Identification of the VirB4-VirB8-VirB5-VirB2 Pilus Assembly Sequence of Type IV Secretion Systems
2005; Elsevier BV; Volume: 280; Issue: 28 Linguagem: Inglês
10.1074/jbc.m502347200
ISSN1083-351X
AutoresQing Yuan, Anna Carle, Chan Gao, Durga Sivanesan, Khaled A. Aly, Christoph Höppner, Lilian Krall, Natalie Domke, Christian Baron,
Tópico(s)Bacteriophages and microbial interactions
ResumoType IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gram-negative bacteria into eukaryotic cells. A complex of 11 conserved proteins (VirB1-VirB11) spans the inner and the outer membrane and assembles extracellular T-pili in Agrobacterium tumefaciens. Here we report a sequence of protein interactions required for the formation of complexes between VirB2 and VirB5, which precedes their incorporation into pili. The NTPase Walker A active site of the inner membrane protein VirB4 is required for virulence, but an active site VirB4 variant stabilized VirB3 and VirB8 and enabled T-pilus formation. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 complex formation depended on VirB4, which identified a novel T-pilus assembly step. Bicistron expression demonstrated direct interaction of VirB4 with VirB8, and analyses with purified proteins showed that VirB5 bound to VirB8 and VirB10. VirB4 therefore localizes at the basis of a trans-envelope interaction sequence, and by stabilization of VirB8 it mediates the incorporation of VirB5 and VirB2 into extracellular pili. Type IV secretion systems mediate the translocation of virulence factors (proteins and/or DNA) from Gram-negative bacteria into eukaryotic cells. A complex of 11 conserved proteins (VirB1-VirB11) spans the inner and the outer membrane and assembles extracellular T-pili in Agrobacterium tumefaciens. Here we report a sequence of protein interactions required for the formation of complexes between VirB2 and VirB5, which precedes their incorporation into pili. The NTPase Walker A active site of the inner membrane protein VirB4 is required for virulence, but an active site VirB4 variant stabilized VirB3 and VirB8 and enabled T-pilus formation. Analysis of VirB protein complexes extracted from the membranes with mild detergent revealed that VirB2-VirB5 complex formation depended on VirB4, which identified a novel T-pilus assembly step. Bicistron expression demonstrated direct interaction of VirB4 with VirB8, and analyses with purified proteins showed that VirB5 bound to VirB8 and VirB10. VirB4 therefore localizes at the basis of a trans-envelope interaction sequence, and by stabilization of VirB8 it mediates the incorporation of VirB5 and VirB2 into extracellular pili. Gram-negative bacteria use secretion systems to translocate macromolecules across their cell envelope of two membranes and the murein cell wall. The term type IV secretion system (T4SS) 1The abbreviations used are: T4SS, type IV secretion system; AS, acetosyringone; ARA, arabinose; DDM, dodecyl-β-d-maltoside; MES, 4-morpholineethanesulfonic acid. was introduced for a group of protein machineries, which translocate proteins or protein-DNA complexes from donor to recipient cells. T4SSs are used by many bacterial pathogens for the translocation of virulence factors, e.g. by Agrobacterium tumefaciens, Bartonella henselae, Bordetella pertussis, Brucella suis, Helicobacter pylori, and Legionella pneumophila (1Cascales E. Christie P.J. Nat. Rev. Microbiol. 2003; 1: 137-149Crossref PubMed Scopus (523) Google Scholar, 2Christie P.J. Biochim. Biophys. Acta. 2004; 1694: 219-234Crossref PubMed Scopus (181) Google Scholar, 3Llosa M. O'Callaghan D. Mol. Microbiol. 2004; 53: 1-8Crossref PubMed Scopus (31) Google Scholar). T4SSs from different bacteria translocate a wide variety of macromolecules to different types of recipients (bacteria, fungi, mammalian, and plant cells) (4Ding Z. Atmakuri K. Christie P.J. Trends Microbiol. 2003; 11: 527-535Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Nevertheless, the basic mechanism and structure of the translocation machinery are likely conserved (5Yeo H.-J. Waksman G. J. Bacteriol. 2004; 186: 1919-1926Crossref PubMed Scopus (65) Google Scholar). The best-characterized model is the plant pathogen A. tumefaciens. The T4SS of the closely related animal pathogen B. suis is encoded by an operon of similar organization, and it is essential for survival and multiplication inside mammalian cells (6Celli J. Gorvel J.P. Curr. Opin. Microbiol. 2004; 7: 93-97Crossref PubMed Scopus (111) Google Scholar). The T4SSs of these bacteria share 11 proteins, which can be divided into three groups. The first group comprises two inner membrane-associated NTPases (VirB4 and VirB11), which reside mainly in the cytoplasm but may traverse the inner membrane and contact periplasmic T4SS components (7Cascales E. Christie P.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17228-17233Crossref PubMed Scopus (124) Google Scholar, 8Dang T.A. Zhou X.-R. Graf B. Christie P.J. Mol. Microbiol. 1999; 32: 1239-1253Crossref PubMed Scopus (81) Google Scholar, 9Dang T.A. Christie P.J. J. Bacteriol. 1997; 179: 453-462Crossref PubMed Google Scholar). They contain Walker A nucleotide-binding motifs and are believed to energize T4SS assembly or substrate transfer. The second group consists of inner membrane VirB6 and periplasmic VirB7, VirB8, VirB9, and VirB10. They form the T4SS core and may constitute the translocation channel (10Cascales E. Christie P.J. Science. 2004; 304: 1170-1173Crossref PubMed Scopus (287) Google Scholar, 11Jakubowski S.J. Krishnamoorthy V. Cascales E. Christie P.J. J. Mol. Biol. 2004; 341: 961-977Crossref PubMed Scopus (95) Google Scholar, 12Hapfelmeier S. Domke N. Zambryski P.C. Baron C. J. Bacteriol. 2000; 182: 4505-4511Crossref PubMed Scopus (62) Google Scholar, 13Das A. Xie Y.-H. J. Bacteriol. 2000; 182: 758-763Crossref PubMed Scopus (106) Google Scholar). The third group comprises the major T-pilus component VirB2, the minor component VirB5, and the pilus-associated protein VirB7 (14Jakubowski S.J. Krishnamoorthy V. Christie P.J. J. Bacteriol. 2003; 185: 2867-2878Crossref PubMed Scopus (80) Google Scholar, 15Schmidt-Eisenlohr H. Domke N. Angerer C. Wanner G. Zambryski P.C. Baron C. J. Bacteriol. 1999; 181: 7485-7492Crossref PubMed Google Scholar, 16Lai E.-M. Kado C.I. Trends Microbiol. 2000; 8: 361-369Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 17Eisenbrandt R. Kalkum M. Lai E.M. Lurz R. Kado C.I. Lanka E. J. Biol. Chem. 1999; 274: 22548-22555Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). VirB3 has not been firmly assigned, but its outer membrane localization and binding to VirB5 suggest that it is a pilus-associated protein (18Shamaei-Tousi A. Cahill R. Frankel G. J. Bacteriol. 2004; 186: 4796-4801Crossref PubMed Scopus (28) Google Scholar, 19Jones A.L. Shirasu K. Kado C.I. J. Bacteriol. 1994; 176: 5255-5261Crossref PubMed Google Scholar). Biochemical experiments based on extraction of VirB proteins with a mild detergent followed by separation under native conditions led to a model for T-pilus assembly (20Krall L. Wiedemann U. Unsin G. Weiss S. Domke N. Baron C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11405-11410Crossref PubMed Scopus (109) Google Scholar). This model is refined here based on improved separation methods and the analysis of the contribution of VirB4. VirB4 is the largest T4SS component (A. tumefaciens, 87 kDa; B. suis, 94 kDa) and exhibits the highest degree of conservation among T4SS components (31% identity and 52% similarity between A. tumefaciens and B. suis). It is homodimeric or homomultimeric and is essential for virulence (8Dang T.A. Zhou X.-R. Graf B. Christie P.J. Mol. Microbiol. 1999; 32: 1239-1253Crossref PubMed Scopus (81) Google Scholar, 21Watarai M. Makino S. Shirahata T. Microbiology. 2002; 148: 1439-1446Crossref PubMed Scopus (50) Google Scholar). An important feature is its Walker A nucleotide-binding site, which is essential for virulence and plasmid transfer (22Sagulenko E. Sagulenko V. Chen J. Christie P.J. J. Bacteriol. 2001; 183: 5813-5825Crossref PubMed Scopus (84) Google Scholar, 23Fullner K. Stephens K.M. Nester E.W. Mol. Gen. Genet. 1994; 245: 704-715Crossref PubMed Scopus (54) Google Scholar). ATPase activity of purified A. tumefaciens VirB4 was previously reported (24Shirasu K. Koukolikova-Nicola Z. Hohn B. Kado C.I. Mol. Microbiol. 1994; 11: 581-588Crossref PubMed Scopus (58) Google Scholar), but a more recent study argues against such an activity of the purified protein (25Rabel C. Grahn A.M. Lurz R. Lanka E. J. Bacteriol. 2003; 185: 1045-1058Crossref PubMed Scopus (58) Google Scholar). Coordinated action of VirB4 with VirB11 and VirD4 was proposed to mediate the early DNA transfer reactions (26Atmakuri K. Cascales E. Christie P.J. Mol. Microbiol. 2004; 54: 1199-1211Crossref PubMed Scopus (174) Google Scholar). In addition, VirB4 stabilized VirB3 and was required for its localization in the outer membrane (19Jones A.L. Shirasu K. Kado C.I. J. Bacteriol. 1994; 176: 5255-5261Crossref PubMed Google Scholar). A study using the yeast two-hybrid system suggested that VirB4 binds to VirB1, VirB8, VirB10, and VirB11, but this was not substantiated with biochemical methods (27Ward D. Draper O. Zupan J.R. Zambryski P.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11493-11500Crossref PubMed Scopus (119) Google Scholar). Interestingly, the VirB4 NTPase active site is essential for virulence, but it is not required for interactions with VirB11 and VirD4, for self-association, and for the stimulation of IncQ plasmid transfer into A. tumefaciens by the T4SS in the recipient (8Dang T.A. Zhou X.-R. Graf B. Christie P.J. Mol. Microbiol. 1999; 32: 1239-1253Crossref PubMed Scopus (81) Google Scholar). To unravel the role of VirB4, we constructed a virB4 deletion mutant and assessed the complementation with A. tumefaciens and B. suis VirB4 and NTPase active site variants. We refined the protocol for the separation of detergent-extracted VirB proteins and identified a novel step in T-pilus assembly. Purified components were used to study interactions in vitro, and this revealed an interaction sequence from the VirB4-VirB8 complex to the pilus component VirB5 and the core component VirB10. This work gives fundamental insights into the contribution of VirB4 to T4SS complex stabilization and T-pilus assembly. Cultivation of Bacteria—The strains and plasmids used in this study are given in Table I. Cultures of Escherichia coli JM109 for cloning experiments were grown at 37 °C in LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl). Antibiotics were added for plasmid propagation (50 μg/ml spectinomycin, 50 μg/ml streptomycin, 100 μg/ml carbenicillin). Overnight cultures of A. tumefaciens were grown in YEB (0.5% beef extract, 0.5% peptone, 0.1% yeast extract, 0.5% sucrose, 2 mm MgSO4) in the absence of antibiotics (wild-type strains) or with spectinomycin (300 μg/ml) and streptomycin (100 μg/ml) for the propagation of pVSBADNco and pVSBAD, followed by virulence gene induction in liquid AB glycerol minimal medium (0.5% glycerol, 0.4% morpholinoethansulfonic acid, 1 mm sodium potassium phosphate, 0.1% NH4Cl, 0.03% MgSO4 × 7H2O, 0.001% CaCl2, 0.00025% FeSO4 × 7H2O, pH 5.5) for 18 h or on AB agar plates for 3 days at 20 °C in the presence of acetosyringone (AS; 200 μm) and arabinose (ARA, 0.2%). For protein overproduction, E. coli strain GJ1158 was grown under aerobic conditions at 37 °C in LBON medium (1% tryptone, 0.5% yeast extract) to an A600 of 0.4-0.8, followed by the addition of NaCl at 0.3 m. Cultivation under aerobic conditions proceeded at different temperatures and times to assure maximal solubility and yield of the fusion proteins (VirB4s and its bicistron constructs, VirB5sp, VirB9sp, and VirB10sp: 16 h, 26 °C; VirB8sp: 4 h, 37 °C).Table IBacterial strains and plasmidsGenotype or descriptionSource/ref. no.StrainsE. coli JM109endA1 gyr96 thi hsdR71 supE44 recA1 relA1 (Δlac-proAB) (F' traD36 proAB+ lacIq lacZΔM15)45E. coli GJ1158ompT hsdS gal dcm ΔmalAp510 malP::(proUp-T7 RNAP) malQ::lacZhyb11 Δ(zhf-900::Tn10ΔTet)46A. tumefaciens C58Wild type, pTiC5847A. tumefaciens A348Wild type, pTiA6NC47A. tumefaciens CB1004pTiC58 carrying an in-frame deletion of virB4This workA. tumefaciens PC1004pTiA6NC carrying an in-frame deletion of virB448A. tumefaciens UIA143(pTiA6)A348, eryr, recA ery14035S. cerevisiae AH109MATa trp1-901 leu2-3 ura3-52 his3-200 gal4Δ gal80Δ LYS2::GAL1UAS-GAL1TATA-HIS3 GAL2UAS-GAL2TATA-ADE2 URA3::MEL1UAS-MEL1TATA-lacZClontechPlasmidspVSBADNcostrr, spcr, pVS1 origin, AraC-controlled promoter expression vector49pVSBADstrr, spcr, pVS1 origin, AraC-controlled promoter expression vector49pT7-7StrepIIcarr, T7 promoter expression vector for N-terminal StrepII affinity peptide fusions34pT7-H6TrxFuscarr, T7 promoter expression vector, for N-terminal H6 TrxA affinity peptide fusions33pLS1carr, IncQ plasmid for VirB/D4-mediated conjugative transfer experiments50pGK217carr, pUC18 with 11-kb virB fragment from A. tumefaciens51pUCvirBcarr, pUC18 with 12-kb virB fragment from B. suis52pVSBADVirB4pVSBAD carrying a 2.4-kb Acc65I/PstI virB4 fragment from A. tumefaciens C58This workpVSBADVirB4K439RpVSBADVirB4, mutated A. tumefaciens virB4 gene, encoding Walker A site K439R variantThis workpVSBADNcoVirB4spVSBADNco carrying 2.5-kb NcoI/XbaI virB4 fragment from B. suisThis workpVSBADNcoVirB4sK464RpVSBADNcoVirB4s, mutated B. suis virB4 gene, encoding Walker A site K464R variantThis workpT7-7StrepIIVirB4spT7-7StrepII carrying 2.5-kb Acc65I/EcoRI virB4 fragment from B. suisThis workpT7-7StrepIIVirB4s-VirB8sppT7-7StrepIIVirB4s bicistron with 492-bp EcoRI/PstI virB8 fragment from B. suis (encoding 163-amino acid periplasmic domain)This workpT7-7StrepIIVirB4s-VirB8spT7-7StrepIIVirB4s bicistron with 720-bp EcoRI/PstI virB8 fragment from B. suis (encoding 239-amino acid, full-length protein)This workpT7-7StrepIIVirB5sppT7-7StrepII carrying 666-bp Acc65I/PstI virB5 fragment from B. suis (encoding 221-amino acid periplasmic domain)This workpT7-7StrepIIVirB8sppT7-7StrepII carrying 492-bp Acc65I/PstI virB8 fragment from B. suis (encoding 163-amino acid periplasmic domain)53pT7-7StrepIIVirB9sppT7-7StrepII carrying 813-bp Acc65I/PstI virB9 fragment from B. suis (encoding 271-amino acid periplasmic domain)This workpT7-7StrepIIVirB10sppT7-7StrepII carrying 1020-bp Acc65I/PstI virB10 fragment from B. suis (encoding 339-amino acid periplasmic domain)This workpT7-H6TrxVirB5sppT7-H6TrxFus carrying 666-bp Acc65I/PstI virB5 fragment from B. suis (encoding 221-amino acid periplasmic domain)54pT7-H6TrxVirB8sppT7-H6TrxFus carrying 492-bp Acc65I/PstI virB8 fragment from B. suis (encoding 163-amino acid periplasmic domain)54pT7-H6TrxVirB9sppT7-H6TrxFus carrying 813-bp Acc65I/PstI virB9 fragment from B. suis (encoding 271-amino acid periplasmic domain)This workpT7-H6TrxVirB10sppT7-H6TrxFus carrying 1020-bp Acc65I/PstI virB10 fragment from B. suis (encoding 339-amino acid periplasmic domain)This workpGADT7carr, GAL4 AD fusion vector, LEU2 marker for selection in yeastClontechpGBKT7kanr, GAL4 DNA-BD fusion vector, TRP1 marker for selection in yeastClontechpGADT7-TpGADT7 construct; encodes fusion of GAL4 AD and SV40 T-antigen; positive controlClontechpGBKT7-53pGBKT7 construct; encodes fusion of GAL4 DNA-BD and mouse-p53, positive controlClontechpGADT7-VirB5sppGADT7 carrying 657-bp NdeI/BamHI virB5 fragment from B. suis (encoding 218-amino acid periplasmic domain)This workpGADT7-VirB8sppGADT7 carrying 510-bp NdeI/BamHI virB8 fragment from B. suis (encoding 170-amino acid periplasmic domain)This workpGADT7-VirB10sppGADT7 carrying 1020-bp NdeI/BamHI virB10 fragment from B. suis (encoding 339-amino acid periplasmic domain)This workpGBKT7-VirB5sppGBKT7 carrying 657-bp NdeI/BamHI virB5 fragment from B. suis (encoding 218-amino acid periplasmic domain)This workpGBKT7-VirB8sppGBKT7 pGADT7 carrying 510-bp EcoRI/PstI virB8 fragment from B. suis (encoding 170-amino acid periplasmic domain)This workpGBKT7-VirB9sppGBKT7 carrying 816-bp NcoI/PstI virB9 fragment from B. suis (encoding 272-amino acid periplasmic domain)This workpGBKT7-VirB10sppGBKT7 carrying 1020-bp EcoRI/PstI virB10 fragment from B. suis (encoding 339-amino acid periplasmic domain)This work Open table in a new tab Plasmid and Strain Constructions and Mutagenesis—DNA manipulations followed standard procedures (28Maniatis T.A. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). A. tumefaciens virB genes were amplified from pGK217, and B. suis virB4 genes were amplified from pUCvirB with oligonucleotides, cleaved with restriction sites, and cloned into vectors with compatible sites (Table II). The codons determining the ATP-binding site Lys residues in VirB4 and VirB4s were changed to Arg using the in vitro site-directed mutagenesis system (Promega). The sequences of PCR-amplified genes were confirmed by DNA sequencing.Table IIOligonucleotide sequencesName/restrictionSequenceConstructed plasmidvirB4 expressionVirB4-5′/Acc65I5′-CGGGGTACCCACAGGAAACAGACCATGCTCGGAGCA-3′pVSBADVirB4VirB4-3′/PstI5-GCGGCTGCAGTCAATCTTTTGCCTCGTGGTAA-3′VirB4s-5′/NcoI5′-GCGGCCATGGTGGGCGCTCAATCCAAATACGCGCAA-3′pVSBADNcoVirB4sVirB4s-3′/XbaI5′-GGCGTCTAGATCACCTTCCTGTTGATTTGGACGACG-3′VirB4M55′-CCCATCGGGAGGGGTAGAACGACGCTCATGACCTTT-3′pVSBADVirB4K439RVirB4sM55′-CCAGTCCGGCGCTGGTAGAACTGTCCTGATGAACTTCTGC-3′pVSBADNcoVirB4sK464RVirB protein overproductionT7B4suis5′/Acc65I5′-GCGCGCGGTACCCATGATGGGCGCTCAATC-3′pT7-7StrepIIVirB4sT7B4suis3′/EcoRI5′-GCGCGAATTCTCATTGCAGGTTCTCCCCGGGC-3′T7B8suisBCsp-5′/EcoRI5′-CCAGAATTCGAAGGAGATATATATATGCGCGTCAACGCACAGACAGGTGCGCCT-3′T7B8suisBCsp-3′/PstI5′-AAAACTGCAGTCATTGCACCACTCCCATTTCTGGATCAAC-3′pT7-7StrepIIVirB4s-VirB8spT7B8suisBCs-5′/EcoRI5′-CCAGAATTCGAAGGAGATCTAATCATGTTTGGACGCAAACAATC-3′T7B8suisBCs-3′/PstI5′-AAAACTGCAGTCATTGCACCACTCCCATTTCTGGATCAAC-3′pT7-7StrepIIVirB4s-VirB8sT7B5suis5′/Acc65I5′-CAGGGTACCCGCGCACGCGCAGCTCC-3′ (54Rouot B. Alvarez-Martinez M.-T. Marius C. Mentanteau P. Guilloteau L. Boigegrain R.-A. Zumbihl R. O'Callaghan D. Domke N. Baron C. Infect. Immun. 2003; 71: 1075-1082Crossref PubMed Scopus (62) Google Scholar)pT7-7StrepIIVirB5sp andT7B5suis3′/PstI5′- GAGCTGCAGCTAATAGGCGGCTTCCAGTGC-3′ (54Rouot B. Alvarez-Martinez M.-T. Marius C. Mentanteau P. Guilloteau L. Boigegrain R.-A. Zumbihl R. O'Callaghan D. Domke N. Baron C. Infect. Immun. 2003; 71: 1075-1082Crossref PubMed Scopus (62) Google Scholar)pT7H6TrxFusVirB5spT7B8suis5′/Acc65I5′-CAGGGTACCCCGCGTCAACGCACAGAC-3′ (54Rouot B. Alvarez-Martinez M.-T. Marius C. Mentanteau P. Guilloteau L. Boigegrain R.-A. Zumbihl R. O'Callaghan D. Domke N. Baron C. Infect. Immun. 2003; 71: 1075-1082Crossref PubMed Scopus (62) Google Scholar)pT7-7StrepIIVirB8sp andT7B8suis3′/PstI5′- GAGCTGCAGCTATTGCACCACTCCCATTTCTGG-3′ (54Rouot B. Alvarez-Martinez M.-T. Marius C. Mentanteau P. Guilloteau L. Boigegrain R.-A. Zumbihl R. O'Callaghan D. Domke N. Baron C. Infect. Immun. 2003; 71: 1075-1082Crossref PubMed Scopus (62) Google Scholar)pT7H6TrxFusVirB8spT7B9suis5′/Acc65I5′-CAGGGTACCCCCGTCCGGCTCAAAATACG-3′pT7-7StrepIIVirB9sp andT7B9suis3′/PstI5′-GAGCTGCAGCTATTGCAGGTTCTCCCCGGGC-3′pT7H6TrxFusVirB9spT7B10suis5′/Acc65I5′-CAGGGTACCCCACATGAGGGGCAATGCAG-3′pT7-7StrepIIVirB10sp andT7B10suis3′/PstI5′-GAGCTGCAGCTACTTCGGTTGGACATCATACAC-3′pT7H6TrxFusVirB10spYeast two-hybrid analysisBKT7-B5sp-5/NdeI5′-CGCGCGCATATGCAGCTCCCGGTGACAGATG-3′pGBKT7-VirB5sp andBKT7-B5sp-3/BamHI5′-GCGCGCGGATCCATAGGCGGCTTCCAGTGCTTT-3′pGADT7-VirB5spBKT7-B8sp-5/EcoRI5′-GCGCGCGAATTCCAACATGTGCCCTACCTGGTG-3′pGBKT7-VirB8spBKT7-B8sp-3/PstI5′-CGCGCGCTGCAGTTGCACCACTCCCATTTCTGGATG-3′BKT7-B9sp-5′/NcoI5′-CGCGCGCCATGGACCAAAATCCCGTCCGGCTCA-3′pGBKT7-VirB9spBKT7-B9sp-3/PstI5′-CGCGCGCTGCAGTTGCAGTTGCAGGTTCTCCCCGGGC-3′BKT7-B10sp-5/EcoRI5′-GCGCGCGAATTCCACATGAGGGGCAATGCAGAG-3′pGBKT7-VirB10spBKT7-B10sp-3/PstI5′-CGCGCGCTGCAGCTTCGGTTGGACATCATACACACT-3′ADT7-B8sp-5/NdeI5′-CGCGCGCATATGCAACATGTGCCCTACCTGGTG-3′pGADT7-VirB8spADT7-B8sp-3/BamHI5′-GCGCGCGGATCCTTGCACCACTCCCATTTCTGGATG-3′ADT7-B10sp-5/NdeI5′-CGCGCGCATATGCACATGAGGGGCAATGCAGAG-3′pGADT7-VirB10spADT7-B10sp 3/BamHI5′-GCGCGCGGATCCCTTCGGTTGGACATCATACACACT-3′ Open table in a new tab Analysis of T4SS Functions: T-pilus Isolation, Conjugation, and Virulence Assays—Assays for T4SS functionality (T-pilus isolation, conjugation, and virulence assays) were performed as previously described (29Höppner C. Liu Z. Domke N. Binns A.N. Baron C. J. Bacteriol. 2004; 186: 1415-1422Crossref PubMed Scopus (42) Google Scholar). Transmission Electron Microscopy—A. tumefaciens strains to be examined were cultivated on AB agar plates in the presence or absence of AS and ARA for gene induction. Cells were collected with 5 ml of 50 mm sodium potassium phosphate buffer, pH 5.5, and the cell density was adjusted to A600 of 1.5-2. 10 μl were applied onto UV-sterilized 200 mesh carbon-coated formvar copper electron microscopy grids and air dried in a laminar flow hood for 10 min. The grids were then stained with 2% phosphotungstic acid-0.01% glucose, pH 6, for 15 s prior to examination. Specimen images were taken with a JEOL 1200EX II transmission electron microscope. T-pili on 300 cells from three independent virulence induction experiments of each strain were counted (10 cells per visual field were analyzed). Membrane Isolation, Detergent Extraction, Blue Native Electrophoresis, and Gel Filtration—Isolation of membranes and detergent extraction with 2% dodecyl-β-d-maltopyranoside (DDM) were performed as previously described (20Krall L. Wiedemann U. Unsin G. Weiss S. Domke N. Baron C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11405-11410Crossref PubMed Scopus (109) Google Scholar). Blue native gradient gel electrophoresis (7-14% acrylamide, 16-cm-long gel) was performed for 20 h and 5 mA at 4 °C. Alternatively, for separation of small proteins, blue native gel electrophoresis was performed in a linear 15% polyacrylamide gel for 5-6 h and 5 mA at 4 °C in a mini-gel system (10-cm-long gel). Calibration was achieved by separation of reference proteins of known molecular masses: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (14 kDa, Amersham Biosciences). For gel filtration, 100 μl of 2% DDM-solubilized membrane proteins were applied to a Superdex 200 or Superdex 75 (XK 16/40; Amersham Biosciences), and chromatography was performed in 6-amino-caproic acid buffer (750 mm 6-amino-caproic acid, 50 mm BisTris, pH 7) containing 0.03% DDM performed at 4 °C with a flow rate of 0.5 ml/min in an Äkta fast protein liquid chromatography purifier (Amersham Biosciences). 100 μl of calibration kit proteins (ferritin, 1 mg/ml; all other proteins, 10 mg/ml) were separated in 6-amino-caproic acid buffer with 0.03% DDM. SDS-PAGE and Western Blotting—Cells were incubated in Laemmli sample buffer for 5 min at 100 °C followed by SDS-PAGE. Chromatography samples were incubated in Laemmli sample buffer buffer for 30 min at 37 °C to avoid aggregate formation followed by SDS-PAGE using the Laemmli (for proteins larger than 20 kDa) or the Schägger and von Jagow system (for proteins smaller than 20 kDa) (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar, 31Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar). Western blotting was performed following standard protocols (32Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar), with VirB protein-specific antisera. Purification of Fusion Proteins—N-terminally hexahistidyl-thioredoxin (H6TrxA)-tagged proteins were overproduced in GJ1158 using pT7-H6TrxFus constructs (33Kromayer M. Wilting R. Tormay P. Böck A. J. Mol. Biol. 1996; 262: 413-420Crossref PubMed Scopus (98) Google Scholar). Cells from 400-ml cultures were suspended in 5 ml of lysis buffer (50 mm Hepes, 200 mm KCl, 5 mm MgCl2, pH 7.5) with 0.5 mm phenylmethylsulfonyl fluoride and lysed by a French Pressure Cell (Aminco) at 18,000 p.s.i. The lysate was centrifuged twice (SS34 rotor, 30 min, 13,000 rpm at 4 °C), and the supernatant was applied to a high pressure liquid chromatography system (Äkta Purifier; Amersham Biosciences) with a Ni2+-charged IMAC column (Talon™ Superflow; Clontech). Tagged proteins were eluted using a step gradient. At a flow rate of 0.5 ml/min, the column was first washed for 5 column volumes in buffer A (50 mm Hepes, 0.3-1 m NaCl, pH 7.5), followed by washing with buffer A with 20 mm imidazole (2.5 column volumes) and elution with buffer B (buffer A with 400 mm imidazole). The fractions were dialyzed overnight in H1B (50 mm Hepes, 200 mm NaCl, pH 7.5), followed by a second dialysis overnight in H2B (H1B with 50% glycerol and 2 mm dithiothreitol) and storage at -20 °C. N-terminally StrepII-tagged proteins were overproduced in GJ1158 using pT7-7StrepII (34Balsinger S. Ragaz C. Baron C. Narberhaus F. J. Bacteriol. 2004; 186: 6824-6829Crossref PubMed Scopus (26) Google Scholar), and 400-ml cultures were lysed in 10 ml of S2B (0.1 m Tris-HCl, pH 8, 0.15 m NaCl, 1 mm EDTA, pH 8) with 0.5 mm phenylmethylsulfonyl fluoride. The cells were lysed, and the supernatant was collected as described above. Fusion proteins were purified with a 1-ml Strep-Tactin Superflow® column (IBA, Göttingen, Germany) following the instructions of the manufacturer using 2.5 mm desthiobiotin in the elution buffer. The fractions were subsequently purified by size exclusion chromatography using S2B at a flow rate of 0.5 ml/min. Superdex 75 or Superdex 200 (Amersham Biosciences) was used, depending on the molecular mass of the protein. The proteins were then dialyzed overnight in PSB (S2B buffer with 50% glycerol and 2 mm dithiothreitol) and stored at -20 °C. Assays for Protein-Protein Interactions: Pull-down and Cross-linking Experiments—For pull-down experiments, 10 μl each of purified StrepII- and H6TrxA-tagged proteins concentrated at 5 pmol/μl in PSB were mixed, followed by the addition of 80 μl of S2B and incubation for 30 min at 22 °C. Next, 20 μl of Strep-Tactin Sepharose beads (50% suspension in S2B; IBA) were added, followed by a 15-min incubation at 22 °C. The Sepharose beads were subsequently sedimented by centrifugation and washed three times with 200 μl of S2B. Bound proteins were eluted with 50 μl of S2B with 1 mm biotin, mixed with 1 volume of Laemmli sample buffer, and analyzed by SDS-PAGE and Western blotting. For chemical cross-linking, 5 μl each of 10 pmol/μl stock solutions of purified StrepII-tagged proteins in PSB (or 5 μl of PSB as negative control) were mixed for 5 min at 22 °C, 90 μl of CLB (50 mm MES-KOH, 150 mm NaCl, 1 mm EDTA, pH 6.5) were added, and the mixture was incubated for 30 min. The cross-linking agent disuccinimidyl suberate (10 mm stock in Me2SO; Pierce) was added in different concentrations (0.05 and 0.1 μm), and the samples were incubated for 1 h at 22 °C, followed by the addition of 1 volume of Laemmli sample buffer and analysis by SDS-PAGE and Western blotting. Yeast Two-hybrid Assay—The Matchmaker 3 system (Clontech) was used for the analysis of protein-protein interactions with the yeast two-hybrid system following the manufacturer's protocols. The genes encoding the periplasmic domains VirB5sp, VirB8sp, VirB9sp, and VirB10sp were cloned into pGADT7 (GAL4 activation domain fusion) and pGBKT7 (DNA binding domain fusion) as described above. The plasmids were transformed into Saccharomyces cerevisiae AH109 using the lithium acetate method (Clontech Manual; Clontech), and plasmid-carrying cells were selected on minimal medium without leucine and tryptophan. Interactions between VirB proteins, which tethered both domains of GAL4 together, were identified by growth of plasmid-carrying cells on minimal medium without histidine and adenine and by β-galactosidase activity. VirB4 Stabilizes VirB3 and VirB8—To study the role of VirB4, we deleted virB4 in the Ti plasmid of wild-type C58, resulting in strain CB1004 (ΔvirB4). Next, virulence genes were induced by cultivation on acidic minimal medium with acetosyringone followed by SDS-PAGE and Western blotting. Specific antisera were applied to compare the VirB protein levels in cells of non-induced and virulence gene-induced C58 (controls) and in CB1004. The levels of most VirB proteins were comparable in C58 and CB1004 (Fig. 1B), but VirB3, VirB8, and VirB4 were not detected or were detected only in very small quantities in CB1004 (Fig. 1A). Whereas the absence of VirB4 was expected, and reduced levels of VirB3 had been reported previously (19Jones A.L. Shirasu K. Kado C.I. J. Bacteriol. 1994; 176: 5255-5261Crossref PubMed Google Scholar), the reduced levels of VirB8 showed that VirB4 plays a role for the accumulation of this protein. A previous study in a different A. tumefaciens strain suggested that the structure of VirB4, but not its NTPase activity, is required for VirB protein stabilization (8Dang T.A. Zhou X.-R. Graf B. Christie P.J. Mol. Microbiol. 1999; 32: 1239-1253Crossref PubMed Scopus (81) Google Scholar). To assess this possibility, A. tumefaciens virB4 was cloned behind the tightly controlled E. coli arabinose (BAD) promoter of broad host range vector pVSBAD. An active site change was engineered, and the cloning vector pVSBAD and constructs encoding VirB4 and its Walker A derivative (VirB4K439R) were introduced into CB1004. Analysis of the resulting strains demonstrated that production of VirB4 as well as production of VirB4K439R restored the levels of VirB3 and VirB8 (Fig. 1A). To further study the requirement of VirB4 sequence and structure, we cloned the gene encoding the B. suis VirB4 homolog (VirB4s) into pVSBADNco and engineered an active site change (VirB4sK464R). The production of both VirB4s and VirB4sK464R fully restored levels of VirB3 and VirB8 (Fig. 1A). When CB1004 complemented with plasmids encoding VirB4 or VirB4s (data not shown) was
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