ml of the crude lysate were partially purified by sedimentation onto a 1 ml 65% sucrose (wt/vol. in 50 mM sodium phosphate buffer [pH 7.4]) cushion overlaid with 1 ml of 20% sucrose by centrifugation for 1 hr at 36,000 rpm and 4°C in a Beckman SW41 rotor. The envelopes were harvested in a minimum volume and resuspended in 10 ml. of PBS. One milliliter of the partially purified envelopes was applied to the top of a sucrose step gradient formed with 0.55 ml 65% and 0.95 ml ea. of 60%, 58%, 56%, 54%, 52%, 50%, 48%, 46%, 44%, 42%, and 40% sucrose (w/v in 50 mM sodium phosphate buffer [pH 7.4]). Membranes were separated by centrifugation for 36 hr at 36,000 rpm and 4°C in a Beckman SW41 rotor. Successive 0.5 ml fractions were collected from the bottom of the gradient and were analyzed. Ten percent of each fraction was used to determine the activity of B-NADH oxidase by the method of Osborne et. al. (1972). For determination of the distribution of LPS across the gradient, 0.15 ml of each fraction was brought to a volume of 1 ml in 10% tricholoracetic acid. After incubation for 15 min. on ice, acid-precipitated material was collected by centrifugation for 15 min. at 16,000 rpm and 4°C in a Beckman JA-20 rotor, and washed with one 1 ml volume of distilled water without resuspension to remove residual sucrose. The amount of LPS in the samples was determined essentially as described by Lee and Tsai (1999). Acid-precipitated material was resuspended in 200 III water and 100 pl 64 mM NaIO4 was added. After incubation for 20 min. at RT, 200 pl 136 mM Purpald (Sigma-Aldrich, St. Louis, MO) in 2N NaOH was added. After further incubation for 20 min. at RT, 200 pl 72 64 mM NaIO4 was added and incubated for another 20 min. at RT. Eighty microliters of isopropanol was added to eliminate the resulting foam, and the absorbance of each sample was recorded and plotted against a standard curve constructed with V. cholerae 569B LPS (Sigma). Fractions were then assayed for the distribution of EpsC, EpsD, EpsG, EpsL, and EpsM by western blotting. Resufls Purification of EpsM(His)5 The V. cholerae gene epsM was efficiently cloned into the expression vector pQE70, to create a fusion protein with a C-terminal oligohistidine tag. The resulting construct, pMMB587, was transformed into E. coli TG1 and expression of the recombinant fusion protein was induced with IPTG. Large amounts of the recombinant fusion protein were produced and were then solubilized from the membrane fraction of the cell with the detergents Triton X-100 and octyl-B-D- glucopyranoside (OG). Since Triton X-100 is known to solublize primarily CM proteins (Schnaitman, 1971b), this suggests that the EpsM(His)5 fusion protein localizes to the CM in E. coli. EpsM(His)5 was purified from both 0.5% Triton X- 100 and 30 mM OG extracts of E. coli membranes as described in the materials and methods. EpsM(His)6 was found to elute from the column at approximately 300 mM imidazole, which suggests that EpsM(His)6 may be a dimer or multimer (Qiagen, 1997), under these conditions (Figure 3.1) To determine whether the C-terminal oligohistidine tag affected the function of EpsM in viva, the recombinant gene was cloned into the broad-host range, low copy number vector, pMMB603, resulting in construction pMMB606. 73 Fraction Number I I 14 16 18 20 22 24 26 28 30 32 34 36 kDa 43— , 3' mm Extract 29 — 18.4 — E 14.3 — 6.2 — Figure 3.1 Purification of EpsM(His)6. SDS-PAGE analysis of 10 III samples of protein from the 1% Triton X-100 extract of E. col/TG1 [pMMBS87], and each fraction after purification by metal chelate affinity chromatography as described in the materials and methods. The arrow indicates the position of EpsM(His)6. This plasmid was transferred to V. cholerae VB9, a derivative of TRH7000 in which Tn5 had inserted into the epsM gene (Overbye eta/., 1993; Sandkvist et al., 1997), and the strain was tested for restoration of protease secretion on Luria agar (LA) plates supplemented with 1% skim milk. Halos formed around colonies carrying either the WT epsM gene or the recombinant oligohistidine tagged epsM, indicating that the C-terminal fusion did not significantly alter or affect the function of EpsM in vivo. To verify the identity of the purified protein as EpsM the first 15 N-terminal amino acids sequenced by the Edman degradation. The sequence of the first 15 amino acids matched exactly the predicted sequence of EpsM from the nucleotide sequence determined previously (Overbye et al., 1993; Sandkvist et al., 1997). However, there are two tandem Met codons at the beginning of epsM. Since the putative ribosome binding site (UAAGGAG) is only separated from the 74 first Met codon by 2 nucleotides, the second Met codon is predicted to be the start codon for epsM. Thus, it is still unknown which Met codon is the start codon of WT EpsM is, but since the recombinant oligohistidine fusion protein complements the epsM::Tn5 mutation in V. cholerae VB9, two N-terrninal Met residues are not essential for proper EpsM function in viva. Purification of EpsG The V. cholerae epsG gene was also amplified and cloned into pQE70. The resulting construct, pMMB671, was transformed in E. coli XL1-Blue MRF’ and induced with IPTG to express a recombinant fusion protein with a C-terminal polyhisitidine tag. In contrast to the Klebsiella oxytoca EpsG homolog, PulG (Pugsley and Possot, 1993), EpsG(His)6 was found to be completely extracted from the membranes of E. coli in solutions containing 0.5% Triton X-100 , suggesting a CM location for the recombinant protein. Purification of EpsG was performed as described in the materials and methods. EpsG(His)6 eluted from the column across the range of imidazole concentrations from 10 to 200 mM, with a minor peak at ~30 mM and a major peak at approximately 100 mM imidazole (Figure 3.2). This elution pattern suggests that at least some fraction of EpsG(His)5 molecules may be monomeric under these conditions. Since it was known at this time that PreEpsG was processed by a signal peptidase in V. cholerae (see chapter 2 and Sandkvist etal., 1997), it was decided to see if the C-terminal oligohistidine-tagged protein could complement an epsG defect in V. cholerae, to see if the recombinant EpsG(His)6 was processed in V. cholerae, and to see if processing of EpsG resulted in any 75 Fraction Number -1 3‘ 5 -7 ’9 11.13 15 17 18 19 20 21 22 ZBI '1 Flowthrough Figure 3.2 Purification of EpsG(His)6. SDS-PAGE analysis of 10 ul samples of protein from the 1% Triton X-100 extract of E. co/iXL1-MRF’ [pMM8670] and each fraction after purification by metal chelate affinity chromatography as described in the materials and methods. The arrow indicates the position of EpsG(His)6. difference in the detergent solubilization of EpsG(His)5. The recombinant gene for EpsG(His)6 was subcloned into the broad-host range, low-copy number expression vector pMMB603, resulting in construction pMMB674, and transferred by conjugation to V. cholerae VB37. V. cholerae VB37 is an epsG mutant of V. cholerae TRH7000 constructed by inserting a gene for KmR into epsG in vitro and introducing it into the chromosome of V. cholerae by homologous recombination (Sandkvist et al., 1997). The presence of EpsG(His)6 in VB37 restored secretion of protease evidenced by the formation of halos around colonies growing on LA + 1% skim milk (results not shown). When expression of EpsG(His)6 was Induced by the addition of IPTG in V. cholerae VB37, EpsG(His)6 could be purified from both Triton X-100 and CG extracts of the membrane fraction. When the purified EpsG(His)6 was analyzed 76 0 E Q .Q 0 g E kD LLI >' 34 ”T“ 52 mg 39 26 " W 21 7 7 _ ,. PreEpsG(Hi56) 15 'm - EpsG(Hlss) 9 Figure 3.3 Comparison of precusor EpsG(His)6 purified from E. coli DH10B [pMMBS74] and processed EpsG(His)6 purified from V. cholerae VB37 [pMMBS74]. A small scale purification was performed as described in the materials and methods. by SDS-PAGE on an 18% polyacrylamide gel and compared with the EpsG(His)6 purified from E. call, it could be clearly seen that the recombinant fusion protein was processed in V. cholerae (Figure 3.3). The bulk of EpsG was present in the Triton X-100 extract. These results indicate that the majority of processed EpsG remains Triton X-100 soluble, suggesting that processing does not affect subcellular localization. Purification of EpsD The gene for EpsD was amplified by PCR. The PCR amplified fragment was eventually cloned into the expression vector pMMB706. The expression vector pMMB706 is a derivative of pT7-5 in which the MCS was replaced with the RBS, MCS, C-terminal oligohistidine tag, and stop codon of pQE70. The resulting construction, pMMB710, could be maintained in E. coli hosts carrying the T7 77 Fraction Number Extract Flowthrough I 13 5 7 911131517192123252729 Figure 3.4 Purification of EpsD(His)6. SDS-PAGE analysis of 10 ul samples of protein from the 0.5% SDS extract of E. coli MC1061 [pGP1-2 pMMB710] and each fraction after purification by metal chelate affinity chromatography as described in the materials and methods. The arrow indicates the position of EpsD(His)5. RNA polymerase under stringent control, such as MC1061 [pGP1-2]. The clone of epsD could not be maintained in strains such as BL21 (ADE3) without also including the plasmid pLysS to lower the basal level of expression. EpsD(His)6 expressed in E. coli MC1061 was produced as an insoluble protein which was not solubilized in PBS containing 1% Triton X-100 or 1% Triton X-100 supplemented with 10 mM EDTA. EpsD(His)6 was however efficiently extracted from the membrane (insoluble) fraction of E. coli with 0.5% SDS. EpsD(His)6 was purified from a 0.5% SDS extract of E. coli membranes as described in the materials and methods (Figure 3.4). EpsD(His)6 eluted from the column at less than 50 mM imidazole. 78 Fraction Number Flowthrough 4—0 U N h ‘2 LL] I 3 5 7 911131517192123252729 Figure 3.5 Purification of EpsF(His)6. SDS-PAGE analysis of 10 III samples of protein from the 0.5% SDS extract of E. co/iXL1-Blue MRF’ [pMM8688] and each fraction after purification by metal chelate affinity chromatography as described in the materials and methods. The arrow indicates the position of EpsF(His)6. Purification of EpsF(His)5 The epsF gene was amplified and cloned into pQE70 and transformed into E. co/iXL1-Blue MRF’. Expression of EpsF(His)6 was induced with IPTG and was found in the insoluble fraction of the cell. Although, similar to EpsD(His)5, EpsF(His)6 was not extractable in 1% Triton X-100 or 1% Triton X-100 supplemented with 10 mM EDTA, but could be solubilized by 0.5% SDS. EpsF(His)5 was purified from a 0.5% SDS extract of E. coli insoluble protein by metal chelate affinity chromatography as described in the materials and methods (Figure 3.5). Similar to the purification of EpsD(His)6 but not to quite the same extent, some amount of EpsF(His)e eluted from the column at the beginning of the gradient. This elution pattern is attributed to the use of the anionic detergent 79 SDS in the purification, which is known to leach Ni+2 from the column (Qiagen, 1 997). EpsM(His)6 and EpsL farm dimers. In gel filtration experiments purified EpsM(His)6 eluted in a single peak, and its elution was compared with that of known molecular mass markers. The results indicated that EpsM(His)6 was present in solution as a 35 kDa protein (Figure 3.6A). This is in good agreement with the expected size for a dimer of the 18.5 kDa molecule predicted from the translation of the epsM gene sequence (Sandkvist et al., 1997). The molecular size of EpsL in solution could not be determined by analysis of purified material, since EpsL has only been purified under denaturing conditions (Sandkvist et al., 1999). Therefore, gel filtration analysis was performed on EpsL extracted from the membrane of E. coli expressing the epsL gene from a low-copy number vector. Figure 3.6B shows the result from a gel filtration through Sephacryl S-300 in which the molecular mass of EpsL was 91 kDa, which is in good agreement for the expected size of an EpsL dimer of the 45.4 kDa monomer predicted from the nucleotide sequence of epsL (Sandkvist et al., 1997). These results suggest that EpsL and EpsM both form dimers when produced in E. coli in the absence of other Eps proteins. Detergent Solubility of Eps Proteins from V. cholerae TRH7000. Since the majority of the proteins that comprise the type II secretory apparatus are located in either the CM or the OM, the ability of various detergents and conditions to solublize these proteins was tested. The insoluble fraction of sanicated V. cholerae TRH7000 cells was resuspended in a 1% 80 Figure 3.6 Size determination of EpsM(His)5 and EpsL. Fractionation of purified EpsM(His)6 and EpsL present in a Triton X-100 extract of membranes from an E. coli expressing epsL was performed on a Sephacryl S-300 HR column (A) and a Superdex 200 HR column (B), respectively. Fractions of 1.0 ml (A) or 3.0 ml (B) were analyzed by ODzeo as well as SDS-PAGE and Coomassie brilliant blue staining or immunoblotting with anti-EpsL(His)5 antibodies. Elution of EpsM(His)5 and EpsL was compared with the elution of standard proteins of known molecular mass. The volume of eluent at which the individual standard proteins emerged from the column was plotted against the logarithm of their molecular mass and fit by linear regression. The elution peak of EpsM(His)5 and EpsL is indicated by a triangle, and the apparent molecular mass for these proteins was calculated from this data. 81 1000.____ H _ . ,3"- _.___--_____ MW (kDa) T _TTinalbumin 45 T . I l _ E.;—.—___ _., E “1 _.._, 202i i—h—..__ 40 50 60 70 Elution Volume (ml) 10 100 *Um;:‘ :2 ;: I::‘ ‘j:—”‘i: UBSA6‘7‘ 7 7* I 7 I‘— — I , - —- OvatbumCIImdAEpsMfiS I ~ -- ~ Carbonic anhydrase 29 MW (kDa) I L Myoglobin 17.6 I . i l 25 30 35 40 45 50 Elution Volume (ml) 10 82 solution of each of the detergents and allowed to sit for 15 min.at RT. The results presented in Table 3.5 indicate whether the protein was completely, partially, or not extracted in relation to amount of protein that could be detected in a subsequent extraction with 1% SDS. As expected EpsD, putatively an OM protein, was not extracted by 1% Triton X-100 supplemented with 10 mM MgCl2, but was efficiently extracted from the membranes of V. cholerae by 1% Triton X- 100 supplemented with10 mM EDTA. Also as expected, the proteins EpsC, EpsG, EpsL, and EpsM, predicted to be CM protein were all extracted in 1% Triton X-100 supplemented with 10 mM MgCl2 suggesting that these proteins are indeed CM proteins. However, the extraction of EpsG in 1% Triton X-100 with 10 mM MgClz is in contrast with the solubilization of the K. oxytoca PulG, an EpsG homolog, which was only partially released from the membrane by 1% Triton X- 100 and 5 mM EDTA (Pugsley and Possot, 1993). Also interesting is the lack of solubilization of EpsD by the detergent octylpolyoxyethylene, which effectively Table 3.5 Detergent solubility of EpsC, EpsD, EpsG, EpsL, and EpsM from the membranes of V. cholerae TRH7000. Legend: (++) protein is completely extracted by the detergent, (+) protein is partially extracted by the detergent, (-) protein is not extracted by indicated detergent. Detergent EpsC EpsD EpsG EpsL EpsM Nan-Ionic Detergents 4% Octylpolyoxyethylene ++ - ++ ++ ? 1% Triton X-100/10 mM MgCl2 ++ - ++ ++ H 1% Triton X-100/10 mM EDTA ++ ++ ++ ++ ++ 1% Thesit + — + + H. 1% Sucrose Monolaurate ++ ? ++ ++ ++ Z witterianic Detergents 1% Lauryl Dimethylamine Oxide ++ ++ ++ ++ ++ 1% Zwittergent 3-12 ++ ++ + ++ ++ Ionic Detergents 1% SDS I ++ ++ ++ ++ ++ 83 solubilized the EpsD homolog,the M13 bacteriophage pIV from the OM of E. coli (Linderoth et al., 1997). Density Gradient Separation of V. cholerae Membranes. In order to confirm the specificity of selective detergent solubilization of CM and OM proteins in V. cholerae the membranes were separated by density gradient centrifugation through a sucrose step gradient. The results presented in Figure 3.7 shows the sedimentation profile of EpsC, EpsD, EpsG, EpsL, and EpsM. The results demonstrate that EpsD is firmly associated with the OM, and that it appears to fractionate with the more dense fragments of the OM since the peak for EpsD was lower in the gradient than the peak for LPS and protein. EpsG demonstrate a nearly homogeneous distribution between the two membranes, which was also seen in the distribution of the Pseudomonas aeruginosa G homolog (Nunn and Lory, 1993). EpsC and EpsM both demonstrate a bimodal distribution between the two membranes with the majority of each protein localized to the CM. A similar distribution of the K. oxytoca homolog of EpsC, PulC was recently demonstrated (Possot et al., 1999). However, there has been no report of such a distribution for EpsM or any of its homologs. Finally, the results show that EpsL is primarily located in the CM but some traces of EpsL can be detected fractionating with the OM. This could be contamination of the OM fractions with fragments of the CM, or could be an indication of a weak association with the OM, EpsD, or another OM component of the secretion apparatus. Taken together these results suggest that there is an 84 association between the CM components, either directly or indirectly, and the OM component(s) of the secretion apparatus. 85 Figure 3.7 Sucrose density gradient separation of cytoplasmic and outer membrane proteins of V. cholerae. Legend (filled circles) % sucrose determined by refractive index; (open squares) Ongo; (open triangles) % NADH Oxidase activity; (open circles) LPS determined by Purpald Assay as described in the materials and methods. Samples from fractions 5 to 20 were precipitated with TCA separated by SDS-Page and immunoblotted with anti-sera specfic for the protein indicated. The blots correlate with the fractions represented in the graph. 86 QSBPIXO HGVN JO SdW % 2000 3000 0000 0000 0000 4 . .IIIIIEM" AIIIIE'I'I‘EI I... I. I" t. .I .40 - 5"!»I A LOQEDZ Cozumel or m I I l I I I I I I | I I I I 1' I I I l I I I < v