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• W1965902126 abstract "Vibrio cholerae colonize the small intestine where they secrete cholera toxin, an ADP-ribosylating enzyme that is responsible for the voluminous diarrhea characteristic of cholera disease. The genes encoding cholera toxin are located on the genome of the filamentous bacteriophage, CTXφ, that integrates as a prophage into the V. cholerae chromosome. CTXφ infection of V. cholerae requires the toxin-coregulated pilus and the periplasmic protein TolA. This infection process parallels that of Escherichia coli infection by the Ff family of filamentous coliphage. Here we demonstrate a direct interaction between the N-terminal domain of the CTXφ minor coat protein pIII (pIII-N1) and the C-terminal domain of TolA (TolA-C) and present x-ray crystal structures of pIII-N1 alone and in complex with TolA-C. The structures of CTXφ pIII-N1 and V. cholerae TolA-C are similar to coliphage pIII-N1 and E. coli TolA-C, respectively, yet these proteins bind via a distinct interface that in E. coli TolA corresponds to a colicin binding site. Our data suggest that the TolA binding site on pIII-N1 of CTXφ is accessible in the native pIII protein. This contrasts with the Ff family phage, where the TolA binding site on pIII is blocked and requires a pilus-induced unfolding event to become exposed. We propose that CTXφ pIII accesses the periplasmic TolA through retraction of toxin-coregulated pilus, which brings the phage through the outer membrane pilus secretin channel. These data help to explain the process by which CTXφ converts a harmless marine microbe into a deadly human pathogen. Vibrio cholerae colonize the small intestine where they secrete cholera toxin, an ADP-ribosylating enzyme that is responsible for the voluminous diarrhea characteristic of cholera disease. The genes encoding cholera toxin are located on the genome of the filamentous bacteriophage, CTXφ, that integrates as a prophage into the V. cholerae chromosome. CTXφ infection of V. cholerae requires the toxin-coregulated pilus and the periplasmic protein TolA. This infection process parallels that of Escherichia coli infection by the Ff family of filamentous coliphage. Here we demonstrate a direct interaction between the N-terminal domain of the CTXφ minor coat protein pIII (pIII-N1) and the C-terminal domain of TolA (TolA-C) and present x-ray crystal structures of pIII-N1 alone and in complex with TolA-C. The structures of CTXφ pIII-N1 and V. cholerae TolA-C are similar to coliphage pIII-N1 and E. coli TolA-C, respectively, yet these proteins bind via a distinct interface that in E. coli TolA corresponds to a colicin binding site. Our data suggest that the TolA binding site on pIII-N1 of CTXφ is accessible in the native pIII protein. This contrasts with the Ff family phage, where the TolA binding site on pIII is blocked and requires a pilus-induced unfolding event to become exposed. We propose that CTXφ pIII accesses the periplasmic TolA through retraction of toxin-coregulated pilus, which brings the phage through the outer membrane pilus secretin channel. These data help to explain the process by which CTXφ converts a harmless marine microbe into a deadly human pathogen. Vibrio cholerae are Gram-negative, rod shaped bacteria that cause the gastrointestinal disease cholera. There are >200 known V. cholerae serogroups, yet only two, O1 and O139, cause pandemic disease. Pathogenic serogroups are distinguished from non-pathogenic strains by the acquisition of two mobile genetic elements: the Vibrio pathogenicity island (1Karaolis D.K. Johnson J.A. Bailey C.C. Boedeker E.C. Kaper J.B. Reeves P.R. A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 3134-3139Crossref PubMed Scopus (375) Google Scholar) and the CTX element, which is a prophage of the filamentous bacteriophage CTXφ (2Pearson G.D. Woods A. Chiang S.L. Mekalanos J.J. CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 3750-3754Crossref PubMed Scopus (184) Google Scholar, 3Waldor M.K. Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin.Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1345) Google Scholar). The Vibrio pathogenicity island contains the tcp operon encoding the toxin-coregulated pilus (TCP), 4The abbreviations used are: TCPtoxin-coregulated pilusTEMtransmission electron microscopyLBLuria-BertaniSmstreptomycinKmkanamycinApampicillinTctetracyclinePBSphosphate buffered salineBSAbovine serum albuminSeMetselenomethionineBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolNi-NTAnickel-nitrilotriacetic acid. which is necessary for V. cholerae colonization of the human intestine (4Herrington D.A. Hall R.H. Losonsky G. Mekalanos J.J. Taylor R.K. Levine M.M. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans.J. Exp. Med. 1988; 168: 1487-1492Crossref PubMed Scopus (530) Google Scholar, 5Taylor R.K. Miller V.L. Furlong D.B. Mekalanos J.J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 2833-2837Crossref PubMed Scopus (766) Google Scholar), and TcpF, a soluble colonization factor of unknown function (6Kirn T.J. Bose N. Taylor R.K. Secretion of a soluble colonization factor by the TCP type 4 pilus biogenesis pathway in Vibrio cholerae.Mol. Microbiol. 2003; 49: 81-92Crossref PubMed Scopus (95) Google Scholar, 7Megli C.J. Yuen A.S. Kolappan S. Richardson M.R. Dharmasena M.N. Krebs S.J. Taylor R.K. Craig L. Crystal structure of the Vibrio cholerae colonization factor TcpF and identification of a functional immunogenic site.J. Mol. Biol. 2011; 409: 146-158Crossref PubMed Scopus (14) Google Scholar). The CTX element contains repetitive sequences involved in site-specific recombination of the CTXφ genome and a core region encoding the phage assembly proteins and the cholera toxin subunits A and B. Expression of tcp and the cholera toxin genes ctxA and ctxB are regulated by the same transcriptional activator, ToxT (8DiRita V.J. Parsot C. Jander G. Mekalanos J.J. Regulatory cascade controls virulence in Vibrio cholerae.Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 5403-5407Crossref PubMed Scopus (342) Google Scholar, 9Skorupski K. Taylor R.K. Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli.Mol. Microbiol. 1997; 25: 1003-1009Crossref PubMed Scopus (199) Google Scholar). toxin-coregulated pilus transmission electron microscopy Luria-Bertani streptomycin kanamycin ampicillin tetracycline phosphate buffered saline bovine serum albumin selenomethionine 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol nickel-nitrilotriacetic acid. CTXφ has a 7-kb single-stranded circular DNA genome that integrates into the V. cholerae chromosome as a prophage. Phage particles are produced from extrachromosomal DNA replicated as a plasmid (10Davis B.M. Moyer K.E. Boyd E.F. Waldor M.K. CTX prophages in classical biotype Vibrio cholerae. Functional phage genes but dysfunctional phage genomes.J. Bacteriol. 2000; 182: 6992-6998Crossref PubMed Scopus (107) Google Scholar). The CTXΦ genome is similar in gene size and organization to Ff family filamentous phage that infect Escherichia coli (coliphage), including M13 and fd (3Waldor M.K. Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin.Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1345) Google Scholar). The core region of the CTXφ prophage contains genes cep, orfU, ace, zot, ctxA, and ctxB. Although not homologous in sequence, the cep, orfU, ace, and zot genes are similar in size and synteny to the M13 genes VIII, III, VI, and I, which encode the structural proteins pVIII, pIII, pVI, and pI, respectively (3Waldor M.K. Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin.Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1345) Google Scholar, 11Heilpern A.J. Waldor M.K. CTXφ infection of Vibrio cholerae requires the tolQRA gene products.J. Bacteriol. 2000; 182: 1739-1747Crossref PubMed Scopus (88) Google Scholar). In M13, the major coat protein pVIII forms the long, cylindrical phage coat that packages the phage genome. pIII, pVI, and pI are minor coat proteins located at the phage tips (12Grant R.A. Lin T.C. Konigsberg W. Webster R.E. Structure of the filamentous bacteriophage fl. Location of the A, C, and D minor coat proteins.J. Biol. Chem. 1981; 256: 539-546Abstract Full Text PDF PubMed Google Scholar, 13Lopez J. Webster R.E. Minor coat protein composition and location of the A protein in bacteriophage f1 spheroids and I-forms.J. Virol. 1982; 42: 1099-1107Crossref PubMed Google Scholar, 14Wen Z.Q. Overman S.A. Thomas Jr., G.J. Structure and interactions of the single-stranded DNA genome of filamentous virus fd. Investigation by ultraviolet resonance raman spectroscopy.Biochemistry. 1997; 36: 7810-7820Crossref PubMed Scopus (73) Google Scholar). Although pVI and pI are very small, pIII is a large protein, present in four-five copies at one end of the phage particle. pIII (also called g3p for gene 3 protein) mediates phage binding, uptake, and assembly (15Crissman J.W. Smith G.P. Gene-III protein of filamentous phages. Evidence for a carboxyl-terminal domain with a role in morphogenesis.Virology. 1984; 132: 445-455Crossref PubMed Scopus (72) Google Scholar, 16Deng L.W. Malik P. Perham R.N. Interaction of the globular domains of pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli.Virology. 1999; 253: 271-277Crossref PubMed Scopus (46) Google Scholar, 17Rakonjac J. Model P. Roles of pIII in filamentous phage assembly.J. Mol. Biol. 1998; 282: 25-41Crossref PubMed Scopus (51) Google Scholar, 18Riechmann L. Holliger P. The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.Cell. 1997; 90: 351-360Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 19Stengele I. Bross P. Garcés X. Giray J. Rasched I. Dissection of functional domains in phage fd adsorption protein. Discrimination between attachment and penetration sites.J. Mol. Biol. 1990; 212: 143-149Crossref PubMed Scopus (99) Google Scholar). pIII is well characterized in Ff phage, in part because of its application in phage display technology (20Scott J.K. Craig L. Random peptide libraries.Curr. Opin. Biotechnol. 1994; 5: 40-48Crossref PubMed Scopus (87) Google Scholar, 21Rakonjac J. Bennett N.J. Spagnuolo J. Gagic D. Russel M. Filamentous Bacteriophage. Biology, phage display, and nanotechnology applications.Curr. Issues Mol. Biol. 2011; 13: 51-76PubMed Google Scholar). fd and M13 pIII amino acid sequences are almost identical, with an 18-amino acid signal peptide and a 406-amino acid mature protein organized into three distinct functional domains, N1, N2, and C (also called D1, D2, and D3 or CT), linked by glycine-rich segments of low structural complexity, LCR1 and LCR2 (19Stengele I. Bross P. Garcés X. Giray J. Rasched I. Dissection of functional domains in phage fd adsorption protein. Discrimination between attachment and penetration sites.J. Mol. Biol. 1990; 212: 143-149Crossref PubMed Scopus (99) Google Scholar, 22Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p.Nat. Struct. Biol. 1998; 5: 140-147Crossref PubMed Scopus (98) Google Scholar, 23Holliger P. Riechmann L. Williams R.L. Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 Å. Evidence for conformational lability.J. Mol. Biol. 1999; 288: 649-657Crossref PubMed Scopus (86) Google Scholar)). The hydrophobic C-terminal segment is required for insertion of the virion into the inner membrane and for excision after phage assembly (15Crissman J.W. Smith G.P. Gene-III protein of filamentous phages. Evidence for a carboxyl-terminal domain with a role in morphogenesis.Virology. 1984; 132: 445-455Crossref PubMed Scopus (72) Google Scholar, 24Bennett N.J. Rakonjac J. Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII.J. Mol. Biol. 2006; 356: 266-273Crossref PubMed Scopus (24) Google Scholar). CTXφ pIII has very little sequence homology to the Ff pIII proteins but is predicted to have a 14-amino acid signal peptide and 3 domains separated by serine/proline-rich linkers (25Heilpern A.J. Waldor M.K. pIIICTX, a predicted CTXφ minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae.J. Bacteriol. 2003; 185: 1037-1044Crossref PubMed Scopus (53) Google Scholar), and CTXφ pIII-C contains a very hydrophobic segment that likely represents an inner membrane anchor. Ff coliphage bind to E. coli using their minor coat protein, pIII, in a two-step process to initiate infection. First, the central N2 domain of pIII binds to the F pilus tip (26Gray C.W. Brown R.S. Marvin D.A. Adsorption complex of filamentous fd virus.J. Mol. Biol. 1981; 146: 621-627Crossref PubMed Scopus (83) Google Scholar, 27Russel M. Whirlow H. Sun T.P. Webster R.E. Low frequency infection of F-bacteria by transducing particles of filamentous bacteriophages.J. Bacteriol. 1988; 170: 5312-5316Crossref PubMed Google Scholar, 28Deng L.W. Perham R.N. Delineating the site of interaction on the pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli.J. Mol. Biol. 2002; 319: 603-614Crossref PubMed Scopus (43) Google Scholar), which spontaneously retracts (29Marvin D.A. Filamentous phage structure, infection. and assembly.Curr. Opin. Struct. Biol. 1998; 8: 150-158Crossref PubMed Scopus (290) Google Scholar, 30Jacobson A. Role of F pili in the penetration of bacteriophage fl.J. Virol. 1972; 10: 835-843Crossref PubMed Google Scholar, 31Clarke M. Maddera L. Harris R.L. Silverman P.M. F-pili dynamics by live-cell imaging.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 17978-17981Crossref PubMed Scopus (95) Google Scholar) to bring the pIII N-terminal domain, pIII-N1, into contact with the C-terminal domain of the periplasmic protein, TolA (32Sun T.P. Webster R.E. Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli. J Bacteriol.J. Bacteriol. 1987; 169: 2667-2674Crossref PubMed Google Scholar, 33Levengood S.K. Beyer Jr., W.F. Webster R.E. TolA. A membrane protein involved in colicin uptake contains an extended helical region.Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 5939-5943Crossref PubMed Scopus (119) Google Scholar, 34Holliger P. Riechmann L. A conserved infection pathway for filamentous bacteriophages is suggested by the structure of the membrane penetration domain of the minor coat protein g3p from phage fd.Structure. 1997; 5: 265-275Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 35Click E.M. Webster R.E. The TolQRA proteins are required for membrane insertion of the major capsid protein of the filamentous phage f1 during infection.J. Bacteriol. 1998; 180: 1723-1728Crossref PubMed Google Scholar). The TolA binding site on pIII-N1 is buried by pIII-N2 (36Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. Filamentous phage infection. Crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.Structure. 1999; 7: 711-722Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and the interaction with the F pilus is required not only for bringing the phage to the bacterial surface but for inducing a conformational change in pIII that exposes the TolA binding site (18Riechmann L. Holliger P. The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.Cell. 1997; 90: 351-360Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 22Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p.Nat. Struct. Biol. 1998; 5: 140-147Crossref PubMed Scopus (98) Google Scholar, 23Holliger P. Riechmann L. Williams R.L. Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 Å. Evidence for conformational lability.J. Mol. Biol. 1999; 288: 649-657Crossref PubMed Scopus (86) Google Scholar). The mechanisms by which the F pilus retracts and the bacteriophage gains entry into the periplasm to bind to TolA are not understood. The F pilus is required for efficient infection, but Ff phage can infect E. coli lacking the F pilus, albeit much lower levels than for F+ strains (18Riechmann L. Holliger P. The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.Cell. 1997; 90: 351-360Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 27Russel M. Whirlow H. Sun T.P. Webster R.E. Low frequency infection of F-bacteria by transducing particles of filamentous bacteriophages.J. Bacteriol. 1988; 170: 5312-5316Crossref PubMed Google Scholar). In contrast, E. coli lacking TolA are resistant to phage infection (18Riechmann L. Holliger P. The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.Cell. 1997; 90: 351-360Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 35Click E.M. Webster R.E. The TolQRA proteins are required for membrane insertion of the major capsid protein of the filamentous phage f1 during infection.J. Bacteriol. 1998; 180: 1723-1728Crossref PubMed Google Scholar). The CTXφ phage utilize a similar mechanism to infect V. cholerae, as both TCP and TolA are required for maximal infectivity (3Waldor M.K. Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin.Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1345) Google Scholar, 11Heilpern A.J. Waldor M.K. CTXφ infection of Vibrio cholerae requires the tolQRA gene products.J. Bacteriol. 2000; 182: 1739-1747Crossref PubMed Scopus (88) Google Scholar). Heilpern and Waldor (25Heilpern A.J. Waldor M.K. pIIICTX, a predicted CTXφ minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae.J. Bacteriol. 2003; 185: 1037-1044Crossref PubMed Scopus (53) Google Scholar) generated recombinant fd phage in which CTXφ pIII domains N1, N2, or N1N2 were fused to the N terminus of fd pIII or to deletion mutants lacking N1N2 and assessed the ability of these hybrid phage to infect V. cholerae. fd hybrid phage displaying CTXφ pIII-N1N2 fused to domain D3 of fd pIII showed high levels of infectivity for V. cholerae. When only CTXφ pIII-N1 was present, infectivity was reduced but measurable, but when only CTXφ pIII-N2 was present, infectivity was undetectable, demonstrating that whereas N2 is important for efficient phage uptake, domain N1 is critical. Crystal structures are available for the N-terminal domains of both M13 and fd pIII, which are 99% identical in amino acid sequence (22Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p.Nat. Struct. Biol. 1998; 5: 140-147Crossref PubMed Scopus (98) Google Scholar, 23Holliger P. Riechmann L. Williams R.L. Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 Å. Evidence for conformational lability.J. Mol. Biol. 1999; 288: 649-657Crossref PubMed Scopus (86) Google Scholar). These structures reveal two discrete domains of similar, predominantly β-sheet fold joined by a linker and by a crossover of the C-terminal strand of N2 onto N1. N1 has a short N-terminal α-helix followed by a four-stranded β-barrel motif, and N2 is dominated by a twisted β-sheet. Not surprisingly, the glycine-rich LCR1 is disordered and is not resolved in the crystal structure. However, rather than acting as a linker between N1 and N2, this segment lies within N1 and is followed by an ordered loop and a β-strand, β5, that are part of the N1 domain. A short linker connects N1 to N2. The two domains also interact via non-covalent contacts between their β-sheet loops and by the C-terminal strand of N2, which extends across to N1 as a β-strand, β13, to form a 2-stranded β-sheet with β5 that lies across the N1 β-barrel. A crystal structure was also determined of a fusion protein consisting of the M13 pIII-N1 and LCR1 fused to the C-terminal domain of TolA (36Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. Filamentous phage infection. Crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.Structure. 1999; 7: 711-722Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The N1 fold is similar in both the pIII-N1/TolA-C fusion protein structure and the N1N2 structure but terminates just C-terminal to the disordered LCR1 in the fusion protein and, hence, lacks strand β5 and the remaining N2 domain. TolA-C interacts with N1 on a face of the β-barrel that is occupied by the β5β13 sheet and part of the N2 domain in the N1N2 structure. Thus, for TolA to bind to pIII, pIII must partially unfold, separating N1 and N2 and removing the β5β13 strand to expose the TolA binding site. The trigger that induces this conformational change is the interaction of N2 with the F pilus (18Riechmann L. Holliger P. The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.Cell. 1997; 90: 351-360Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 22Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p.Nat. Struct. Biol. 1998; 5: 140-147Crossref PubMed Scopus (98) Google Scholar, 23Holliger P. Riechmann L. Williams R.L. Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 Å. Evidence for conformational lability.J. Mol. Biol. 1999; 288: 649-657Crossref PubMed Scopus (86) Google Scholar). This movement is proposed to involve isomerization of the Gln-212–Pro-213 bond immediately after β13 (37Martin A. Schmid F.X. A proline switch controls folding and domain interactions in the gene-3-protein of the filamentous phage fd.J. Mol. Biol. 2003; 331: 1131-1140Crossref PubMed Scopus (30) Google Scholar). Commonalities and differences between the pIII proteins of CTXφ and the Ff phage prompted us to investigate the interaction between CTXφ and V. cholerae at a molecular level. Here we show by transmission electron microscopy (TEM) that phage binding to V. cholerae does not require TolA, but phage uptake does. We demonstrate a direct interaction between pIII-N1 and TolA-C and describe crystal structures of pIII-N1 alone and in complex with TolA-C. Our data reveal similarities in structure but surprising differences between CTXφ and the coliphage in their interactions with TolA, advancing our understanding of CTXφ infection of V. cholerae. Bacterial strains, plasmids, and primers are listed in Table 1. E. coli strains were grown in Luria-Bertani (LB) broth at 37 °C with appropriate antibiotics. V. cholerae strains were grown in LB, pH 6.5, Sm at 30 °C on a Ferris wheel rotator (TCP-inducing conditions). Antibiotics were used at a final concentration of 200 μg/ml streptomycin (Sm), 50 μg/ml kanamycin (Km), 100 μg/ml ampicillin (Ap), and 12 μg/ml tetracycline (Tc). Anti-TcpA antibodies were a gift from Ronald Taylor (Geisel School of Medicine).TABLE 1List of bacterial strains, plasmids, and primersReagentDescription or nucleotide sequenceSource/ReferenceStrainsE. coli BL21 (DE3)F− ompT hsdSB (rB− mB−) gal dcm (DE3)NovagenE. coli Rosetta-gami B (DE3)F− ompT hsdSB (rB− mB−) gal dcm lacY1 ahpC (DE3) gor522::Tn 10 trxB pRARE (CmR, KmR, TcR)NovagenE. coli DH5α-λpirendAl hsdR17 glnV44 thi-1 recA1 gyrA relA1 Δ(lacIZYA-argF)U169 deoR [80dlacΔ(lacZ)M15] λpir+53Woodcock D.M. Crowther P.J. Doherty J. Jefferson S. DeCruz E. Noyer-Weidner M. Smith S.S. Michael M.Z. Graham M.W. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants.Nucleic Acids Res. 1989; 17: 3469-3478Crossref PubMed Scopus (639) Google ScholarV. cholerae O395O1 classical Ogawa, SmRR. TaylorV. cholerae DH3O395 ΔtolA::pDH149 (tolA bp 79–530 inserted into pGP704)11Heilpern A.J. Waldor M.K. CTXφ infection of Vibrio cholerae requires the tolQRA gene products.J. Bacteriol. 2000; 182: 1739-1747Crossref PubMed Scopus (88) Google ScholarV. cholerae TCP2O395 ΔtcpA4Herrington D.A. Hall R.H. Losonsky G. Mekalanos J.J. Taylor R.K. Levine M.M. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans.J. Exp. Med. 1988; 168: 1487-1492Crossref PubMed Scopus (530) Google ScholarV. cholerae CL101O1 El Tor, pCTX-Km3Waldor M.K. Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin.Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1345) Google ScholarPlasmidspET-15bT7 promoter, His-tag coding sequence, T7 terminator, lacI coding sequence, pBR322 origin, bla (ApR)NovagenpET-15b·pIII-N1pET vector with insertion of CTXφ pIII DNA encoding residues − 5 to + 96 at NdeI and BamH1 sites downstream of His-tag coding sequenceThis studypET-15b·pIII-ΔTMpET vector with insertion of CTXφ pIII DNA encoding residues − 5 to + 355 at NdeI and BamH1 sites downstream of His-tag coding sequence; contains a mutation encoding a S65T substitution in pIII-ΔTMThis studypET-15b·pIII-N1+pET vector with insertion of CTXφ pIII DNA encoding residues − 5 to + 134 at NdeI and BamH1 sites downstream of His-tag coding sequence; contains a mutation encoding a S65T substitution in pIII-ΔTMThis studypET-15b·TolA-CpET vector with insertion V. cholerae TolA DNA encoding residues 241–356 at NdeI and BamH1 sites downstream of His-tag coding sequenceThis studypCTX-KmReplicative form of CTX-Kmφ DNA derived from the chromosomal CTX element of V. cholerae strain P27459, with ctxAB replaced with a KmR cassette66Goldberg I. Mekalanos J.J. Effect of a recA mutation on cholera toxin gene amplification and deletion events.J. Bacteriol. 1986; 165: 723-731Crossref PubMed Google Scholar, supplied by R. TaylorPhage, fdΔ1-pIIICTX(15–274)Gene fragment encoding pIII residues − 5 to 269 (corresponds to old numbering 15–274, which assumed the signal peptide spanned the first 14 residues of the unprocessed pIII) cloned into phage display vector fd-DOG in place of gene fragment encoding fd pIII domains N1 and N225Heilpern A.J. Waldor M.K. pIIICTX, a predicted CTXφ minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae.J. Bacteriol. 2003; 185: 1037-1044Crossref PubMed Scopus (53) Google ScholarPrimerspIII-N1-forGGGAATTCCATATGCCATCGGTAACGGCTTCCGThis studypIII-N1-revCGTAGGATCCTTAGCACTCTTCCCCCTCAGGThis studypIII-ΔTM-forGGGAATTCCATATGCCATCGGTAACGGCTTCCGThis studypIII-ΔTM-revCGTAGGATCCTTAGTGCAGGTTTTCAGAAAAGAGGGAGThis studypIII-N1+-forGGCTTCGACTCTCTAACATGTCAGTGGTCAGGThis studypIII-N1+-revCCTGACCACTGACATGTTAGAGAGTCGAAGCCThis studyTolA-C-forCGTAGAAGACTACTCGAATGATATTTTTGGCAGCTTGAGTGAAGThis studyTolA-C-revCGTAGGATCCTTATTCAGGTGCTACGGTTAAATTAATATTCTTTAGThis studyT7 promoterTAATACGACTCACTATAGGGNovagenT7 terminatorGCTAGTTATTGCTCAGCGGNovagen Open table in a new tab V. cholerae strains O395, DH3, and TCP2 used in the infection assays were grown overnight in TCP-inducing conditions. To prepare CTXφ, V. cholerae CL101 cells were grown under the same conditions but with Km in addition to Sm. CL101 cells produce CTX-KmΦ in which the ctxA gene is replaced with the Km resistance marker (3Waldor M.K. Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin.Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1345) Google Scholar). To produce fdΔ1-pIIICTX(15–274) phage, E. coli DH5α-λpir(fdΔ1-pIIICTX(15–274)) cells were grown at 37 °C overnight with Tc, 1.2 ug/ml. CL101, and E. coli DH5α-λpir cells were removed from the supernatant containing the phage by centrifugation and filtration on a 0.2-μm pore filter. Transduction assays were performed by mixing 75 μl of phage with 75 μl of V. cholerae overnight culture and shaking for 20 min at room temperature. Serial dilutions of the infection mixture were plated on LB-Sm/Km agar plates to enumerate the transductants and on LB-Sm plates to enumerate the input bacteria. Cells were grown overnight at 37 °C, and colony-forming units (cfu) were counted. The phage transduction frequency was calculated as the ratio of transductants to input V. cholerae cells. For the transfection inhibition assay, the filtered cell supernatant containing CTXφ or fdΔ1-pIIICTX(15–274) (50 μl) was mixed with 3 μl of His-TolA-C (100 μm) or with buffer only and incubated at room temperature for 30 min. Seventy-five microliters of V. cholerae O395 overnight culture was added to 75 μl of the phage mixture and incubated for another 20 min. The infection mixtures were serially diluted, plated on LB-Sm/Km and LB-Sm agar plates, grown overnight at 37 °C, then counted for cfu. Sample grids were prepared by floating glow discharged carbon-coated Formvar copper grids on transfection solutions (described above) for 10 min and then fixing with 2% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4 for 5 min. The grids were then washed twice on drops of PBS containing 0.15% glycine, blocked for 5 min with 1% bovine serum albumin (BSA) in PBS, and then incubated with polyclonal rabbit anti-TcpA antibodies (1:100 dilution) for 30 min. After 2 washes on PBS-glycine and 1 on PBS-BSA, the grids were incubated with 10- or 15-nm gold-conjugated protein A (diluted 1:70) for 20 min. After two washes on PBS, grids were fixed with 1% glutaraldehyde in PBS for 5 min, washed twice on PBS-glycine, and then blocked for 5 min" @default.
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• W1965902126 title "Crystal Structures of a CTXφ pIII Domain Unbound and in Complex with a Vibrio cholerae TolA Domain Reveal Novel Interaction Interfaces" @default.
• W1965902126 cites W1503309822 @default.
• W1965902126 cites W1514501127 @default.
• W1965902126 cites W1520730837 @default.
• W1965902126 cites W1531405061 @default.
• W1965902126 cites W1540496882 @default.
• W1965902126 cites W1574094463 @default.
• W1965902126 cites W1580820940 @default.
• W1965902126 cites W1595641092 @default.
• W1965902126 cites W1620801087 @default.
• W1965902126 cites W1769588871 @default.
• W1965902126 cites W1881598967 @default.
• W1965902126 cites W1913106712 @default.
• W1965902126 cites W1948606996 @default.
• W1965902126 cites W1950883248 @default.
• W1965902126 cites W1965277349 @default.
• W1965902126 cites W1966328487 @default.
• W1965902126 cites W1970225178 @default.
• W1965902126 cites W1970645837 @default.
• W1965902126 cites W1977151336 @default.
• W1965902126 cites W1977648667 @default.
• W1965902126 cites W1983410419 @default.
• W1965902126 cites W1986191025 @default.
• W1965902126 cites W1987537357 @default.
• W1965902126 cites W1991373417 @default.
• W1965902126 cites W1999383345 @default.
• W1965902126 cites W2000840258 @default.
• W1965902126 cites W2001048874 @default.
• W1965902126 cites W2001641653 @default.
• W1965902126 cites W2008282998 @default.
• W1965902126 cites W2012031105 @default.
• W1965902126 cites W2014694459 @default.
• W1965902126 cites W2015181445 @default.
• W1965902126 cites W2017191600 @default.
• W1965902126 cites W2020315094 @default.
• W1965902126 cites W2026217873 @default.
• W1965902126 cites W2031928642 @default.
• W1965902126 cites W2033289414 @default.
• W1965902126 cites W2038840577 @default.
• W1965902126 cites W2050418435 @default.
• W1965902126 cites W2050443196 @default.
• W1965902126 cites W2053785011 @default.
• W1965902126 cites W2060101911 @default.
• W1965902126 cites W2078078891 @default.
• W1965902126 cites W2078184263 @default.
• W1965902126 cites W2080426420 @default.
• W1965902126 cites W2086154563 @default.
• W1965902126 cites W2087166919 @default.
• W1965902126 cites W2112502782 @default.
• W1965902126 cites W2122339645 @default.
• W1965902126 cites W2131457995 @default.
• W1965902126 cites W2131458704 @default.
• W1965902126 cites W2135183599 @default.
• W1965902126 cites W2143694829 @default.
• W1965902126 cites W2144081223 @default.
• W1965902126 cites W2147645629 @default.
• W1965902126 cites W2150010294 @default.
• W1965902126 cites W2151687803 @default.
• W1965902126 cites W2152093372 @default.
• W1965902126 cites W2157392818 @default.
• W1965902126 cites W2159393451 @default.
• W1965902126 cites W2163341755 @default.
• W1965902126 cites W2163395290 @default.
• W1965902126 cites W2164263567 @default.
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