Molecular Dissection of VirB, a Key Regulator of the Virulence Cascade of Shigella flexneri
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m111429200
ISSN1083-351X
AutoresChristophe Beloin, Sorcha McKenna, Charles J. Dorman,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe VirB protein is a key regulator of virulence gene expression in the facultative enteroinvasive pathogenShigella flexneri. While genetic evidence has shown that it is required for activation of transcription of virulence genes located on a 230-kb plasmid in this bacterium, hitherto, evidence that VirB is a DNA-binding protein has been lacking. Although VirB shows extensive homology to proteins involved in plasmid partitioning, it does not resemble any known conventional transcription factor. Here we show for the first time that VirB binds to the promoter regions of the virulence genes in vivo. We also show that VirB forms dimeric and higher oligomeric structures both in vivo andin vitro and that this property is independent of DNA binding. The oligomerization activity of VirB is distributed over two domains: a leucine zipper-like motif and a carboxyl-terminal domain likely to form triple coiled structures. VirB possesses a helix-turn-helix motif, which is required for DNA binding. The amino-terminal domain of the protein is also required for DNA binding and virulence gene activation. The possibility that VirB requires a co-factor for specific interaction with target promoters in vivo is discussed. The VirB protein is a key regulator of virulence gene expression in the facultative enteroinvasive pathogenShigella flexneri. While genetic evidence has shown that it is required for activation of transcription of virulence genes located on a 230-kb plasmid in this bacterium, hitherto, evidence that VirB is a DNA-binding protein has been lacking. Although VirB shows extensive homology to proteins involved in plasmid partitioning, it does not resemble any known conventional transcription factor. Here we show for the first time that VirB binds to the promoter regions of the virulence genes in vivo. We also show that VirB forms dimeric and higher oligomeric structures both in vivo andin vitro and that this property is independent of DNA binding. The oligomerization activity of VirB is distributed over two domains: a leucine zipper-like motif and a carboxyl-terminal domain likely to form triple coiled structures. VirB possesses a helix-turn-helix motif, which is required for DNA binding. The amino-terminal domain of the protein is also required for DNA binding and virulence gene activation. The possibility that VirB requires a co-factor for specific interaction with target promoters in vivo is discussed. helix-turn-helix chloramphenicol carbenicillin tetracycline kanamycin phosphate-buffered saline dithiobis(succinimidyl propionate) large virulence plasmid leucine zipper Shigella flexneri is a Gram-negative, facultative intracellular pathogen of humans and primates and is the causative agent of bacillary dysentery. This extremely infectious disease is widespread in the developing world, where it is responsible for around 600,000 deaths per annum, most particularly affecting children (1Sansonetti P.J. Am. Soc. Microbiol. News. 1999; 65: 611-617Google Scholar). The gene products that mediate the invasion of the lower intestine byShigella are located on a 230-kb large virulence plasmid, where they are clustered in a 31-kb segment called the entry region. Here are found the ipa genes, which encode secreted invasins responsible for macrophage apoptosis, epithelial cell invasion, and vesicle escape (2High N. Mounier J. Prévost M.C. Sansonetti P.J. EMBO J. 1992; 11: 1991-1999Crossref PubMed Scopus (249) Google Scholar, 3Ménard R. Sansonetti P.J. Parsot C. EMBO J. 1996; 13: 5293-5302Crossref Scopus (303) Google Scholar, 4Wassef J. Karen D.F. Mailloux J.L. Infect. Immun. 1989; 57: 858-863Crossref PubMed Google Scholar); the mxi and spa genes, encoding the type III secretion system for export of the ipagene products (5Dorman C.J. Porter M.E. Mol. Microbiol. 1998; 29: 677-684Crossref PubMed Scopus (120) Google Scholar); and the icsA, icsB, andvirA genes required for cell-to-cell spread (5Dorman C.J. Porter M.E. Mol. Microbiol. 1998; 29: 677-684Crossref PubMed Scopus (120) Google Scholar, 6Blocker A. Gounon P. Larquet E. Niebuhr K. Cabiaux V. Parsot C. Sansonetti P.J. J. Cell Biol. 1999; 147: 683-696Crossref PubMed Scopus (396) Google Scholar). It is likely that expression of these structural genes represents a large metabolic burden for the bacteria. Therefore, it is unsurprising that the bacterium has evolved a complex regulatory system that integrates several environmental signals to prevent inappropriate expression. At the transcriptional level, a cascade that involves both chromosomally encoded proteins including IHF (integrationhost factor) and H-NS (histone-likenucleoid structuring protein) and plasmid-encoded regulatory proteins, VirF and VirB, restricts expression of the structural genes to conditions that approximate those in the lower intestine (i.e. a pH optimum of 7.4, moderate osmolarity, and a temperature of 37 °C (see Refs. 7Maurelli A.T. Baudry B. d'Hauteville H. Hale T.L. Sansonetti P.J. Infect. Immun. 1985; 49: 164-171Crossref PubMed Google Scholar, 8Porter M.E. Dorman C.J. J. Bacteriol. 1994; 176: 4187-4191Crossref PubMed Google Scholar, 9Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar, 10Tobe T. Yoshikawa M. Mizuno T. Sasakawa C. J. Bacteriol. 1993; 175: 6142-6149Crossref PubMed Google Scholar; for a review, see Ref. 11Dorman C.J. McKenna S. Beloin C. Int. J. Med. Microbiol. 2001; 290: 89-96Crossref Scopus (46) Google Scholar)). VirF is an AraC-like transcription factor responsible for activation of regulatory gene virB and structural gene icsA and autorepression of virF (12Falconi M. Colonna B. Prosseda G. Micheli G. Gualerzi C.O. EMBO J. 1998; 17: 7033-7043Crossref PubMed Scopus (229) Google Scholar, 13Nakayama S.I. Watanabe H. J. Bacteriol. 1998; 180: 3522-3528Crossref PubMed Google Scholar, 14Porter M.E. Dorman C.J. J. Bacteriol. 2002; 184: 531-539Crossref PubMed Scopus (37) Google Scholar). The virB gene product in turn activates expression of the remaining structural genes required for virulence via an unknown mechanism. VirB expression is also regulated by H-NS, which, together with levels of negative DNA supercoiling, appears to be responsible for the temperature dependence of virB transcription (8Porter M.E. Dorman C.J. J. Bacteriol. 1994; 176: 4187-4191Crossref PubMed Google Scholar, 10Tobe T. Yoshikawa M. Mizuno T. Sasakawa C. J. Bacteriol. 1993; 175: 6142-6149Crossref PubMed Google Scholar, 15Tobe T. Yoshikawa M. Sasakawa C. J. Bacteriol. 1995; 177: 1094-1097Crossref PubMed Google Scholar). Sensitivity to levels of negative DNA supercoiling also appears to be responsible for the osmoregulation of virB expression (8Porter M.E. Dorman C.J. J. Bacteriol. 1994; 176: 4187-4191Crossref PubMed Google Scholar, 15Tobe T. Yoshikawa M. Sasakawa C. J. Bacteriol. 1995; 177: 1094-1097Crossref PubMed Google Scholar). Recently,virB expression was shown to be regulated by quorum sensing (16Day W.A.J. Maurelli A.T. Infect. Immun. 2001; 69: 15-23Crossref PubMed Scopus (82) Google Scholar). The VirB protein was first identified through transposon mutagenesis of the virulence plasmid (17Watanabe H. Arakawa E. Ito K.I. Kato J.I. Nakamura A. J. Bacteriol. 1990; 172: 619-629Crossref PubMed Google Scholar), when it was shown to be essential for the expression of almost all of the structural virulence genes. VirB possesses no homology to previously described conventional transcriptional activators. Small and basic, VirB (35.4 kDa) shows most homology at the amino acid sequence level to ParB and SopB, proteins that are involved in plasmid partition and the maintenance of stable plasmid copy number, on the P1/P7 and F plasmids, respectively (17Watanabe H. Arakawa E. Ito K.I. Kato J.I. Nakamura A. J. Bacteriol. 1990; 172: 619-629Crossref PubMed Google Scholar, 18Abeles A.L. Friedman S.A. Austin S.J. J. Mol. Biol. 1985; 185: 261-272Crossref PubMed Scopus (144) Google Scholar, 19Bignell C. Thomas C.M. J. Biotechnol. 2001; 91: 1-34Crossref PubMed Scopus (165) Google Scholar, 20Porter M.E. The Regulation of Virulence Gene Expression in Shigella flexneriPh.D. thesis. Trinity College, Dublin1998Google Scholar, 21Radnedge L. Davis M.A. Austin S.A. EMBO J. 1996; 15: 1155-1162Crossref PubMed Scopus (42) Google Scholar). This homology is most pronounced in the first two-thirds of the proteins that includes, in ParB, a helix-turn-helix (HTH)1 motif (22Surtees J.A. Funnell B.E. J. Biol. Chem. 2001; 276: 12385-12394Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), whereas the C-terminal parts, encompassing in ParB its major oligomerization domain, are more divergent (20Porter M.E. The Regulation of Virulence Gene Expression in Shigella flexneriPh.D. thesis. Trinity College, Dublin1998Google Scholar, 23Surtees J.A. Funnel B.E. J. Bacteriol. 1999; 181: 5898-5908Crossref PubMed Google Scholar). The domain structure and activity of the VirB protein are unknown, and it has been assumed that VirB is in some way acting as a conventional transcriptional activator at the promoters of the structural virulence genes. Thus far, evidence that VirB activates structural gene expression directly is lacking, as is evidence that it is a DNA-binding protein. The aim of this work is to identify the important structural domains within the VirB protein and to understand how this unusual protein regulates virulence gene expression in S. flexneri. Extensive mutational and deletion analyses were carried out. VirB derivatives harboring point mutations or truncations were analyzed for their ability to activate gene expression and to bind DNA and for theirtrans-dominant phenotype, and they were also used inin vivo cross-linking analysis to determine their ability to oligomerize. Here we present evidence that VirB possesses separate structural domains responsible for its oligomerization, DNA binding, and transcription activation properties. Moreover, evidence is presented that VirB is able to bind in vivo to the promoter regions of the structural virulence genes, supporting the hypothesis that VirB activates these genes directly. The bacterial strains and plasmids used are listed in TableI. Antibiotics used in selective media were chloramphenicol (Cm; 20 μg/ml), carbenicillin (Ca; 50 μg/ml), tetracycline (Tet; 10 μg/ml), and kanamycin (Km; 50 μg/ml). Cells were grown in Luria Broth (LB). LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) at a concentration of 40 μg/ml, and MacConkey Lactose agar plates were used to indicate levels of β-galactosidase activity.Table IBacterial strains, plasmids, and phageStrain or plasmidRelevant characteristicsSource/ReferenceStrains S. flexneriBS184mxiC::MudI1734, KmrRef. 39Maurelli A.T. Blackmon B. Curtis III, R. Infect. Immun. 1984; 1984: 195-201Crossref Google ScholarCJD1018BS184 virB::Ap, CarKmrRef. 9Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar E. coliXL-1 BluesupE44hsdR17recA1endA1gyrA46thi relA1lac− F[proAB+ lacIqlacZΔM15Tn10], TetrStratageneDH5αsupE44ΔlacU169(φ80lacZΔM15)hsdR17recA1endA1gyrA96thi-1relA1Ref. 51Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8407) Google ScholarAG1688hsdRmcrBaraD139Δ(araABC-leu)7679ΔlacX7 4galUgalKrpsLthiF′128lacIqlacZ::Tn5, KmrE. coli GCGJH372AG1688([λ]202), KmrRef.26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google ScholarJH607AG1688 ([λ]1120sPs), KmrRef.26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google ScholarBL21DE3hsdSgal(λcIts857ind1Sam7nin5lacUV5-T7gene1)Ref.52Studier F.W. Meffel B.W. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (5083) Google ScholarPlasmids pBCCloning vector; ColE1 replicon, CmrStratagene pACYC184Cloning vector; p15A replicon, CmrTetrStratagene pET22bHis tag expression vector, CarNovagen pBCP378Expression vector; ColE1 replicon, CarRef.53Veletrop J.S. Dijkhuizen M.A. van't Hof R. Postma P.W. Gene (Amst.). 1995; 153: 63-65Crossref PubMed Scopus (11) Google Scholar pMEP539cat gene in pBCP378, Car CmrRef. 9Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar pMEP538virB gene in pMEP539, CarCmrRef. 9Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar pBCPK152EpMEP538 with K152E mutation, Car CmrThis work pBCPK164EpMEP538 with K164E mutation, CarCmrThis work pBCPL196EpMEP538 with L196E mutation, Car CmrThis work pBCPL203PpMEP538 with L203P mutation, CarCmrThis work pBCPL203VpMEP538 with L203V mutation, Car CmrThis work pBCPN207HpMEP538 with N207H mutation, CarCmrThis work pBCPN207IpMEP538 with N207I mutation, Car CmrThis work pBCPL210HpMEP538 with L210H mutation, CarCmrThis work pBCPL210PpMEP538 with L210P mutation, Car CmrThis work pBCPP215ApMEP538 with P215A mutation, CarCmrThis work pBCPL217SpMEP538 with L217S mutation, Car CmrThis work pBCPL224RpMEP538 with L224R mutation, CarCmrThis work pBCPK152E L203PpMEP538 with K152E + L203P, Car CmrThis work pBCPK152E L210PpMEP538 with K152E + L210P, Car CmrThis work pBCPΔ1–30pMEP538 with Δ1–30 deletion, CarCmrThis work pBCPΔ1–65pMEP538 with Δ1–65 deletion, Car CmrThis work pBCPΔ244–309pMEP538 with Δ244–309 deletion, CarCmrThis work pBCPΔ262–309pMEP538 with Δ262–309 deletion, Car CmrThis work pBCPΔ292–309pMEP538 with Δ292–309 deletion, CarCmrThis work pBCPΔLZpMEP538 with ΔLZ deletion, Car CmrThis work pJH391"Stuffer" plasmid: pJH370, withSalI-SacI lacZ fragment from pMC1871, deletion between BamHI, CarRef.26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google Scholar pFG157pZ150 with intact λ cI repressor, CarRef. 26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google Scholar pZ150pBR322 with M13 ssDNA ori, CarRef. 54Zagursky R.J. Berman M.L. Gene (Amst.). 1984; 27: 183-191Crossref PubMed Scopus (151) Google Scholar pJH370pFG157 1–115 of λ cI + GCN4 LZ onHindIII-EcoRV fragment, CarRef.26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google Scholar pKH101pFG157 Δ115–237 λ cI repressor, Car CmrRef. 26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google Scholar pMC1871Multipurpose cloning vector, CarRef.55Pouwels P.H. Cloning Vectors. North-Holland, Amsterdam1985: 451-460Google Scholar pSMvirBpJH391 with virB fused to λ cI1–115, CarThis work pSML203PpSMvirB with L203P mutation, CarThis work pSMK152EpSMvirB with K152E mutation, CarThis work pSMΔ1–144pSMvirB with Δ1–144 deletion, CarThis work pSMΔ1–184pSMvirB with Δ1–184 deletion, CarThis work pSMΔ1–225pSMvirB with Δ1–225 deletion, CarThis work pSMΔ232–309pSMvirBwith Δ232–309 deletion, CarThis work pSMΔ176–309pSMvirB with Δ176–309 deletion, CarThis work pSMΔ147–309pSMvirBwith Δ147–309 deletion, CarThis work pSMHTHpSMvirB with VirB HTH, CarThis work pSMLZpSMvirB with VirB LZ, CarThis workPhage λ virKH54 h80Ref.26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google Scholar λ cI−KH54 ΔcI−Ref. 26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google Scholar Open table in a new tab The virB gene previously cloned in pMEP538 (9Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar) was sequenced to confirm its integrity. This plasmid is derived from pMEP539 (9Porter M.E. Dorman C.J. J. Bacteriol. 1997; 179: 6537-6550Crossref PubMed Google Scholar), a derivative of expression vector pBC378 that contains acat gene (Table I). Derivatives of pMEP538 with mutations in the virB gene were constructed using the Stratagene QuikChange™ kit. Each mutant was sequenced to confirm the presence of the mutation. Truncates of VirB were constructed by amplifying truncated forms of the virB gene by PCR and cloning into theNdeI and SalI restriction sites of pMEP539. The leucine zipper (LZ) motif deletion truncate was constructed by three-way cloning into the same sites. Putative clones of each truncate were sequenced, and expression of truncates was confirmed by western immunoblotting. The program HTH (24Dodd I.B. Egan B.J. Nucleic Acids Res. 1990; 18: 5019-5026Crossref PubMed Scopus (454) Google Scholar) was used for detection of a possible HTH DNA-binding motif in VirB. The programs COILS, MULTICOIL, and PARCOIL from the Expasy Web server (www.expasy.ch/) were used to analyze the tertiary structure of the LZ motif and the C terminus of VirB. Transcription of the mxiC-lacZ fusion was monitored by β-galactosidase assay of cells cultured overnight, according to Miller (25Miller J.H. Press C.S.H. Experiments in Molecular Genetics. Cold Spring Harbor, NY1972Google Scholar). Assays were performed at least in triplicate, and the data are expressed as the mean of two measurements. N-terminal His-tagged VirB was overexpressed in BL21DE3 cells from the pET22b Novagen vector. Expression was induced in exponentially growing 500-ml cultures with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside. After 3 h, the cells were harvested, and lysates were prepared by repeated passage through a French pressure cell. The lysate (∼15 ml) was applied to a His-bind® Quick column (Novagen), which had been preequilibrated with binding buffer. The column was then washed with binding buffer (50 ml) and wash buffer (25 ml). The protein was then eluted in 1-ml fractions of 2 × 15 ml of elution buffer (10% glycerol, 50 mmTris-HCl, pH 7.9, 0.5 m NaCl, 0.1 mmphenylmethylsulfonyl fluoride) containing 100 or 500 mm imidazole. Fractions were analyzed by SDS-PAGE, and those containing VirB were pooled and dialyzed three times against 1 liter of 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, pH 8, 300 mm NaCl, 5% glycerol, 0.1 mmphenylmethylsulfonyl fluoride, 1 mm dithiothreitol. VirB was estimated to be ∼95% pure. Rabbit polyclonal antibodies were conventionally prepared using purified VirB. Prior to immunodetection, the serum was adsorbed against a crude protein extract of avirB-S. flexneri strain. Total protein extracts were separated through 12% SDS-PAGE. The separated proteins were electroblotted onto a nitrocellulose membrane using the Bio-Rad miniprotean II system for 1 h at 80 V. Nitrocellulose membranes were stained with Ponceau (0.2% Ponceau dye, 3% trichloroacetic acid) to check the efficiency of transfer before being blocked overnight with 5% dried skimmed milk in phosphate-buffered saline (PBS). Detection of VirB was performed in PBS containing 1% dried skimmed milk with a primary polyclonal anti-VirB antiserum (1:500) and a secondary goat anti-rabbit horseradish peroxidase-conjugated antiserum (1:10,000). Membranes were developed using the chemiluminescent Pierce West Pico Super Signal kit. Cells were grown overnight at 37 °C in 3 ml of LB medium and diluted to A600 0.05 in 50 ml of LB medium. At A600 0.6, 1 ml of cells were transferred to a microcentrifuge tube and incubated at 37 °C for 30 min with 50 mm iodoacetamide, with or without 25 mm dithiobis(succinimidyl propionate) (DSP). The reaction was quenched by adding 100 mm Tris-HCl, pH 7.A600 of the sample was monitored, and the equivalent of 1 ml of A600 0.5 was harvested, washed twice in PBS, and resuspended in 20 μl of buffer containing 50 mm Tris-HCl, pH 7, 1 mm EDTA, pH 8, 10% glycerol, 200 mm NaCl, 1 mmphenylmethylsulfonyl fluoride (added fresh). 10 μl of 3× Laemmli buffer (without dithiothreitol or 2-β-mercaptoethanol) was added, and samples were boiled for 3 min at 100 °C. Prior to loading, samples were treated during 20 min at 37 °C with 20 units of benzonase. Cross-linked proteins were then separated on a 12% SDS-PAGE. VirB was then immunodetected. Purified VirB was treated in 100-μl reaction mixes consisting of 50 ng of protein and 50 mmiodoacetamide in PBS. At time 0, 10 μl of a 0.1 mm DSP solution in Me2SO was added. The control tube received an equal volume of Me2SO alone. The reactions were incubated at 37 °C. 10-μl samples were removed at fixed time intervals and quenched by the addition of 20 μl of 3× Laemmli buffer followed by boiling for 10 min. Samples were electrophoresed on a 12% SDS-PAGE, and VirB was then detected by Western blotting. In order to determine the oligomerization properties of VirB and parts of VirB, fusion proteins were constructed by cloning the appropriate fragment of VirB into the SalI–BamHI site in pJH391, thereby creating an in-phase translational fusion to the N terminus (DNA-binding region) of λ cI (see Fig. 6). Each construction was sequenced to verify its integrity. To assess the ability of cloned fragments to oligomerize, two assays were carried out, a phage sensitivity assay and β-galactosidase repression assay (25Miller J.H. Press C.S.H. Experiments in Molecular Genetics. Cold Spring Harbor, NY1972Google Scholar, 26Hays L.B. Chen Y.S. Hu J.C. BioTechniques. 2000; 29: 288-290Crossref PubMed Scopus (17) Google Scholar). Plasmids expressing the chimeric proteins were transformed into Escherichia coli strain AG1688, and the strains were infected with λ cI−. Strains immune to this phage, as judged by no plaque formation, possess a fragment capable of dimerization fused to the N terminus of cI. Similarly, plasmids were transformed into the strain JH372, and β-galactosidase assays were performed as described above; in this case, a repression of β-galactosidase activity indicated a dimerizing fragment. Cells were grown in conditions of VirB induction (i.e. in LB medium at 37 °C, up toA600 ∼0.6). Cross-linking and sample preparation were based on chromatin immunoprecipitation assays (27Orlando V. Trends Biochem. Sci. 2000; 25: 99-104Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar,28Strahl-Bolsinger S. Hecht A. Luo K. Grunstein M. Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (594) Google Scholar). 10 ml of cells was then transferred to a new vial, and samples were treated with formaldehyde (final concentration 0.1%) for 30 min at 37 °C with shaking. Cells were pelleted, washed twice with PBS, resuspended in 0.5 ml of lysis buffer (10 mm Tris-HCl, pH 8, 20% sucrose, 50 mm NaCl, 10 mm EDTA, pH 8) containing 2 mg/ml of lysozyme and incubated during 30 min at 37 °C. After freeze-thawing, 0.5 ml of 2× immunoprecipitation buffer (100 mm Tris-HCl, pH 7, 300 mm NaCl, 2% Triton X-100, 0.2% deoxycholic acid) and phenylmethylsulfonyl fluoride (final concentration 1 mm) was added to the samples, and the cell extract was incubated an additional 10 min at 37 °C. The DNA was then sheared by sonication using a Ca MSE Soniprep 150 sonicator (Sanyo). Insoluble cell debris was removed by centrifugation, and the supernatant was transferred to a new microcentrifuge tube. Protein and protein-DNA complexes were immunoprecipitated with absorbed polyclonal anti-VirB antibodies (1 h at room temperature on a rotating wheel) followed by incubation with 30 μl of a 50% protein A-Sepharose slurry (1 h at room temperature with mixing). Complexes were collected by centrifugation and washed five times with 1 ml of 1× immunoprecipitation buffer and twice with 1 ml of 1× TE (10 mm Tris-HCl, pH 8, 0.1 mm EDTA, pH 8). The slurry was then resuspended in 50 μl of 1× TE. Formaldehyde cross-links were reversed by incubation at 65 °C for 6 h. PCR was performed with Taq DNA polymerase using 2 μl of the immunoprecipitated DNA and a constant amount of either purifiedS. flexneri chromosomal or large virulence plasmid (LVP) DNA as controls. PCRs were carried out with a master-mix containing the primers. The only difference between the samples was the DNA added. All assays were performed several times, and reproducible results were obtained. The identities of the PCR products generated by these primers were confirmed previously by DNA sequencing. Primers were ∼25 bp in length and amplified ∼300–400-bp products. Sequences of all primers are available upon request. Relative affinities of VirB to different sites were determined by comparing the intensity of bands from the immunoprecipitate and the chromosomal/LVP DNA. As a first approach to identify structural domains of the VirB protein, we analyzed its predicted secondary structure in silico. Since VirB is regarded as a putative transcriptional regulator, it was first scanned for the presence of a putative DNA-binding motif. The program HTH (24Dodd I.B. Egan B.J. Nucleic Acids Res. 1990; 18: 5019-5026Crossref PubMed Scopus (454) Google Scholar) predicted at 100% probability the region 148–171 of VirB to contain a typical HTH DNA-binding motif (Fig.1A) as observed in different prokaryotic transcriptional regulators such as λ Cro, LacR, or CRP (29Brennan R.G. Matthews B.W. J. Biol. Chem. 1989; 264: 1903-1906Abstract Full Text PDF PubMed Google Scholar). The HTH motif consists of two helices separated by a short extended chain of amino acids, which are held at fixed angles by electrostatic interactions between side chains on each helix. The second helix, known as the recognition helix, fits into the major groove of DNA to participate in sequence specific interactions. The first helix, known as the positioning helix, stabilizes this complex by interacting nonspecifically with the DNA backbone. As eukaryotic and prokaryotic HTH DNA-binding motifs are known to interact with their targets as dimers, we also tried to identify possible oligomerization domains in VirB. Coiled-coil domains have been described as common features allowing oligomerization of proteins (30Baxevanis A.D. Vinson C.R. Curr. Opin. Genet. Dev. 1993; 3: 278-285Crossref PubMed Scopus (181) Google Scholar, 31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). A coiled-coil structure was predicted between positions ∼190 and ∼230 of VirB by the COILS program (Expasy; www.expasy.ch). In this domain, the ScanProsite program (Expasy) detected the pattern of an LZ motif commonly associated with dimerization in eukaryotic proteins such as the GCN4 yeast transcription factor protein and less frequently in prokaryotic proteins such as the IS911 transposase or antiterminator protein, BglG (32Boss A. Nussbaum-Shochat A. Amster-Choder O. J. Bacteriol. 1999; 181: 1755-1766Crossref PubMed Google Scholar, 33Haren L. Polard P. Ton-Hoang B. Chandler M. J. Mol. Biol. 1998; 283: 29-41Crossref PubMed Scopus (39) Google Scholar, 34O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1306) Google Scholar). The putative LZ in VirB possessed some characteristics of previously described LZ motifs (Fig.1, A and B; Refs. 35Alber T. Cur. Opin. Gen. Dev. 1992; 2: 205-210Crossref PubMed Scopus (255) Google Scholar and 36Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2796) Google Scholar). It consisted of 5 leucines (in position d) repeated every seventh residue. Residues in position a are also hydrophobic and, together with the leucine repeat, line up along the face of an α-helix to form a hydrophobic interface where two monomers can interact (Fig.1B). In the central a position of the putative LZ, VirB contains an asparagine that is conserved in the LZ family and is thought to favor the positioning of the two coils and to help in the determination of dimerization specificity (34O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1306) Google Scholar, 37Gonzalez L.J. Woolfson D.N. Alber T. Nat. Struct. Biol. 1996; 3: 1011-1018Crossref PubMed Scopus (218) Google Scholar). Residues in positions e and g normally carry opposite charges and are potentially able to form intersubunit salt bridges that stabilize the dimeric structure (31Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). This last characteristic was not conserved in the VirB LZ motif, suggesting that a dimeric form of this LZ motif might not be stabilized by intersubunit salt bridges. In addition, the putative LZ contains a proline residue, which is unusual in a coiled-coil structure. Finally, the C-terminal region of VirB was predicted to form a coiled-coil structure. Indeed, MULTICOIL and PARCOIL programs (Expasy) predicted positions ∼260 to 309 of VirB to form a trimeric coiled-coil region. The presence of putative coiled-coil structures in VirB was consistent with the possibility that the protein could oligomerize. Oligomerization was evaluated in vivo using a cross-linking method. First, we determined the best conditions for VirB production in order to facilitate its detection. Virulence gene expression was induced in bacteria grown at 37 °C in growth media with an osmolarity similar to that of physiological saline and at a pH close to neutrality (8Porter M.E. Dorman C.J. J. Bacteriol. 1994; 176: 4187-4191Crossref PubMed Google Scholar, 38Hromockyj A.E. Maurelli A.T. Infect. Immun. 1989; 57: 2963-2970Crossref PubMed Google Scholar, 39Maurelli A.T. Blackmon B. Curtis III, R. Infect. Immun. 1984; 1984: 195-201Crossref Google Scholar). S. flexneri 2a strain BS184 was cultured in the complex medium LB at 30 or 37 °C, and crude protein extracts were prepared at the midexponential or late stationary phase of growth. VirB was identified by immunodetection using a polyclonal antiserum raised against a purified N-terminal His-tagged VirB protein (Fig.2A). 2C. Beloin, S. McKenna, and C. J. Dorman, unpublished data.Whereas VirB was detectable at 30 °C, its production was increased by about 8-fold at 37 °C, in keeping with the well known thermoregulation of virulence gene expression in S. flexneri. At this temperature, VirB appeared to be somewhat more abundant in midexponential compared with late stationary growth phase. These growth conditions were then employed when performing the in vivo cross-linking experiments. The
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