Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA1
Author for correspondence: Milton H. Saier, Jr. Tel: +1 858 534 4084. Fax: +1 858 534 7108. e-mail: msaier{at}ucsd.edu
Keywords: conjugation, virulence, DNA transfer, protein secretion, Agrobacterium
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Overview |
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Background |
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In addition to VirB211 homologues, VirD4 homologues are thought to play a role as coupling proteins, linking DNAprotein substrates to the membrane pore (Hamilton et al., 2000 ). The X-ray structure of the VirD4 homologue, TrwB from plasmid R388, has recently been solved (Gomis-Ruth et al., 2001
). Homologues of this protein are found in H. pylori and Ric. prowazekii, organisms that do not possess a full complement of the VirB homologues (C. Baron, personal communication). Although VirD4 and several other peripheral constituents are important for the secretory process, we will nevertheless focus on the VirB2VirB11 constituents that are believed to play a direct role in translocation by providing the structural constituents of the secretory apparatus.
Although previous efforts have resulted in the identification of common type IV secretory system constituents in a variety of Gram-negative bacteria (Christie, 2001 ; Krause et al., 2000
; Li et al., 1999
), no previously published work has described systematic phylogenetic analyses of these proteins. We have therefore initiated a comprehensive analysis of these proteins using the agrobacterial VirB system as a starting point. We identify all recognizable homologues of the VirB211 proteins, characterize these homologues with respect to organismal source and size, and align the sequences for the purposes of (1) constructing phylogenetic trees, (2) deriving mean hydropathy and amphipathicity plots that lead to estimates of topology, (3) determining regions of high conservation, and (4) identifying well-conserved residues that are likely to be of structural or functional significance.
Only representative summary tables and data figures are presented to document our most important conclusions. The complete list of protein homologues in the ten families (the VirB2 to VirB11 families), including protein abbreviations, database descriptions, organismal sources, protein sizes and database accession numbers that allow easy access to the sequences, can be found as supplementary data (see http://mic.sgmjournals.org). The multiple alignments, mean hydropathy, amphipathicity and similarity plots and phylogenetic trees can also be found at this site.
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Computer methods |
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Two programs were used to align the protein sequences retrieved as described above: CLUSTALX (Thompson et al., 1997 ) and TREE (Feng & Doolittle, 1990
) (see Young et al., 1999
for comparison and evaluation of these and other available programs). The gap penalty and gap extension values used with the CLUSTALX program were 10 and 0·1, although other combinations were tried. This set of values consistently gave similar phylogenetic trees to those calculated with values of 8 and 2, respectively. Default parameters were used for the TREE program. The multiple alignments from which the results are derived, as well as many of the derived figures are not presented here, can be found at http://mic.sgmjournals.org. Both the CLUSTALX and TREE programs were used to derive phylogenetic trees although only CLUSTALX-derived trees are presented. Both alignments were also used to derive mean hydropathy, similarity and amphipathicity plots as outlined previously (Kyte & Doolittle, 1982
; Le et al., 1999
). A sliding window of 21 residues was generally used, although for identification of amphipathic ß structure a sliding window of 7 or 9 residues was sometimes used with the TREEMOMENT program (Le et al., 1999
) or the AveHAS program (Zhai & Saier, 2001a
). For calculation of mean amphipathicity, the angle used was 100° for
structure or 180° for ß structure (Le et al., 1999
; Zhai & Saier, 2001a
). Topology was estimated using the TMPRED (Hofmann & Stoffel, 1993
) and DAS (Cserzo et al., 1997
) programs on individual proteins (Zhai & Saier, 2001b
) as well as by viewing the mean hydropathy plots.
In the case of the large VirB11 family of (putative) ATPases, the phylogenetic trees were derived both with the complete sequences and with the most conserved regions of these proteins. These latter regions were selected by visual inspection and were spliced according to the alignment of their full sequences generated with CLUSTALX. This method obviates the problem of artifactually distorted branch lengths due to association with non-homologous or poorly aligned sequences.
The organismal abbreviations for organisms that clearly encode parts of IVSP systems include (1) Atu, A. tumefaciens; (2) Bab, Brucella abortus; (3) Bhe, Bartonella henselae; (4) Bpe, Bordetella pertussis; (5) Bsu, Brucella suis; (6) Eae, Enterobacter aerogenes; (7) Eco, Escherichia coli; (8) Hpy, H. pylori; (9) Lpn, L. pneumophila; (10) Reu, Ralstonia eutropha; (11) Ret, Rhizobium etli; (12) Rsp, Rhizobium sp.; (13) Rpr, Rickettsia prowazekii; (14) Sti, Salmonella typhi; (15) Sty, Salmonella typhimurium; (16) Xfa, Xylella fastidiosa. Plasmids encoding such systems include RK2, RP4 and IncF.
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VirB protein homologues: overview |
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Functions suggested for these constituents are shown in Table 2. VirB4 and VirB11 are ATPases. VirB11 may function in biogenesis/assembly of the transporter complex while VirB4 probably energizes the translocation process. VirB6 and VirB4 may together comprise the inner membrane channel. Alternatively, VirB8, B9 and B10, which we suggest form a transperiplasmic channel as well as the outer membrane pore, could comprise the cytoplasmic channel. VirB7 is a lipoprotein that may anchor and stabilize the VirB810 complex and/or the pilus to the outer membrane (Sagulenko et al., 2001
). VirB2, B3 and B5 may be constituents of a pilus or pilus-like structure (Christie, 2001
; Sagulenko et al., 2001
; Shirasu & Kado, 1993
).
Our phylogenetic analyses revealed 10 overall clusters of sequence-related proteins (clusters 110; see Tables 1 and 3
). All 10 IVSP constituents were found in clusters 1, 3, 4, 5, 7 and 8, suggesting that all constituents of at least one represented system in each of these clusters have been identified and sequenced. However, nine constituents of cluster 6, five constituents of clusters 2 and 9, and only one of cluster 10 are represented. Proteins in these clusters may serve functions not requiring all protein components of an IVSP.
Table 3 presents the names of representative proteins in each of the 10 clusters. Clusters 7 and 8 include VirB proteins of Agrobacterium and of Bartonella, respectively. Clusters 1, 4 and 5 include the Trb proteins of plasmid IncP, the Tra proteins of plasmid IncN and the Trw proteins of IncW, respectively. Cluster 6 includes the Ptl pertussis toxin export proteins of Bor. pertussis. Only a VirB5 homologue is missing in this system. Because the Ptl system does not transport DNA and functions exclusively to export a toxin, this deficiency may have functional significance. All other clusters (clusters 2, 9 and 10) are not represented in several of the phylogenetic trees. Proteins included in these clusters may be constituents of incompletely sequenced IVSP systems or constituents of other types of systems. The latter possibility, clearly valid for some constituents such as the VirB4 and VirB11 ATPase homologues, is consistent with the notion that IVSP systems were originally constructed in part from pre-existing proteins that were constituents of other types of systems (see Conclusions).
A list of all of the proteins identified in each of the ten protein families is available at http://mic.sgmjournals.org. A few of these entries either are putative fragments or are homologues of strikingly dissimilar size as compared with the other homologues. These few proteins were excluded from our analyses. Table 1 should be used as a guide for the analyses reported below dealing with the ten recognized constituents of the IVSP family.
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VirB2 |
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Mean amphipathicity plots revealed that when the angle was set at 100°, as is appropriate for an -helix, peak 2 is preceded by a region of striking amphipathicity, while a region of lesser amphipathicity separates putative TMSs 2 and 3. With the angle set at 180°, as for a ß-strand, the region immediately preceding putative TMS1 exhibits a strong peak. This information suggests that VirB2 homologues exhibit ß structure in their N-terminal regions but
structure in remaining parts.
No fully conserved residues were identified in the VirB2 multiple alignment. However, regions of striking sequence similarity were observed in putative TMSs 2 and 3. At several positions within these two secondary structural elements, only hydrophobic residues were found. Well-conserved glycyl residues were also present in both regions, suggesting structural flexibility. At the end of helix 3, a well-conserved hydroxy amino acid (serine or threonine) was found. The results suggest that although no residue is essential for a single function shared by all of these proteins, certain residue types are important to maintain a requisite structure/function relationship.
The phylogenetic tree for the VirB2 family shows that eight major clusters are present; clusters 2 and 10 are missing (see Tables 1 and 3
). The clustering of proteins within each major cluster is undoubtedly of phylogenetic significance. This is particularly relevant for large clusters 1, 6 and 9. Other clusters or branches are either represented by a single protein (clusters 3, 4, 5 and 8) or by a pair of close orthologues (cluster 7).
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VirB3 |
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The phylogenetic tree for VirB3 homologues revealed that the proteins in clusters 1, 6 and 9 exhibit relative distances from each other similar to those observed for the corresponding proteins in the VirB2 tree. Also, the Esc. coli Tra and Trw proteins of clusters 4 and 5 (Table 3) are more closely related to each other than to other homologues. Thus, it appears that VirB2 and B3 homologues have evolved in parallel without shuffling of constituents between systems.
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VirB4 |
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The multiple alignment of the VirB4 homologues revealed several regions of sequence conservation separated by gaps. Most striking was the well-conserved BBGX(ST)G(AS)GK(ST)XBBXBB motif at alignment positions 710725 where B is a hydrophobic residue and X is any residue. This motif presumably serves for nucleotide binding. Adjacent to this motif is another well-conserved region: BBBDXDXGX25BNPLB (alignment positions 764802). Other regions (positions 9801050) also proved to be highly conserved. Thus, these strongly conserved residues occur in the C-terminal portions of the VirB4 homologues where the enzyme active sites responsible for substrate recognition are likely to be found.
The phylogenetic tree for VirB4 homologues is most revealing. First, clusters 1, 6 and 9 are strikingly similar to those shown for VirB2 and VirB3 homologues. Second, the Tra and Trw proteins (clusters 4 and 5) are again adjacent to each other. Third, unexpectedly, these two proteins cluster loosely with cluster 6, a fact that may reflect the higher phylogenetic resolution possible with proteins of long sequence. Finally, clusters 3, 4, 5, 7 and 8 each generally consists of a single protein (or a pair of orthologues) as observed for the VirB2 and VirB3 family trees.
The only significant difference between this tree and those for the VirB2 and VirB3 families is the presence of clusters 2 and 10, which were lacking from the latter trees. Cluster 2 includes proteins exclusively from H. pylori while cluster 10 includes proteins exclusively from low G+C Gram-positive bacteria. Since cluster 10 is found only in the VirB4 family, it can be concluded that the homologous Gram-positive bacterial ATPases serve a function that is very different from those exhibited by their homologues in Gram-negative bacteria. At least some of the H. pylori homologues, of widely differing sequences, are likely to serve non-IVSP functions that, however, may include other VirB homologues.
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VirB5 |
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The phylogenetic tree for the VirB5 family reveals interesting similarities and differences with those discussed before. Thus, in cluster 1, only three proteins instead of four or five are found, but the same organisms are represented and the relative distances of the included homologues are in accordance with expectation. Cluster 2 is missing, as for the VirB2 and VirB3 trees. In cluster 3, the L. pneumophila proteins are present in all four trees. Clusters 47 are as expected, but cluster 8 is absent from the VirB5 tree. Particularly noteworthy is the loose clustering of the TraC and TrwJ proteins of E. coli (clusters 4 and 5) in this and many of the other VirB211 family trees.
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VirB6 |
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Mean hydropathy and similarity plots for the VirB6 homologues reveal two well-conserved N-terminal peaks of hydrophobicity, followed by a poorly conserved hydrophilic region, a second hydrophobic region of about 40 residues (probably two close TMSs separated by a ß turn) and two final hydrophobic peaks, each about equidistant from the other and from the preceding region of hydrophobicity. They are somewhat less well conserved than the previous ones. There are thus six putative TMSs (Table 1).
VirB6 homologues are relatively poorly conserved with no fully conserved residues. Most of the well-conserved residues either are hydrophobic in nature or are glycyl residues within the transmembrane regions. However, the mean amphipathicity plots revealed that preceding, in between, and following all putative TMSs (except between TMSs 3 and 4, and between TMSs 5 and 6) are regions of very strong amphipathicity when the angle is set at 100° for helix. We therefore predict that VirB6 proteins are largely
-helical, both in the membrane and in the inter-TMS loop regions.
The phylogenetic tree for the VirB6 family resembles those discussed previously, although PtlD Bpe (cluster 6) is more distant from the other members of its cluster than expected. The Tra and Trw proteins of Esc. coli again cluster loosely together. Clusters 2, 9 and 10 are not represented (Table 2). This is surprising in view of the large number of cluster 9 homologues. This fact leads us to suggest that cluster 9 VirB homologues function by mechanisms different from those used by most IVSP family members. Alternatively, some of the IncF-like plasmid transfer genes may have not been sequenced, or some of the constituents may exhibit too great a degree of sequence divergence to be recognized.
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VirB7 |
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The mean hydrophobicity plot of the 11 established members of the VirB7 family reveals a single N-terminal TMS of less than 20 residues followed by a 30 residue hydrophilic peptide. This hydrophilic region did not exhibit a striking amphipathic character when the angle was set to 100°, but its C-terminal portion showed three sharp peaks when the angle was set at 180° and the sliding window was set at seven residues. This suggests that the C-terminal regions of VirB7 lipoproteins may contain substantial ß structure.
Multiple alignments revealed positively charged residues preceding and a fully conserved cysteyl residue following the N-terminal leader sequence motif as expected for these lipoproteins. A well-conserved motif including this cysteyl residue is (LIV)(SAG)(GA)C. The hydrophilic C-termini of these proteins are proline rich but otherwise poorly conserved. The phylogenetic tree resembles those described previously for other VirB family homologues in virtually all respects, a fact that is surprising in view of the small sizes of these homologues.
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VirB8 |
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Except for well-conserved R/K residues preceding the putative TMS and one or two well-conserved prolyl residues following this putative TMS, the N-terminal region shows little sequence conservation. However, in the C-terminal portion we found a Y(VI)X2RE motif that is fully conserved except in the Trb proteins (cluster 1). Further downstream of this conserved motif are well-conserved hydroxy amino acids (ST), aromatic amino acids (YF) and hydrophobic residues (LIV) interspersed with hydrophilic residues. These latter regions are responsible for the ß-structure amphipathicity. The phylogenetic tree shown in Fig. 1 reveals clustering of VirB8 family proteins in accordance with expectation assuming that IVSP systems have evolved without shuffling of constituents between systems.
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VirB9 |
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Fig. 2 shows the VirB9 family tree. There are no surprises with respect to the clustering of most of the proteins. However, four H. pylori proteins comprise cluster 2. Cluster 2 is represented in only five of the VirB families. The two VirB9 homologues from Ric. prowazekii cluster loosely with the LvhB protein of L. pneumophila. It is of considerable interest that Ric. prowazekii homologues of VirB4, B8, B9, B10 and B11 were identified, but homologues of the other VirB proteins were not found in this organism. A similar scenario is observed for H. pylori but not for L. pneumophila. Since the genomes of H. pylori and Ric. prowazekii have been completely sequenced, we suggest that these proteins may serve common functions in virulence that do not require a complete IVSP system and possibly comprise part of a different type of transporter. A unified function for the latter four of these five VirB constituents can be suggested. It is interesting to note that Ric. prowazekii displays only one VirB4, VirB8, VirB10 or VirB11 homologue, but two sequence-divergent VirB9 homologues. Because their sequences are so different, it seems unlikely that the latter two paralogues serve the same function.
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VirB10 |
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The multiple alignment of the VirB10 homologues supports this supposition. Thus, their N termini, preceding the single TMS, are cationic. This is followed by a region of poor conservation (the proposed transperiplasmic region), and finally, in the C-termini one finds alternating hydrophilic and hydrophobic residues. Within this C-terminal putative ß-sheet region are four fully conserved and an additional nine well-conserved glycyl residues. They occur at irregular intervals of between 3 and 17 residues, most at odd-numbered intervals. Well-conserved cationic residues (R and K) are also found. Finally, five well-conserved blocks of three to five hydrophobic residues as well as occasional aromatic residues and proline can be found in this C-terminal domain. These observations undoubtedly have both structural and functional significance in the formation of a transenvelope structure.
The phylogenetic tree for the VirB10 proteins (Fig. 3) shows a configuration that illustrates the proposed co-evolution of the 10 VirB family homologues. However, the loose clustering of ComB3 Hpy with VirB10 Rpr suggests that these two proteins may serve a similar function. ComB3 Hpy is assigned to cluster 2, corresponding to the clusters 2 found in previously mentioned phylogenetic trees.
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VirB11 |
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The mean hydropathy and similarity plots for the VirB11 proteins reveal a strongly hydrophilic character with the hydrophilic domains showing the greatest sequence similarity. The mean hydropathy plot indicates the presence of a single region sufficiently long and hydrophobic to traverse the membrane, but this could be the hydrophobic core of a water-soluble protein.
Mean amphipathicity plots reveal three broad regions of putative amphipathic ß strand (alignment positions 80130, 200270 and 320390). It seems likely that VirB11 homologues include considerable ß structure.
The multiple alignment reveals several well-conserved motifs. At position 516528 is a well-conserved motif: (B)3(AST)G*(PG)T(AGS)SG*K*(ST)T (asterisks indicate fully conserved residues). A second well-conserved motif, centred at alignment position 550, is B2(ST)BED(ST)EB. The ED motif is almost fully conserved. A third well-conserved motif is centred at alignment position 598. This large region exhibits the motif B(ST)BX2B2(KR)B3RX2P(DE)XB3GE*BR. Finally, a well-conserved TGH motif occurs at alignment positions 624626.
Phylogenetic trees were derived both for the full length VirB11 proteins and for the best conserved regions of these proteins (about 120 residues; alignment positions 512628 in the full-length sequence alignment). Branching patterns in the two trees proved to be in excellent agreement. The former tree is reproduced in Fig. 4. All of the proteins that comprise the IVSP family constituents are found in cluster 8. Moreover, within cluster 8, eight of the ten subclusters identified in the other VirB family trees are found with configurations as expected assuming that these VirB11 homologues co-evolved in parallel with the other VirB proteins. This observation provides convincing evidence for the lack of appreciable shuffling of protein constituents between IVSP systems during their evolution. All remaining clusters in the VirB11 tree (clusters 17, 9 and 10) are not represented in the other VirB family trees. The proteins comprising most or all of these clusters may serve a unified function in energizing export and/or promoting assembly of extracellular protein complexes. It is suggested that VirB11 was borrowed from pre-existing systems for the assembly of more recently arising IVSP systems.
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Conclusions |
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(2) These proteins may be primarily cytoplasmically localized (VirB4 and VirB11), and/or they may be localized to the inner membrane as integral membrane proteins (VirB4 and VirB6). Three of them (VirB8, B9 and B10) are predicted to traverse both membranes and the periplasm with their N termini in the cytoplasm and their C termini embedded in the outer membrane (Kumar & Das, 2001 ). VirB7 is an outer membrane lipoprotein that may anchor both the VirB8B9B10 channel and the pilus to the outer membrane (Sagulenko et al., 2001
). VirB2, B3 and B5 may be constituents of the pilus (or pilus-like organelle) that mediates cellcell interactions or functions as a piston to drive protein export (Christie, 2001
).
(3) The two putative cytoplasmic constituents are ATPases; one (VirB11) may function in biogenesis of the IVSP system (Yeo et al., 2000 ) while the other (VirB4) may energize transport of the substrate protein or nucleoprotein complex (Christie, 2001
). The six-TMS integral inner membrane VirB6 protein may, together with VirB4, comprise an oligomeric pore, providing the protein translocation pathway across the inner membrane, but it may also play a role in pilus stabilization. The outer-membrane-associated proteins VirB8, B9 and B10 may comprise the envelope pore, and VirB7, a small outer-membrane lipoprotein, may stabilize the VirB8B9B10 complex and/or anchor it and the pilus to a specific region of the outer membrane (Sagulenko et al., 2001
). VirB8, B9 and B10 appear to each exhibit a single N-terminal inner transmembrane helix, a central transperiplasmic
-helical region and a C-terminal ß-structured domain that may interact with and/or insert into the outer membrane of the Gram-negative bacterial envelope. Thus, these three proteins may comprise or interact with the inner-membrane channel formed by VirB4VirB6 to allow construction of a continuous channel through the entire envelope (the inner membrane, the periplasm and the outer membrane). Such a structure could be either static or dynamic. In the latter case, its formation could be induced by the presence of a substrate protein as has been demonstrated for ABC (type I secretory pathway) protein export (Binet et al., 1997
). In either case, however, the conduit could allow transport of macromolecules directly across the entire cell envelope in a single energy-coupled step.
(4) All 10 constituents of the IVSP family transporters are found in at least five sequence-divergent systems. These systems, in addition to the agrobacterial and bartonellar VirB systems (phylogenetic clusters 7 and 8, respectively), include the Trb proteins of plasmid IncP (cluster 1), the LvhB proteins of L. pneumophila (cluster 3), the Tra proteins of plasmid IncN (cluster 4) and the Trw proteins of plasmid IncW (cluster 5). Nine of the ten VirB proteins are found in the Ptl system of Bor. pertussis (Table 2), a system involved in export of Bordetella protein toxin(s). Only VirB5 was not identified in this system, and VirB5 homologues have been proposed to be adhesin-like proteins localized to the pilus (fimbrial) tip. Alternatively, they may associate with the pilus in another capacity (see Table 1
). Other systems such as the Tra system of plasmid IncF (cluster 9) seem to lack several constituents, possibly because of incomplete plasmid sequencing. The VirB8, B9 and B10 proteins, and possibly VirB11 as well, that comprise cluster 2 probably form a structure that functions in a unified capacity, possibly in virulence and/or competence in H. pylori (Ramarao et al., 2000
) and Ric. prowazekii by a mechanism that does not utilize a complete IVSP system. This suggestion was recently reinforced by a report demonstrating the presence of an operon in Wolbachia species that encodes VirB8, 9, 10 and 11 as well as VirB4 but not other IVSP constituents. This operon is required for arthropod sexual alteration by this intracellular symbiont (Masui et al., 2000
). If these three proteins comprise the transenvelope conduit as has recently been suggested (Kumar & Das, 2001
), they could together serve a variety of functions in addition to type IV secretion. Finally, the VirB4 ATPase is the only constituent found in cluster 10. These proteins are derived from Gram-positive bacteria where the ATPase must energize a process unrelated to type IV secretion.
(5) Of the ten IVSP protein constituents we have analysed, eight are found only in Gram-negative bacteria. Only the two ATPases, VirB4 and VirB11, are also found in Gram-positive bacteria, and only VirB11 is found ubiquitously in a wide variety of bacteria and archaea. In these other organisms, these ATPases clearly do not function as constituents of IVSP systems.
(6) While the structural analyses lead to clear predictions regarding function (see point 3 above), the phylogenetic analyses suggest that IVSP systems have evolved from a single precursor system with virtually no shuffling of constituents between sequence-divergent systems. This important observation is illustrated by the phylogenetic trees shown in Figs 14. Moreover, these systems have, for the most part, probably diverged in function without gain or loss of protein constituents. The only exceptions are the proteins of clusters 2, 9 and 10 (see Table 2
). The same general conclusion has been reached regarding other macromolecular export systems including the type I secretory pathway (ABC) (Fernandez & de Lorenzo, 2001
) and type III secretory pathway (Vir) (Plano et al., 2001
) systems (see Kuan et al., 1995
; Nguyen et al., 2000
; Paulsen et al., 1997
). In all three cases, the lack of shuffling indicates the occurrence of extensive proteinprotein interactions, allowing construction of transenvelope exporters that can secrete their substrates directly from the cell cytoplasm to the extracellular medium without allowing accumulation of a periplasmic intermediate. It is interesting to note that type II secretory pathway systems do allow accumulation of folded periplasmic intermediates, and that export across the two envelope membranes occurs in two distinct steps (Pugsley, 1993
; Sandkvist, 2001
).
The computational analyses presented in this article provide some confirmatory and some valuable new information about the structures and mechanisms of action of a unique set of protein complexes. The sequence analyses reported should provide guides for detailed molecular genetic analyses. Many of the observations and predictions presented require direct experimental verification. It is hoped that these studies will be forthcoming in the near future.
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Acknowledgements |
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REFERENCES |
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Anderson, L. B., Hertzel, A. V. & Das, A. (1996). Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex. Proc Natl Acad Sci USA 93, 8889-8894.
Baron, C., Thorstenson, Y. R. & Zambryski, P. C. (1997). The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens. J Bacteriol 179, 1211-1218.[Abstract]
Beaupre, C. E., Bohne, J., Dale, E. M. & Binns, A. N. (1997). Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells. J Bacteriol 179, 78-89.[Abstract]
Berger, B. R. & Christie, P. J. (1994). Genetic complementation analysis of the Agrobacterium tumefaciens virB2 through virB11 are essential virulence genes. J Bacteriol 176, 3646-3660.[Abstract]
Binet, R., Létoffé, S., Ghigo, J. M., Delepelaire, P. & Wandersman, C. (1997). Protein secretion by Gram-negative bacterial ABC exporters a review. Gene 192, 7-11.[Medline]
Bohne, J., Yim, A. & Binns, A. N. (1998). The Ti plasmid increases the efficiency of Agrobacterium tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid. Proc Natl Acad Sci USA 95, 7057-7062.
Burns, D. L. (1999). Biochemistry of type IV secretion. Curr Opin Microbiol 2, 25-29.[Medline]
Christie, P. J. (1997). Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. J Bacteriol 179, 3085-3094.
Christie, P. J. (2001). Type IV secretion: intercellular transfer of macromolecules by systems ancestrally related to conjugation machines. Mol Microbiol 40, 294-305.[Medline]
Christie, P. J. & Vogel, J. P. (2000). Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol 8, 354-360.[Medline]
Covacci, A. & Rappouli, R. (1993). Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon. Mol Microbiol 8, 429-434.[Medline]
Covacci, A., Falkow, S., Berg, D. E. & Rappouli, R. (1997). Did the inheritance of a pathogenicity island modify the virulence of Helicobacter pylori? Trends Microbiol 5, 205-208.[Medline]
Covacci, A., Telford, J. L., Del Giudice, G., Parsonnet, J. & Rappuoli, R. (1999). Helicobacter pylori virulence and genetic geography. Science 289, 1328-1333.
de la Cruz, F. & Lanka, E. (1998). Function of the Ti plasmid Vir proteins: T-complex formation and transfer to the plant cell. In The Rhizobiaceae , pp. 281-301. Edited by H. P. Spaink, A. Kondorosi & P. J. J. Hooykasd. Dordrecht:Kluwer Academic Publishing.
Cserzo, M., Wallin, E., Simon, I., von Heijne, G. & Elofsson, A. (1997). Prediction of transmembrane alpha-helices in prokaryotic membrane protein: the Dense Alignment Surface method. Prot Eng 10, 673-676.[Abstract]
Dang, T. A. T. & Christie, P. J. (1997). The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface. J Bacteriol 179, 453-462.[Abstract]
Dang, T. A., Zhou, X.-R., Graf, B. & Christie, P. J. (1999). Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on assembly and function of the T-DNA transporter. Mol Microbiol 32, 1239-1253.[Medline]
Das, A. & Xie, Y.-H. (1998). Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins. Mol Microbiol 27, 405-414.[Medline]
Das, A. & Xie, Y.-H. (2000). The Agrobacterium T-DNA transport pore proteins VirB8, VirB9 and VirB10 interact with one another. J Bacteriol 182, 758-763.
Devereux, J., Haeberli, P. & Smithies, N. O. (1984). A comprehensive set of sequence analyses for the VAX. Nucleic Acids Res 12, 387-395.[Abstract]
Dreiseikelmann, B. (1994). Translocation of DNA across bacterial membranes. Microbiol Rev 58, 293-316.[Abstract]
Feng, D.-F. & Doolittle, R. F. (1990). Progressive alignment and phylogenetic tree construction of protein sequences. Methods Enzymol 183, 375-387.[Medline]
Fernandez, L. A. & de Lorenzo, V. (2001). Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway. Mol Microbiol 40, 332-346.[Medline]
Fernandez, D., Dang, T. A. T., Spudich, G. M., Zhou, Z.-R., Berger, B. & Christie, P. J. (1996a). The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface. J Bacteriol 178, 3156-3167.[Abstract]
Fernandez, D., Spudich, G. M., Zhou, Z.-R. & Christie, P. J. (1996b). The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus. J Bacteriol 178, 3168-3176.[Abstract]
Frost, L. S., Ippen-Ihler, K. & Skurray, R. A. (1994). Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol Rev 58, 162-210.[Abstract]
Fullner, K. J., Lara, J. C. & Nester, E. W. (1996). Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273, 1107-1109.[Abstract]
Gelvin, S. B. (2000). Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Physiol Plant Mol Biol 51, 223-256.
Gomis-Ruth, F. X., Moncalian, G., Perez-Luque, R., Gonzalez, A., Cabezon, E., de la Cruz, F. & Coll, M. (2001). The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase. Nature 409, 637-641.[Medline]
Hamilton, C. M., Lee, H., Li, P. L., Cook, D. M., Piper, K. R., von Bodman, S. B., Lanka, E., Ream, W. & Farrand, S. K. (2000). TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58. J Bacteriol 182, 1541-1548.
Hapfelmeier, S., Domke, N., Zambryski, P. C. & Baron, C. (2000). VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens. J Bacteriol 182, 4505-4511.
Hofmann, K. & Stoffel, W. (1993). Tmbase a database of membrane spanning protein segments. Biol Chem Hoppe-Seyler 347, 166.
Jack, D. L., Paulsen, I. T. & Saier, M. H.Jr (2000). The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations. Microbiology 146, 1797-1814.
Jones, A. L., Shirasu, K. & Kado, C. I. (1994). The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of the VirB3 protein. J Bacteriol 176, 5255-5261.[Abstract]
Jones, A. L., Lai, E.-M., Shirasu, K. & Kado, C. L. (1996). Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins. J Bacteriol 179, 5835-5842.[Abstract]
Kuan, G., Dassa, E., Saurin, W., Hofnung, M. & Saier, M. H.Jr (1995). Phylogenic analyses of the ATP-binding constituents of bacterial extracytoplasmic receptor-dependent ABC-type nutrient uptake permeases. Res Microbiol 146, 271-278.[Medline]
Kuldau, G. A., De Vos, G., Owen, J., McCaffrey, G. & Zambryski, P. (1990). The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames. Mol Gen Genet 221, 256-266.[Medline]
Kumar, R. B. & Das, A. (2001). Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8. J Bacteriol 183, 3636-3641.
Krause, S., Pansegrau, W., Lurz, R., de la Cruz, F. & Lanka, E. (2000). Enzymology of type IV macromolecule secretion systems: the conjugative transfer regions of plasmids RP4 and R388 and the cag pathogenicity island of Helicobacter pylori encode structurally and functionally related nucleoside triphosphate hydrolases. J Bacteriol 182, 2761-2770.
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105-132.[Medline]
Lai, E. M. & Kado, C. I. (2000). The T-pilus of Agrobacterium tumefaciens. Trends Microbiol 8, 361-369.[Medline]
Le, T., Tseng, T.-T. & Saier, M. H.Jr (1999). Flexible programs for the estimation of average amphipathicity of multiply aligned homologous proteins: application to integral membrane transport proteins. Mol Membr Biol 16, 173-179.[Medline]
Lessl, M., Balzer, D., Pansegrau, W. & Lanka, E. (1992). Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem 267, 20471-20480.
Li, P.-L., Hwang, I., Miyagi, H., True, H. & Farrand, S. K. (1999). Essential components of the Ti plasmid trb system, a type IV macromolecular transporter. J Bacteriol 181, 5033-5041.
Masui, S., Sasaki, T. & Ishikawa, H. (2000). Genes for the type IV secretion system in an intracellular symbiont, Wolbachia, a causative agent of various sexual alterations in arthropods. J Bacteriol 182, 6529-6531.
Nguyen, L., Paulsen, I. T., Tchieu, J., Hueck, C. J. & Saier, M. H.Jr (2000). Phylogenetic analyses of the constituents of type III protein secretion systems. J Mol Microbiol Biotechnol 2, 125-144.[Medline]
Nicosia, A., Perugini, M., Franzini, C. & 7 other authors (1986). Cloning and sequencing of the pertussis toxin genes: operon structure and gene duplication. Proc Natl Acad Sci USA 83, 46314635.[Abstract]
Paulsen, I. T., Beness, A. M. & Saier, M. H.Jr (1997). Computer-based analyses of the protein constituents of transport systems catalysing export of complex carbohydrates in bacteria. Microbiology 143, 2685-2699.[Abstract]
Plano, G. V., Day, J. B. & Ferracci, F. (2001). Type III export: new uses for an old pathway. Mol Microbiol 40, 284-293.[Medline]
Pugsley, A. P. (1993). The complete general secretory pathway in Gram-negative bacteria. Microbiol Rev 57, 50-108.[Abstract]
Ramarao, N., Gray-Owen, S. D., Backert, S. & Meyer, T. F. (2000). Helicobacter pylori inhibits phagocytosis by professional phagocytes involving type IV secretion components. Mol Microbiol 37, 1389-1404.[Medline]
Rashkova, S., Spudich, G. M. & Christie, P. J. (1997). Characterization of membrane protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase. J Bacteriol 179, 583-591.[Abstract]
Rashkova, S., Zhou, X. R., Chen, J. & Christie, P. J. (2000). Self-assembly of the Agrobacterium tumefaciens VirB11 traffic ATPase. J Bacteriol 182, 4137-4145.
Sagulenko, V., Sagulenko, E., Jakubowski, S., Spudich, E. & Christie, P. J. (2001). VirB7 lipoprotein is exocellular and associates with the Agrobacterium tumefaciens T pilus. J Bacteriol 183, 3642-3651.
Saier, M. H.Jr (1994). Computer-aided analyses of transport protein sequences: gleaning evidence concerning function, structure, biogenesis, and evolution. Microbiol Rev 58, 71-93.[Abstract]
Saier, M. H.Jr (2000). A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev 64, 354-411.
Sandkvist, M. (2001). Biology of type II secretion. Mol Microbiol 40, 271-283.[Medline]
Schmidt-Eisenlohr, H., Domke, N. & Baron, C. (1999a). TraC of IncN plasmid pKM101 associates with membranes and extracellular high-molecular-weight structures in Escherichia coli. J Bacteriol 181, 5563-5571.
Schmidt-Eisenlohr, H., Domke, N., Angerer, C., Wanner, G., Zambryski, P. C. & Baron, C. (1999b). Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens. J Bacteriol 181, 7485-7492.
Segal, G., Purcell, M. & Shuman, H. A. (1998). Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc Natl Acad Sci USA 95, 1669-1674.
Shirasu, K. & Kado, C. I. (1993). Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens. FEMS Microbiol Lett 111, 287-294.[Medline]
Shirasu, K., Koukolíková-Nicola, Z., Hihn, B. & Kado, C. I. (1994). An inner-membrane-associated virulence protein essential for T-DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes. Mol Microbiol 11, 581-588.[Medline]
Spudich, G. M., Fernandez, D., Zhou, Z.-R. & Christie, P. J. (1996). Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus. Proc Natl Acad Sci USA 93, 7512-7517.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.
Thorstenson, Y. R., Kuldau, G. A. & Zambryski, P. C. (1993). Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure. J Bacteriol 175, 5233-5241.[Abstract]
Tummuru, M. K. R., Sharma, S. A. & Blaser, M. J. (1995). Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells. Mol Microbiol 18, 867-876.[Medline]
Tzfira, T., Rhee, Y., Chen, M.-H., Kunik, T. & Citovsky, V. (2000). Nucleic acid transport in plantmicrobe interactions: the molecules that walk through the walls. Ann Rev Microbiol 54, 187-219.[Medline]
Vergunst, A. C., Schrammeijer, B., den Dulk-Ras, A., de Vlaam, C. M. T., Regensburg-Tuïnk, T. J. G. & Hooykaas, P. J. J. (2000). VirB-D4-dependent protein translocation from Agrobacterium into plant cells. Science 290, 979-982.
Vogel, J. P., Andrews, H. L., Wong, S. K. & Isberg, R. R. (1998). Conjugative transfer by the virulence system of Legionella pneumophila. Science 279, 873-876.
Ward, J. E., Dale, E. M., Nester, E. W. & Binns, A. N. (1990). Identification of a VirB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens. J Bacteriol 172, 5200-5210.[Medline]
Weiss, A. A., Johnson, F. D. & Burns, D. L. (1993). Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci USA 90, 2970-2974.[Abstract]
Winans, S. C., Burns, D. L. & Christie, P. J. (1996). Adaptation of a conjugal transfer system for the export of pathogenic macromolecules. Trends Microbiol 4, 64-68.[Medline]
Yeo, H. J., Savvides, S. N., Herr, A. B., Lanka, E. & Waksman, G. (2000). Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol Cell 6, 1461-1472.[Medline]
Young, G. B., Jack, D. L., Smith, D. W. & Saier, M. H.Jr (1999). The amino acid/auxin:proton symport permease family. Biochim Biophys Acta 1415, 306-322.[Medline]
Zhai, Y. & Saier, M. H.Jr (2001a). A web-based program for the prediction of average hydropathy, average amphipathicity and average similarity of multiply aligned homologous proteins. J Mol Microbiol Biotechnol 3, 285-286.[Medline]
Zhai, Y. & Saier, M. H.Jr (2001b). A web-based program (WHAT) for the simultaneous prediction of hydropathy, amphipathicity, secondary structure and transmembrane topology for a single protein sequence. J Mol Microbiol Biotechnol 3, 501-502.[Medline]
Zhou, Z.-R. & Christie, P. J. (1997). Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins. J Bacteriol 179, 5835-5842.[Abstract]