1 Ludwig-Maximilians-Universität, Department Biologie I, Bereich Mikrobiologie, Maria-Ward-Str. 1a, D-80638 München, Germany
2 McMaster University, Department of Biology, 1280 Main St West, Hamilton, ON, Canada LS8 4K1
3 Ludwig-Maximilians-Universität, Gene Center, Feodor-Lynen Str. 25, D-81377 München, Germany
Correspondence
Christian Baron
baronc{at}mcmaster.ca
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ABSTRACT |
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INTRODUCTION |
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VirB1 is the only non-essential T4SS component. It was previously demonstrated that VirB1 homologues play an important role in the T4SS of A. tumefaciens and Escherichia coli strains harbouring plasmids pKM101 and R1. The efficiency of substrate transfer was reduced 10- to 1000-fold upon non-polar deletion of the encoding genes (Bayer et al., 1995; Berger & Christie, 1994
; Fullner, 1998
; Winans & Walker, 1985
). An infection assay with signature-tagged Brucella abortus mutants demonstrated that mutagenesis of the virB1 gene causes attenuation of virulence (Hong et al., 2000
). A more recent study demonstrated that survival of B. abortus in macrophage cell cultures was attenuated in strains carrying a non-polar virB1 mutation (den Hartigh et al., 2004
). Thus the deletion of genes encoding VirB1 homologues generally has an attenuating effect on T4SS-related functions. The Helicobacter pylori VirB1 homologue HP0523 is an exception to this rule, as it was shown to be essential for bacterial virulence (Odenbreit et al., 2001
; Rohde et al., 2003
). Due to the presence of highly conserved sequence motifs, VirB1 was identified as a putative lytic transglycosylase, but its specific role for T4SS function was not elucidated in detail. A. tumefaciens and Brucella suis VirB1 both possess a signal sequence and are therefore directed to the periplasmic space by the general secretion pathway (Llosa et al., 2000
; O'Callaghan et al., 1999
). Their enzymic activity probably leads to localized cell wall lysis, creating space for accommodation of the T4SS (Bayer et al., 2001
; Mushegian et al., 1996
; Zahrl et al., 2005
). Despite the well-known area of lysozyme biochemistry, proposals for the catalytic mechanism of lytic transglycosylases were published only recently. Whereas the well-known lysozymes like hen egg white lysozyme break up murein by hydrolysis of the
(1
4)-glycosidic bond between the N-acetylmuramic acid (MurNAc)-C1 and the N-acetylglucosamine (GlcNAc)-C4, the lytic transglycosylases lyse this substrate in a transglycosylation reaction utilizing the C6-OH residue of the same MurNAc (Blackburn & Clarke, 2001
; Koraimann, 2003
). Thus, no water is required for the reaction, which produces a 1,6-anhydromuramic acid terminal residue. Special features of the active site that distinguish the lytic transglycosylases from lysozymes must explain the mechanistic difference, and this question is subject to structural biological studies (Lehnherr et al., 1998
; Leung et al., 2001
; Mushegian et al., 1996
; Thunnissen et al., 1994
; van Asselt et al., 1999
, 2000
).
When VirB1 was identified as a lytic transglycosylase, its importance for the A. tumefaciens T4SS was largely attributed to its proposed catalytic activity. This notion was repeatedly confirmed by the observation that active-site mutants of the protein failed to fully complement virB1 deletion strains (Höppner et al., 2004; Mushegian et al., 1996
). After export across the inner membrane, VirB1 of A. tumefaciens is further processed in the periplasm, yielding a processing product of the C-terminal 73 amino acids designated VirB1* (Baron et al., 1997
). VirB1* and the N-terminus, representing the lytic transglycosylase domain, independently enhanced tumorigenicity, which implied an additional function of VirB1* (Llosa et al., 2000
). Further evidence for this hypothesis was generated when it was shown by co-immunoprecipitation that VirB9 interacts with VirB1* in A. tumefaciens (Baron et al., 1997
). A high-resolution dihybrid screen with protein components of the A. tumefaciens T4SS suggested self-interaction and a number of uni- and bidirectional interactions between VirB1 and VirB4, VirB8, VirB9, VirB10 and VirB11 (Ward et al., 2002
). Direct biochemical evidence for the interactions was not presented in that study.
In spite of the low overall amino acid sequence identity of 22 %, the VirB1 homologue from B. suis complemented virB1 gene defects in A. tumefaciens, suggesting that it engages in similar interactions with T4SS components. Similarly, the B. suis VirB4 homologue complemented virB4 gene defects in A. tumefaciens (Yuan et al., 2005), which further supported the notion that the overall architecture of different T4SS is very similar (Christie, 2004
; Yeo & Waksman, 2004
). To directly test interactions of VirB1sp with T4SS core components we chose derivatives of VirB proteins from B. suis, (abbreviated VirBs in the following or VirBsp to indicate periplasmic domains without signal peptides or membrane domains), which are more readily amenable to overproduction and purification than those from A. tumefaciens. Using different biochemical methods (affinity precipitation, gel filtration, bicistron expression, peptide array analysis), we showed that purified B. suis T4SS core components undergo different interactions. VirB1sp was found to interact with VirB9sp, and whereas this interaction was the strongest among those we investigated, VirB1sp also bound to VirB8sp and VirB11s. The binding sites were localized in a structure model of VirB1sp, suggesting that a sequence of transient interactions guides lytic transglycosylase function during T4SS assembly.
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METHODS |
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Molecular biology methods.
Manipulations of DNA for plasmid isolation, PCR amplification, restriction, ligation and sequencing followed standard procedures, using enzymes from New England Biolabs and MBI Fermentas and E. coli JM109 as cloning host (Maniatis et al., 1982; Yanisch-Perron et al., 1985
). PCR fragments were first cloned into pCR2.1-TOPO (Invitrogen), followed by sequencing and further subcloning into expression vectors as described below.
Construction of virB gene expression vectors.
Expression vectors for the production of VirBs proteins (Table 1) were constructed by PCR amplification of the genes with oligonucleotides, which introduced 5' and 3' restriction sites (sequences given in Table 2
), followed by ligation into similarly cleaved vectors. Constructs for the overproduction of N-terminally tagged StrepIIVirB1sp and StrepIIVirB11s were generated by PCR amplification of the genes, cleavage with Acc65I/PstI and Acc65I/HindIII, and ligation into similarly cleaved pT7-7StrepII (pT7-7StrepIIVirB8sp, pT7-7StrepIIVirB9sp and pT7-7StrepIIVirB10sp were described previously; Yuan et al., 2005
). The gene encoding VirB11s was subsequently subcloned using the same restriction sites into pT7-H6TrxFus for expression as an N-terminally His6TrxA-tagged fusion protein (pT7-H6TrxVirB8sp, pT7-H6TrxVirB9sp and pT7-H6TrxVirB10sp were described previously; Yuan et al., 2005
). Similar procedures were applied for the cloning of VirB7s into pT7-H6TrxFus using XbaI/PstI restriction sites. pET24dVirB1spHis6 for overproduction of C-terminally His6-tagged VirB1sp was constructed by PCR cloning of the virB1 gene and introduction of the NcoI/NotI-treated fragment into similarly cleaved pET24d.
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Strep-Tactin Sepharose chromatography.
Bacterial cells were resuspended in 48 ml StrepII buffer (S2B) without DTT (300 mM NaCl, 100 mM Tris/HCl, 1 mM EDTA, pH 7·0) and with 0·5 mM PMSF and passed three times through a French pressure cell (Aminco) at 18 000 p.s.i. The lysate was centrifuged (SS34 rotor, 25 min, 13 000 r.p.m. at 4 °C) to remove cell debris and unbroken cells, and the N-terminal StrepII-fusion protein was purified with a 1 ml Strep-Tactin Superflow column following the instructions of the manufacturer (IBA), 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 min1. Superdex 75 or Superdex 200 (Amersham Pharmacia Biosciences) was used depending on the molecular mass of the protein. The samples were dialysed for >12 h against 1 litre of PSB (S2B with 50 % glycerol) in dialysis tubing (Visking, Roth) at 4 °C and were stored at 20 °C until further use. Protein concentrations were determined using the Bradford dye binding assay (Bio-Rad) with bovine serum albumin as a reference.
Immobilized metal chelate affinity chromatography (IMAC).
Cells were lysed in S2B without DTT (0·5 mM PMSF) and centrifuged as described above; the supernatant was applied to an HPLC system (Äkta Purifier, Amersham Pharmacia Biotech) with a 5 ml Co2+-charged IMAC column (Talon Superflow, Clontech). His6-tagged recombinant protein was eluted according to a step-gradient protocol. At a flow rate of 0·51·0 ml min1 the column was first washed for 5 column volumes. Then, a stringent washing step with 20 mM imidazole proceeded for 2·5 column volumes, before 150 mM imidazole was applied to the column for 2·5 column volumes. Both the stringent wash fractions and the elution fractions were collected in 2 ml aliquots, followed by gel filtration, dialysis in PSB and determination of the protein concentrations as described above.
Strep-Tactin Sepharose pull-down assay.
Samples (10 µl) of purified StrepII-tagged proteins (5 pmol µl1 in PSB) were incubated with 20 µl Strep-Tactin Sepharose (50 % suspension in S2B, IBA) for 15 min. Then 80 µl S2B and 10 µl His6TrxA-fusion protein (5 pmol µl1 in PSB) were added. After 15 min incubation at room temperature, the Sepharose matrix was sedimented (centrifugation at 13 000 r.p.m., 2 min) and washed three times with 500 µl S2B. Bound proteins were eluted with 35 µl 1 mM biotin followed by sedimentation of the matrix, mixing of the supernatant with 1 vol. Laemmli sample buffer, SDS-PAGE, Western blotting and analysis with VirB protein-specific antisera.
Gel filtration chromatography.
Samples generated by affinity chromatography (1 ml maximum) were loaded onto Superdex 200 or Superdex 75 gel filtration columns in S2B; the flow rate was 0·5 ml min1 or 1·0 ml min1. To determine the molecular mass of proteins, the columns were calibrated with the Gel Filtration Calibration Kit (Amersham Pharmacia Biotech), which uses reference proteins in the range between 13·7 and 669 kDa.
SDS-PAGE and Western blotting.
Proteins were separated in denaturing SDS gels using the Laemmli system (Laemmli, 1970) followed by transfer to PVDF membranes (Immobilon-P, Millipore) in a vertical blot device (Trans Blot Cell, Bio-Rad; blot buffer 192 mM glycine, 25 mM Tris, 20 % methanol) at 90 V for 1 h or 30 V for 16 h (Harlow & Lane, 1988
). Proteins attached to peptide array membranes (see below) were transferred in a semi-dry blot device (Fast-Blot, Biometra) onto PVDF membranes following a specialized protocol, followed by regeneration of the membrane as suggested by the manufacturer (Jerini). Proteins were detected with goat anti-rabbit IgG-HRP (Bio-Rad), a chemiluminescence detection system (Lumi Light, Roche Diagnostics) and X-ray film (Harlow & Lane, 1988
).
Peptide array experiments.
The entire sequence of VirB1sp from B. suis (GenBank accession no. NP_699276) without the signal peptide was displayed on a cellulose membrane as seventy 13-mers, covalently bound at the C-terminus and with N-terminal acetylation, shifting three amino acid positions each time, beginning with peptide 1 (AAIVQVESGFNPY), peptide 2 (VQVESGFNPYAIG), etc., to peptide 70 (PPGKDNTDGVVVF). The protocol for Mapping of discontinuous epitopes' from the manual of the supplier (Jerini) was followed. The peptide array membrane, which features all possible linear epitopes of VirB1sp, was preincubated for 30 min in TBS-T (20 mM Tris/HCl; 137 mM NaCl; 0·1 % Tween-20; pH 8·0), transferred into blocking solution (Roche) for 1 h, washed again with TBS-T for 10 min and then incubated in blocking solution containing 15 µg ml1 of different proteins (StrepIIVirB1sp, StrepIIVirB8sp, StrepIIVirB9sp or StrepIIVirB11s) for 12 h at 4 °C. Before transfer of the attached proteins onto PVDF membranes with a semi-dry blot device (see above), the peptide array membrane was washed three times in TBS-T for 10 s to remove non-specifically bound protein.
Generation of polyclonal antisera.
Soluble StrepIIVirB11s and H6TrxAVirB7 were purified by affinity chromatography as described above, whereas StrepIIVirB1sp was obtained from a preparation of inclusion bodies, separated by SDS-PAGE, excised from the gel and subjected to electroelution. Approximately 0·5 mg of each protein was lyophilized and used for immunization of rabbits (BioGenes) to generate specific antisera. The other antisera used in this study were described elsewhere (Yuan et al., 2005).
Graphical data processing.
To capture images of polyacrylamide gels and chemoluminogramms, they were digitized using a UMAX UTC-6400 scanner, followed by processing with Photoshop 6.0 (Adobe) and Canvas 7.0 (Deneba Systems).
Protein sequence analysis.
The CLUSTAL W (version 1.82) algorithm for multiple sequence alignment (Higgins, 1994) (http://www.ebi.ac.uk/clustalw) or EMBOSS for alignment of two less conserved amino acid sequences (Needleman & Wunsch, 1970
; Smith & Waterman, 1981
) (http://www.ebi.ac.uk/emboss/align) were applied. Sequence information was processed with NORSp (Liu & Rost, 2003
) (http://cubic.bioc.columbia.edu/services/NORSp) in order to discover long regions without regular secondary structure. Predictions of secondary structure were obtained with the PHD algorithm (Rost, 1996
) (http://www.embl-heidelberg.de/predictprotein). To create a conservation plot of sequence alignment, the alignment data were transferred to the AMAS server (Livingstone & Barton, 1993
) (http://barton.ebi.ac.uk/servers/amas_server.html) using standard default values. All structure images were generated with DINO 9.0 (http://cobra.mih.unibas.ch/dino/intro.php).
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RESULTS |
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We subsequently followed an alternative strategy based on the observation that recombinant proteins produced in E. coli are often insoluble or misfolded, and one reason for this is that their natural binding partners are absent. One method to circumvent this problem is the co-expression of the genes encoding such proteins together with genes encoding potential interaction partners (Lutzmann et al., 2002). To this end, the potential interactions of VirB1sp with other components of the B. suis T4SS were tested by expression from vectors encoding C-terminally tagged VirB1spHis6 fusion proteins and their putative interaction partners VirB8sp, VirB9sp or VirB10sp. In order to co-produce His6-tagged VirB1sp with putative interaction partners, vector pET21BC was constructed to permit expression of bicistronic mRNAs. The first ORF of the pET21BC series encoded the putative interaction partners VirB8sp, VirB9sp and VirB10sp, and the second one VirB1spHis6. In addition, a series of pET21BC-derived monocistronic constructs for the expression of the non-tagged interaction partners VirB8sp, VirB9sp or VirB10sp without VirB1spHis6 was generated to serve as controls.
The proteins encoded on the pET21BC-derived vectors were overproduced in E. coli, followed by cell lysis, purification over a His6-tag-specific affinity column and Superdex 200 gel filtration for analysis of complex formation. In spite of the two-step separation procedure, the untagged VirBsp proteins from both monocistronic and bicistronic expression experiments were detected in the gel filtration eluates, indicating that VirB8sp, VirB9sp and VirB10sp had non-specific binding affinity to the column. In order to distinguish between co-elution due to similar molecular masses and co-elution as an effect of an interaction, we compared the elution after expression from a bicistronic expression vector with that after expression from a monocistronic vector. VirB8sp eluted as a monomer in fractions 2428 from the gel filtration in both cases, and VirB1spHis6 eluted in fractions 1721, supposedly the tetrameric and dimeric form (Fig. 2a). The co-expression with virB8 thus had no apparent effect on the elution of VirB1spHis6 and vice versa, suggesting that these two proteins did not interact. When VirB1spHis6 was produced from the bicistronic vector with VirB10sp it eluted in fractions 1719, which corresponded to the molecular mass expected for the tetramer (Fig. 2c
). VirB10sp produced in strains carrying the monocistronic as well as the bicistronic vector eluted in fractions 1719 as a dimer. As the molecular masses of VirB1spHis6 and VirB10sp were very similar, this experiment did not give any evidence for an interaction. In contrast, when VirB1spHis6 was produced from the bicistronic vector with VirB9sp it eluted from the column in three forms (Fig. 2b
). First, it was detected in fractions 69, representing a large complex that had a lower molecular mass than that of VirB1spHis6 when it was expressed from a monocistronic vector. Second, it eluted in fractions 1517, corresponding to a complex markedly larger than the hexamer of 160 kDa, and third, it was detected in fractions 2123, corresponding to a size between the dimer and tetramer. VirB9sp eluted in fractions 69 and 1517 but the largest portion of the protein was monomeric (fractions 2225). In contrast, when VirB9sp was expressed from the monocistronic vector, it predominantly eluted in fractions 2225, which corresponded to the supposed monomer. It is evident from the comparison of the elution profiles that the co-expression with VirB9sp affected the oligomeric state of VirB1spHis6 and vice versa. Whereas this method did not permit the unambiguous identification of hetero-oligomer formation, the results support a direct interaction between the two proteins. Alternative methods were employed in the following to further assess this possibility.
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DISCUSSION |
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The purified B. suis VirB proteins were then used to systematically assess the predicted interactions with VirB1sp. Some of those interactions were confirmed here, whereas others were only weak or not observed. Using co-elution and pull-down assays we did not get any evidence for an interaction between VirB1sp and VirB8sp or VirB10sp, which had been predicted from a previous yeast two-hybrid study (Ward et al., 2002). In contrast, data from the peptide array experiment suggested that a set of VirB1sp peptides interacted with StrepIIVirB8sp, which supports the notion that these two proteins interact at least transiently. The evidence is not as substantial as that in case of StrepIIVirB9sp discussed below, suggesting that the interaction is weaker and/or may need additional interaction partners. These results are in line with the supposed role of VirB8 as nucleation factor, which undergoes transient interactions with many T4SS components (Kumar & Das, 2001
; Yuan et al., 2005
). The in vitro data therefore argue against the postulated mechanism implying the VirB8VirB1 interaction as key step for T4SS assembly (Ward et al., 2002
), but we can not rule out that the interaction is stronger in vivo.
Similar to VirB8sp, we obtained evidence for an interaction of the hexameric ATPase VirB11s with VirB1sp using peptide array as well as pull-down experiments. The interaction between VirB1sp and VirB9sp was demonstrated using a variety of different assays such as a pull-down assay and co-elution of the two proteins following their co-expression in a bicistronic construct. Peptide array experiments identified several StrepIIVirB9sp-interacting peptides throughout the VirB1sp sequence, and a considerable portion of them was in the C-terminus. Whereas this might argue for non-specific binding of StrepIIVirB9sp to the VirB1sp-derived peptides, we do not favour this interpretation for the following reasons. First, we have tested the binding of StrepIIVirB9sp to a pepspot membrane displaying the VirB5sp sequence, and, in line with other results showing that it does not bind strongly to VirB5sp, no non-specific binding to the membrane was observed (unpublished observations). Second, we observed non-specific binding of StrepIIVirB1sp as reflected by binding to most peptides on the VirB1sp pepspot membrane. Both the extent and strength of the signal were drastically elevated as compared to the relatively modest signal of bound StrepIIVirB9sp (unpublished observations). Finally, the peptide array data are very much consistent with the results of a previous study in intact cells, which demonstrated the interaction of the VirB1 C-terminus with VirB9 in A. tumefaciens (Baron et al., 1997). We therefore conclude that the binding by StrepIIVirB9sp to many peptides of VirB1sp is likely to be of biological relevance.
The interaction with VirB9sp via the C-terminus is intriguing and this domain appears to play an important role for the functionality of the protein. Sequence analyses of a number of VirB1 homologues demonstrated special properties of the C-terminal part. The processed A. tumefaciens VirB1 C-terminus VirB1* (Baron et al., 1997) and also the C-termini of B. suis VirB1 and pKM101 TraL were classified as NORS regions. These are regions of more than 70 amino acids in length that show less than 12 % secondary structure elements and an amino acid composition different from loop regions. It was demonstrated that these very flexible regions show similar degrees of conservation as other domains in similar proteins, and that they are more abundant in proteins with functions as regulators or transcription factors than in those with functions in biosynthesis or energy metabolism-related proteins (Liu & Rost, 2003
). This implies important functional roles, most likely for transient proteinprotein interactions with different partners. Only 4 % of all prokaryotic proteins contain NORS regions, and among the VirB proteins from A. tumefaciens and B. suis, channel component VirB10, which is supposedly involved in a high number of interactions (Cascales & Christie, 2003
, 2004
), is the only other protein that possesses such a region. Most interacting amino acid stretches identified here constitute loop or NORS regions, which are especially suited for establishing transient proteinprotein interactions, suggesting that the interaction sites are biologically relevant.
To assess the biological relevance of the interactions and binding site(s) identified here, we pursued a modelling approach based on the known X-ray structure of a soluble lytic transglycosylase enzyme. To date, the structures of 18 murein-lytic lysozymes from different organisms have been solved. In addition, the three structures of the lytic transglycosylases LaL from bacteriophage , Slt35 and Slt70 (both from E. coli) are also available (Leung et al., 2001
; Thunnissen et al., 1994
; van Asselt et al., 2000
). The enzymic action of lysozymes and soluble lytic transglycosylases differs, but the protein fold is highly conserved (Mushegian et al., 1996
). Sequence comparison of B. suis VirB1 with two murolytic enzymes, whose X-ray structure was known, yielded intriguing results. The soluble lytic transglycosylase portion of Slt70 from E. coli ranges from amino acid P494 to A620 and has a significant degree of sequence similarity to B. suis VirB1 (identical, 23·1 %; similar, 38·1 %; gaps, 35·6 %). Other proteins like the lysozymes LysG from Anser anser (identical, 18·0 %; similar, 33·1 %; gaps, 23·8 %) and Gallus gallus LysC are less similar, although they were previously chosen to model the structure of A. tumefaciens VirB1 (Mushegian et al., 1996
). Structural superposition shows that despite an almost identical tertiary structure of LysG and Slt70 (Koraimann, 2003
), their sequence similarity (identical, 22·0 %; similar, 36·3 %; gaps, 32·7 %) is less than that between Slt70 and VirB1. It was therefore appropriate to use the surface model of Slt70 to visualize regions of VirB1sp interaction with other VirB proteins. If the amino acids identified here by peptide array analysis were important for interactions they would be expected to localize on the surface of a protein. We indeed localized the binding sites for StrepIIVirB8sp, StrepIIVirB9sp and StrepIIVirB11s on the surface of StrepIIVirB1sp, and these amino acids were not involved in the stabilization of the tertiary structure, which further substantiates the validity of the model. The model suggests that there was no apparent interference with the active-site cleft, but all three proteins bound C-terminally to residues likely to be involved in enzyme activity (regions 2 and 3 boxed in Fig. 5a
). Thus, the binding may modulate enzyme activity; this possibility will be directly addressed in future.
Taken together, the results presented here suggest that VirB1s is a self-interacting protein that establishes transient contacts with other VirB proteins, such as VirB8s, VirB9s and VirB11s. A comparison of these results with predictions and results of previous studies is given in Table 4. A large amount of information on the role of VirB1-like proteins was collected in previous studies (Baron et al., 1997
; Höppner et al., 2004
; Llosa et al., 2000
; Mushegian et al., 1996
; Ward et al., 2002
; Zahrl et al., 2005
). Together with this analysis of protein interactions of VirB1s from the B. suis T4SS and the results from functional studies conducted with both the A. tumefaciens and B. suis T4SS, the following model was designed describing the function(s) of VirB1. Upon expression of the virB operon, all VirB proteins possessing an N-terminal signal peptide are exported into the periplasm or partially traverse the inner membrane. VirB1 may form a 50 kDa homodimer, which may render the active site inaccessible. The predicted pore size of the peptidoglycan layer permits diffusion of globular proteins smaller than 55 kDa, and therefore VirB9, VirB7, VirB5, VirB2 and the VirB1 dimer probably diffuse freely in the periplasm. The contact between VirB1 and VirB9 may be mediated by the C-terminus of VirB1 and lead to activation of the lytic transglycosylase activity of VirB9-bound VirB1. Transient interactions of VirB1 with VirB8 and VirB11 may facilitate this process, which may lead to a conformational change, followed by processing of VirB1 at its VirB1* cleavage site. The enzyme activity may be modulated by binding of VirB8, VirB9 or VirB11 close to active-site residues. Assembly of VirB7 and VirB9, which subsequently recruit other channel components such as VirB10 and VirB8, may accompany the opening of the cell wall. The N-terminal lytic transglycosylase domain of VirB1 (B1N) may subsequently be degraded in order to protect cellular integrity, whereas the C-terminal domain may remain attached to VirB9. VirB1* may exert an additional function in host cell recognition. As VirB1-like proteins can apparently be exchanged between different T4SS (Höppner et al., 2004
; Zahrl et al., 2005
), the results of these studies will probably be applicable to a wide variety of VirB1-like proteins from T4SS and other secretion systems (Koraimann, 2003
).
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ACKNOWLEDGEMENTS |
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Received 6 July 2005;
revised 16 August 2005;
accepted 30 August 2005.
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