1 Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA
2 Unité de Génétique Moléculaire, CNRS URA 2172, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France
Correspondence
Milton H. Saier Jr
msaier{at}ucsd.edu
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ABSTRACT |
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Present address: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
Present address: Department of Life Science, Jeonju University, Chonju, Korea.
Present address: Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan, Republic of China.
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Overview |
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Surprisingly, the T2SD family of proteins (members of the secretin superfamily; TC #1.B.22) (Martinez et al., 1998; Nguyen et al., 2000
; Thanassi, 2002
; Yen et al., 2002
) are the only integral outer-membrane secreton components. Therefore, they are the only ones capable of forming channels in bacterial outer membranes to permit exoprotein efflux (Bitter et al., 1998
; Hardie et al., 1996a
; Nouwen et al., 1999
, 2000
). The well-established multimeric state and low-resolution structures of secretins (Bitter et al., 1998
; Brok et al., 1999
; Collins et al., 2001
, 2003
) are consistent with this idea. A role in pilus biogenesis has been proposed (Sauvonnet et al., 2000
).
The other secreton components include the following. (1) A peripheral outer-membrane lipoprotein (the T2SS protein or pilotin) (Hardie et al., 1996a, b
) that has so far been found only in a small number of secreton systems. (2) A peripheral plasma membrane protein (the T2SE protein), a putative ATP-binding protein that, in one case, is reported to be monomeric and to have both ATPase and autokinase activities (Sandkvist et al., 1995
). T2SE proteins have characteristic signature sequences, including a highly conserved region that is flanked by aspartate residues as well as an essential zinc-finger-like motif (Possot & Pugsley, 1994
, 1997
). They are part of a superfamily of ATPases that includes a subfamily of multimeric proteins (often referred to as the VirB11 subfamily) involved in type IV secretion/bacterial conjugation (Cao & Saier, 2001
; Krause et al., 2000
; Yeo et al., 2000
). (3) Predicted integral plasma membrane proteins (T2SA, B, C, F, G, H, I, J, K, L, M, N and O). T2SG through K (the pseudopilins) have N-terminal domains that are similar to those of type IV pilins (Nunn, 1999
; Pugsley, 1993a
). According to modelling based on the structure of a type IV pilin (Parge et al., 1995
), they may mediate subunit interactions that lead to filament formation. T2SO is the prepilin peptidase that cleaves and then N-methylates pseudopilins/pilins at a conserved site N-terminal to the hydrophobic region (Bleves et al., 1998
; Nunn & Lory, 1992
, 1993
; Pugsley, 1993b
; Pugsley et al., 2001
). T2SL is required for the T2SE protein to associate with the plasma membrane and is stabilized by T2SM (Michel et al., 1998
; Possot et al., 2000
; Py et al., 1999
, 2001
; Sandkvist et al., 1995
, 1999
, 2000
).
The precise functions of the plasma membrane protein constituents of the secreton other than T2SO remain largely a matter of conjecture although, in view of the established similarity with the T4P systems, many of them are probably involved in the assembly of a pilus-like structure (see below). T2SC, T2SL and T2SM have relatively large periplasmic domains, leading to the notion that they might form part of a trans-periplasmic complex that controls the opening of the secretin channel and/or recognizes and directs the substrate exoproteins to this secretin (Possot et al., 2000). Nevertheless, all three of these proteins are required for pilus formation by the T2S. Other proteins, such as the T2SE ATPase and/or a proton-channel-forming constituent (possibly T2SF), could be involved in energizing secreton/pseudopilus assembly or exoprotein transport through the outer membrane (Bleves et al., 1999
; Letellier et al., 1997
; Possot et al., 1997
, 2000
). This latter suggestion is based, in part, on a superficial analogy between protein secretion and the import of bulky ligands (e.g. siderophores and cyanocobalamin) across the outer membrane of Escherichia coli. The latter process is driven by the proton-motive force (pmf) via an integral plasma membrane protein complex, the TonB/ExbBD complex (Postle & Kadner, 2003
). However, it is also possible that ATP hydrolysis plays a direct role in the secretory process, especially in secretons that have two ATPases, like those in Aeromonas species (Schoenhofen et al., 1998
).
The long-recognized similarity between the T2S and T4P systems (Hobbs & Mattick, 1993; Pugsley, 1993a
) was strengthened by the recent observation that increased expression of the major pseudopilin (T2SG) caused bacteria expressing secreton genes to assemble a pilus composed of this protein (Sauvonnet et al., 2000
). The similarities between the T2S and T4F systems extend beyond the pilins/pseudopilins and prepilin peptidase to include T2SD (secretin) (Bitter et al., 1998
; Collins et al., 2001
; Schmidt et al., 2001
) as well as T2SE and T2SF (Nunn et al., 1990
). In addition, a pilotin whose sequence is unrelated to that of identified T2S proteins is required for secretin assembly and stability in T4P systems (Drake et al., 1997
). However, some secreton components that are needed for pilus assembly by the T2S (e.g. T2SC; Sauvonnet et al., 2000
) appear to be absent from the T4P system. Additionally, certain T4P systems have unique components that are required for pilus assembly (see later). These observations probably reflect the ancient separation during divergent evolution of the T2S and T4P systems.
A uniform system of nomenclature for T4P system components remains to be established. In the following sections, we will refer extensively to three relatively well-characterized T4P systems. These are from Pseudomonas aeruginosa (Pil), Neisseria (Pil) and the E. coli EAF plasmid (Bfp) (see footnote 3 in Table 1 for nomenclature of major T4P components in these bacteria). Many T4P systems, including these three, have two or even three ATPases that are related to T2SE. In these bacteria, T4P systems cause twitching motility by cycles of pilus extrusion (assembly) and retraction (disassembly) (Merz et al., 2000
; Skerker & Berg, 2001
). PilT/BfpF have been proposed to be the force-generating proteins (Merz et al., 2000
). The pilus might span the outer membrane by passing though the centre of the secretin channel (Wolfgang et al., 2000
).
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In this paper, we identify recognizable homologues in the current databases of the protein constituents of a generic T2S system that includes all secreton components irrespective of the bacterium in which they were identified, the related T4P systems of P. aeruginosa and other Gram-negative bacteria, and the related archaeal flagellar systems of Methanococcus voltae and other archaea. The sequences of the most conserved of these proteins are analysed for structural and phylogenetic attributes, and the conclusions resulting from these analyses are presented. Tables of proteins as well as the corresponding multiple alignments and some supplementary phylogenetic trees can be found on our website (www-biology.ucsd.edu/msaier/supmat).
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Computer methods |
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In this paper, we use the WHAT (Zhai & Saier, 2001b) and AveHAS (Zhai & Saier, 2001a
) programs in combination to predict transmembrane segments (TMSs). These programs combine several established programs to make structural predictions about transmembrane proteins. For example, the WHAT program examines individual proteins, using JNET (Cuff et al., 1998
) and MEMSAT (Jones et al., 1994
) for secondary structure and transmembrane topology prediction, respectively. Both of these programs are among the best available for these purposes. The AveHAS program first generates a multiple alignment for a collection of homologous sequences (Thompson et al., 1997
) and then averages (1) hydropathy, (2) amphipathicity and (3) similarity plots to provide structural information that is much more reliable than possible when evaluating a single protein sequence (Zhai & Saier, 2001a
). Transmembrane
-strands can thus be accurately predicted because they exhibit (1) predicted
-structure using JNET, (2) increased hydrophobicity, relative to other portions of the polypeptide chain, and (3) increased amphipathicity when the angle is set at 180° as is appropriate for
-strands (Le et al., 1999
; Zhai & Saier, 2002
). This method predicts transmembrane
-strands with about 80 % accuracy.
Supplementary material which can be found on our website (www-biology.ucsd.edu/msaier/supmat) includes: (1) tables of all homologues of the different protein types included in this study, (2) the multiple alignments for these homologues, (3) the phylogenetic trees for these same families of proteins, (4) a 16S rRNA phylogenetic tree for all bacteria from which proteins included in this study were derived and (5) a tabulation of known protein constituents of all T2S systems for which homologues of all or most constituents of the secreton have been identified.
Complementation of the pulF deletion in the complete pul gene cluster was carried out using pBR322 derivatives by homologous genes under lacp control in a compatible plasmid, as described by Possot et al. (2000). gspF was amplified using specific primers that incorporated restriction endonuclease cleavage sites for cloning, as previously described (Possot et al., 2000
).
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T2S, T4P and Fla system constituents |
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T2S systems |
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Examination of the phylogenetic trees for the other constituents of these systems (see Table 3 and www-biology.ucsd.edu/
msaier/supmat) revealed that they exhibit essentially the same configurations and relative branch lengths within experimental error. Thus we conclude that secreton systems have probably evolved by whole gene cluster duplication and by speciation without appreciable exchange of constituents between systems.
The T2SC family of proteins deserves special mention for two reasons. First, the Xanthomonas campestris gene designated xpsN (Lee et al., 2000, 2001
) was clearly misnamed, since it is similar to genes for T2SC proteins (and, therefore, should be called xpsC) and is unrelated to genes for T2SN proteins. This allows one to rationalize recent data showing that XpsC(N) is essential and interacts with proteins D, M and/or L (Lee et al., 2000
, 2001
). PulN is not essential while T2SC proteins are essential and interact with T2SD, L and/or M proteins (Bleves et al., 1999
; Possot et al., 2000
). Second, the T2SC family of proteins can be divided into several distinct clusters depending on whether they possess (1) a coiled-coil segment, (2) a PDZ-type structure (Gerard-Vincent et al., 2002
; Pallen & Ponting, 1997
) or (3) neither, close to the C-terminal ends of the proteins (Fig. 2
). It is interesting to note that at least one member of the last class, HxcC from P. aeruginosa, is apparently functional (Ball et al., 2002
), indicating that neither the coiled-coil domain nor the PDZ domain is essential for secretion. Furthermore, the PDZ domain, predicted to exist in PulC (Pallen & Ponting, 1997
), is also predicted to be a coiled-coil by the algorithms we used (Fig. 2
).
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Pilins (T2SG, H, I, J and K) and the pilin processing enzyme (T2SO) |
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In addition to the authenticated Gram-negative bacterial T2SO proteins, members of the prepilin peptidase family are derived from both high- and low-G+C Gram-positive bacteria as well as very diverse Gram-negative bacteria (e.g. Chlorobium, Deinococcus, Synechocystis and Thermatoga) and might have different functions with related or unrelated substrate specificities. Moreover, a single organism may have multiple paralogues. For example, seven have been identified in E. coli, all very divergent in sequence, branching from points near the centre of the phylogenetic tree. They must have resulted from early gene duplication events or possibly were acquired by lateral transfer. On the other hand, only one prepilin peptidase gene is present in P. aeruginosa [PilD/XcpO(A)] and many other bacteria with fully sequenced genomes.
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T2SE/T4PC/FlaI ATPase phylogeny |
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T2SF/T4PC/FlaJ transmembrane (TM) protein phylogeny |
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It is interesting to note that the two integral membrane proteins TadB and TadC apparently function with a single ATPase, TadA. This observation might suggest that these integral membrane constituents function as homo- or hetero-oligomeric (possibly dimeric) structures. A dimeric structure would be in agreement with the fact that the archaeal homologues are internally duplicated proteins, twice as large as the bacterial homologues with approximately twice as many TMSs (see next section).
Cluster 8 includes proteins derived exclusively from archaea. The functions of cluster 8A and cluster 8D proteins are unknown, and they may or may not have counterparts in Fig. 3. Cluster 8A in Fig. 3
includes only one protein per organism, except for Archaeoglobus fulgidus where three paralogues are found. However, in cluster 8A of Fig. 5
, two sets of homologues are found for most represented organisms. Most of the proteins in cluster 8B are probably constituents of archaeal flagellar systems. These proteins are represented only once per organism, have counterparts in Fig. 3
, and exhibit phylogenetic relationships that reflect those of the 16S rRNAs (compare Figs 3, 4 and 5
). These proteins are therefore likely to be orthologues with a single ATPase per TM protein. It should be noted that many clusters of ATPases found in Fig. 3
are not represented in Fig. 5
. These ATPases probably function in a process and by a mechanism that is independent of a multispanning TM protein homologue. Alternatively, they may act with multispanning TM proteins that are too divergent in sequence for us to recognize.
The data summarized in Table 5 reveal that, as for the ATPases, each major cluster of multispanning TM proteins exhibits its own characteristic size range. However, there is no direct or inverse size relationship between the ATPases tabulated in Table 4
and the membrane proteins tabulated in the corresponding clusters in Table 5
.
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Multispanning TM protein topologies and the occurrence of internal repeats |
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The topology of T2SF homologues in the T4P system is equally unclear. The experimentally determined protein topology of one member of this group, the E. coli protein BfpE, a member of the PilR cluster of pilus-related proteins in Fig. 5, gives four TMSs, with TMSs 2 and 3 being nearly contiguous (Blank & Donnenberg, 2001
). Indeed, most algorithms predict the same four TMSs in this protein (not shown). However, as with GspFEco, the topology of BfpE cannot be predicted from the positive-inside rule. Furthermore, most algorithms predict three TMSs for the closely related V. cholerae protein, TcpE, and three to five TMSs for other proteins in the PilR cluster. Topological predictions for the PilC/G cluster also indicate three and five TMSs, with three TMSs most frequently predicted for the archetypal protein of this cluster, P. aeruginosa PilC (Fig. 6
). Once again, a large domain that is predicted to be periplasmic by the 3 TMS model is predicted to be cytoplasmic by the 4 TMS model, as discussed above for T2SF proteins. Although not as highly conserved as the corresponding segment of the T2SF proteins, this region of almost all proteins under consideration (including the T2SF proteins) contains several highly or absolutely conserved residues. The possible exceptions are all in the PilR cluster, including TcpE and BfpE, in which only some of these highly conserved residues are present. Furthermore, these regions of TcpE and BfpE are almost totally unrelated, which is in marked contrast, for example, to proteins in the T2SF cluster. Therefore, in contrast to GspFEco noted above, it is quite conceivable that BfpE does have a topology different from the predicted 3 TM proteins such as PilC and TcpE.
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The GAP program (Devereux et al., 1984) predicted that regions within the first halves of the TM proteins are homologous to regions in the second halves. One such alignment for P. aeruginosa PilC is shown in Fig. 7(a)
. This 91 residue binary comparison shows 33 % identity and 55 % similarity with an e value of 3x10-8. Comparison scores of 2325 SD for these portions of the two halves of pilc and of 10 SD for corresponding portions of the two halves of PulF of K. pneumoniae were obtained. These values are sufficient to establish that the two halves of these proteins arose from a common origin, probably by an internal gene duplication event. Interestingly, part of this duplicated region includes a diagnostic motif for members of the T2SF-PilC/G-PilR protein clusters, while other residues are well conserved in all clusters (Fig. 7
).
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Based on these results, we suggest that the smaller bacterial proteins arose by one internal gene duplication event, and that a second internal gene duplication event gave rise to the larger archaeal homologues. Judging from the relative degrees of sequence similarity, however, the bacterial duplication event(s) probably occurred after the archaeal duplication event(s). This fact suggests that these duplication events have occurred more than once during the evolution of this family of TM proteins. A similar situation has been observed for other transmembrane protein families (Tseng et al., 1999). It should be noted that the topologies of these proteins can not be deduced with certainty from the hydropathy plots alone.
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Conclusions |
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This report presents sequence comparisons that allow us to establish relationships between several of the protein constituents of the T2S, T4P and Fla systems of bacteria and archaea. Further analyses will be required to establish the functional significance of many of the provocative observations made here.
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ACKNOWLEDGEMENTS |
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