Department of Biology, University of California at San Diego, La Jolla, CA, 92093-0116, USA1
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2UH, UK 2
Author for correspondence: Milton H. Saier, Jr. Tel: +1 619 534 4084. Fax: +1 619 534 7108. e-mail: msaier{at}ucsd.edu
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ABSTRACT |
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Keywords: ion transport, dicarboxylates, TRAP-T family , phylogeny, molecular evolution
Abbreviations: gb, GenBank; IT, ion transporter; p.m.f., protonmotive force; sp, SWISS-PROT; TC, transport classification; TMS, transmembrane spanning -helix; TRAP-T, tripartite ATP-independent periplasmic transporters
a Present address: MPI für marine Mikrobiologie, Celsiustr. 1, D-28359 Bremen, Germany.
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INTRODUCTION |
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As noted above, ATP-driven uptake systems of the ABC superfamily (Ames, 1986 ; Higgins et al., 1990
; Saier, 1998
) function in conjunction with periplasmic solute-binding receptors that release the solute to the integral membrane translocator (Boos & Lucht, 1996
; Higgins et al., 1990
; Hyde et al., 1990
; Fig. 1d
), and equivalent receptors are essential constituents of TRAP-T systems. These systems have been shown to be specific for C4 -dicarboxylates in Rhodobacter capsulatus (Forward et al., 1997
; Shaw et al., 1991
; Shaw & Kelly, 1991
) or glutamate in Rhodobacter sphaeroides (Jacobs et al., 1996
). The former system is encoded within the dctPQM operon (Forward et al., 1997
; Hamblin et al., 1990
) where DctP is the periplasmic C4-dicarboxylate-binding protein (M r 36128), DctQ is a small integral membrane protein (M r 24763) with 4 putative TMSs and DctM is a large integral membrane protein (Mr 46827) with 12 putative TMSs. Insertional mutagenesis of any one of the three dct genes results in complete loss of p.m.f.-dependent transport activity (Forward et al., 1997
). Glutamate uptake in Rhodobacter sphaeroides, which is dependent on a glutamate- binding protein, is also driven by the p.m.f. (Jacobs et al., 1996
).
In the present study we identified all DctP, DctQ and DctM homologues in the current databases and conducted phylogenetic analyses on representative TRAP-T protein constituents. We thus define the three families of functionally related proteins that presumably comprise TRAP- T systems. Phylogenetic analyses lead to the conclusion that such systems are ancient, and that all currently identified systems probably arose from a single primordial system with little or no shuffling of constituents between them. In Bacillus subtilis, an organism with a completely sequenced genome, a DctP homologue may function independently of DctQ and DctM homologues as a transcriptional sensor. TRAP-T DctP proteins are proposed to be homologous to extracytoplasmic solute-binding receptors of ABC uptake systems. We provide evidence that DctM is a member of a large superfamily of ion transporters and suggest that members of this superfamily have the unusual capacity to associate with auxiliary proteins in order to modify their transport characteristics. Some of the data on which our conclusions are based are included on our phylogenetic web site (http://www-biology.ucsd.edu/~msaier/phylo/titlepage.html) rather than in the text of this paper. This site will be referred to as the Phylo web site in the text.
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COMPUTER METHODS |
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Multiple sequence alignments were constructed and phylogenetic analyses were performed using the tree program (Feng & Doolittle, 1990 ) and the clustal x program (Thompson et al., 1997
). In order to correctly align the DctP, DctQ and DctM family proteins using the tree program, N-and C-terminal extensions of non-homology found in some or all of the homologues were artificially removed. Mean hydropathy and mean similarity analyses were conducted for all protein families analysed based on the resultant multiple alignments of the protein members of these families. Well-conserved portions of the alignments generated with the tree program, and the complete alignments generated with the clustal x program, are presented on our Phylo web site (see Introduction). Multiple alignments and bootstrapped phylogenetic trees (Felsenstein, 1985
) of the DctP, DctM and DctQ family proteins obtained using the clustal x program can also be found on this web site. Differences between the trees generated with the tree and the clustal x programs primarily reflect the different assumptions upon which these programs are based (Young et al., 1999
). Bootstrapping, applied to the trees derived by use of the latter program, do not evaluate these assumptions and therefore do not provide a true measure of confidence as discussed previously (Young et al., 1999
).
Mean hydropathy, similarity and amphipathicity plots were generated using a sliding window of 21 residues (Le et al., 1999 ). The hydropathy values described by Kyte & Doolittle (1982)
were used in the first of these three analyses. Motif searches were conducted using the meme and mast programs (Bailey & Gribskov, 1998
; Grundy et al., 1997
) and signature sequences were defined according to Bairoch et al. (1997)
. All signature sequences were screened against the SWISS-PROT and TREMBL databases. Protein abbreviations used in this study as well as accession numbers which allow easy access to the sequences and primary references are provided in Tables 13
.
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RESULTS |
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The DctP family |
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Fig. 2 presents mean hydropathy (a) and mean similarity (b) plots for the 12 analysed N-terminally truncated members of the DctP family. The mean hydropathy plot revealed that DctP family members are hydrophilic with two distinct regions showing partial hydrophobic character (from alignment positions 80110 and from positions 180200). The latter region and the adjacent hydrophilic region (positions 200220) show the highest percentage similarity. The poorly conserved N-terminal leader sequences of these proteins were removed to facilitate correct alignment of the proteins as indicated in the Computer Methods section.
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A phylogenetic tree for the DctP family was also derived using the clustal x program, and bootstrapping was applied in order to evaluate the reliability at each node. This tree (and the corresponding bootstrapped trees for the DctQ and DctM family proteins) are presented on our Phylo web site (see Introduction for address). As expected, the results confirmed the reliability of nodes more distant from the centre of the unrooted tree but revealed a lesser degree of reliability for branch points near the centre.
Short but well-conserved and gap-free portions of the complete multiple alignment upon which the plots and tree shown in Fig. 2 were based are presented on our Phylo web site. From one of these alignments, a potential signature sequence for the DctP family was derived:
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(x=any residue; alternative residues at any position are presented in square brackets)
This signature sequence was screened against the SWISS-PROT and TREMBL databases and was found to retrieve only members of the DctP family. By these criteria, it is thus a bona fide signature sequence and can be used to identify sequences of new members of the DctP family as they become available.
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The DctQ family |
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Fig. 3 presents mean hydropathy (a) and mean similarity (b) plots for the eight analysed members of the DctQ family. The mean hydropathy plot reveals four putative TMSs with greatest sequence similarity between residue positions 80 and 100. The portion of the multiple alignment indicated by the bar in Fig. 3(a)
is presented on our Phylo web site.
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The most conserved portion of the complete multiple alignment is a gap-free 35 residue segment which shows three fully conserved amino acyl residues and several positions with only conservative substitutions (see our Phylo web site). The following signature sequence for the DctQ family was derived from this portion of the multiple alignment:
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It proved to be specific to proteins of the DctQ family.
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The DctM family |
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Fig. 4 presents mean hydropathy (a) and mean similarity (b) plots for the 11 analysed members of the DctM family. The mean hydropathy plot reveals nine regions of hydrophobicity which span 20 residues or more. Three of these regions (residue positions 110150, 250300 and 350400) probably encompass two TMSs each. Thus, the plot is consistent with the presence of 12 TMSs. The mean similarity plot reveals that almost without exception, each peak of high similarity aligns with a corresponding peak of hydrophobicity. Most of these hydrophobic peaks are about 80% similar.
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A portion of the complete multiple alignment upon which the plots and tree shown in Fig. 4 were based is presented on our Phylo web site. This gap-free alignment shows 3 fully conserved amino acyl residues in 34 residue positions with several positions exhibiting only conservative substitutions. The signature sequence for the DctM family is:
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Additional members of the TRAP-T families in bacteria and archaea |
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Three DctM paralogues are encoded within the completely sequenced genome of the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus (Klenk et al., 1997 ) and two more are found in a second archaeon, Aeropyrum pernix (Table 3
). The Archaeoglobus fulgidus proteins proved to be more similar to the Ralstonia DctM protein than to the other bacterial homologues listed in Table 3
. The comparison scores of these proteins with the Ralstonia DctM protein are presented in Table 4
. Because these three proteins exhibit comparison scores with each other in excess of 20 sd, and two of them give comparison scores with the Ralstonia protein of 10 sd or greater, they can all be considered to be homologues. Fig. 5
shows a portion of the complete multiple alignment of the three Archaeoglobus fulgidus paralogues together with three selected bacterial members of the DctM family. Six positions are fully conserved, and only conservative substitutions are observed at many other positions. Consequently, there is little question that these proteins are homologous.
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Immediately adjacent to the genes encoding the putative DctPQM2 transporter of Archaeoglobus fulgidus, we noted the presence of a gene cluster (korBADG) encoding the four subunits of the 2-oxoglutarate:ferridoxin 2-oxidoreductase (Klenk et al., 1997 ). The clustering of these genes suggests a common function. The proposed function of DctPQM2 is therefore that of a 2-oxoglutarate uptake system.
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Relative degrees of sequence divergence among the three TRAP-T family constituents |
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Preliminary evidence that DctM is a member of a large superfamily of ion transporters |
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DISCUSSION |
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The genome of the archaeon Archaeoglobus fulgidus was shown to encode three DctM homologues and all of these possess N-terminal regions that are homologous with each other. These N-terminal regions exhibit sizes and topological features that resemble the bacterial DctQ homologues. In Haemophilus influenzae, one of the three TRAP-T systems identified exhibits a fused DctQM protein of 16 TMSs. Since the Archaeoglobus fulgidus paralogues also exhibit 16 putative TMSs, it is reasonable to propose that the Archaeoglobus fulgidus homologues represent the equivalent of both DctQ and DctM of Rhodobacter capsulatus. This suggestion is in agreement with our observation that the bacterial DctQ homologues have diverged much more rapidly than have the DctM homologues. Adjacent to each of the Dct(Q)M homologues of Archaeoglobus fulgidus is a gene encoding a putative extracytoplasmic solute-binding receptor with significant sequence and motif similarity to the well-characterized glutamine- and glutamate-binding proteins of ABC-type transporters in Escherichia coli. These three archaeal proteins exhibit a single N-terminal hydrophobic peak followed by regions of relative hydrophilicity as expected for an extracytoplasmic solute-binding protein. It seems likely that these three Archaeoglobus fulgidus proteins function as the solute-binding receptors for the Dct(Q)M homologues of this organism. This suggestion is substantiated by the observation that the genes encoding the four subunits of 2-oxoglutarate:ferridoxin 2- oxidoreductase map immediately adjacent to the dctPQM2 genes of Archaeoglobus fulgidus.
The great degree of sequence divergence between the constituents of the bacterial and archaeal TRAP-T systems suggests that they are related by vertical descent, and that horizontal transmission of genetic material between these two domains of living organisms does not account for their occurrences. This observation leads to the suggestion that the TRAP-T family is very old, dating back to before the split between bacteria and archaea.
Encoded within the fully sequenced genome of Treponema pallidum , a DctM homologue was identified. This protein exhibits the same size and apparent topology as the DctQM protein of Haemophilus influenzae with the non-homologous putative DctQ moiety at the N- terminus. Moreover, adjacent to the gene encoding this protein were two genes encoding proteins of about 330 amino acyl residues which exhibited N-terminal peaks of hydrophobicity. Either of these proteins could be the solute-binding receptor for the Dct(Q)M homologue of T. pallidum. Thus T. pallidum may possess a complete DctPQM transporter as is apparently true for many other prokaryotes.
DctP, DctQ and DctM occur in many bacteria as distinct polypeptide chains encoded by three contiguous genes, usually in operons with the gene order dctPQM. However, we found two types of protein (or domain) fusions. One is DctPQ (Y4 mM from Rhizobium sp. strain NGR243) and the other is DctQM (Y147 from Haemophilus influenzae and several other organisms). None of the other possible domain orders (i.e. DctQP, DctMQ, DctPM and DctMP) were found. This observation leads to the prediction that P interacts with Q, and Q with M in all DctPQM homologues. DctQ may serve as a structural anchor for DctP, allowing association of P with M, a suggestion that is consistent with the relative rates of divergence of these three TRAP-T protein constituents. It is also interesting to note that no DctPQM fusions have been detected, and that this fact correlates with the fact that in ABC uptake permeases, the extracytoplasmic receptors are seldom fused to the integral membrane translocator protein (Obis et al. , 1999 ). Possibly this last fact points to additional non-transport-related functions (e.g. chemoreception, regulation) for these proteins.
We have obtained preliminary evidence that DctM may be a member of a large superfamily of ion transporters which we have tentatively designated the ion transporter (IT) superfamily. Among the members of this superfamily are many well-documented secondary transporters that function without the participation of a extracytoplasmic receptor or an integral membrane auxiliary protein. This fact strengthens our conviction that in Rhodobacter capsulatus, DctM alone provides the pathway of solute transport, and that DctQ and DctP function in accessory capacities. In confirmation of this suggestion we note that both Pseudomonas aeruginosa and Synechocystis sp. strain PCC6803 apparently have a surplus of genes encoding DctP homologues with no corresponding dctQ- or dctM-like genes mapping nearby. Either these proteins serve as auxiliary binding receptors for the recognized TRAP-T family systems in these organisms, or they serve entirely different functions as we have proposed for Bacillus subtilis.
Another member of the putative IT superfamily (see Tables 5 and 6
) is the ArsB protein of Escherichia coli, a constituent of the Ars family (TC no. 3.4). In this organism, ArsB can function together with ArsA to form an ATP- dependent anion translocator. However, in the absence of ArsA, ArsB transports arsenite and antimonite in a p.m.f.-driven process (Kuroda et al., 1997
; Silver et al., 1993
). The auxiliary protein, ArsA, has evidently been superimposed on the integral membrane secondary transporter ArsB, allowing the use of ATP to energize transport. It seems that members of the IT superfamily exhibit the unusual characteristic of allowing superimposition of additional protein constituents on the basic transport process that modify transport characteristics by allowing high-affinity solute reception (e.g. using DctP), by altering the energy coupling mechanism (e.g. using ArsA) or by providing an unknown function (e.g. DctQ). The molecular basis for this unusual characteristic remains to be elucidated.
DctP is a periplasmic solute-binding protein with many characteristics that typify the extracytoplasmic receptors of ABC transporters (Tam & Saier, 1993 ). For example, DctP is the same size as many of the latter proteins and it confers high- affinity substrate binding and recognition to its transporter (Walmsley et al., 1992
). We have noted that DctP exhibits regions of limited sequence similarity to binding receptors of ABC systems including the citrate-binding protein of Salmonella typhimurium (Tam & Saier, 1993
). We therefore propose that although sequence divergence is very considerable, the DctP proteins, as well as the putative Archaeoglobus fulgidus solute-binding receptors, represent authentic constituents of the solute-binding receptor superfamily that includes all extracytoplasmic receptors that feed into ABC transporters (Kuan et al., 1995
; Quiocho & Ledvina, 1996
; Tam & Saier, 1993
). In this connection it is interesting to note that neither ABC uptake systems nor TRAP-T uptake systems, both of which are dependent on extracytoplasmic receptors, are found in eukaryotes. Either binding-protein-dependent transport arose after prokaryotes and eukaryotes diverged from each other during the great split, or such a transport mode is inconsistent with eukaryotic life-styles. In either case, it seems that horizontal transmission of genetic material encoding transport proteins between prokaryotes and eukaryotes has been exceptionally rare (Saier, 1998
, 1999
). This postulate also leads to the possibility that ABC uptake systems arose from ABC-type efflux systems that function independently of binding receptors and are found ubiquitously throughout the three domains of life (Saurin et al., 1999
).
Several members of the IT superfamily arose by an intragenic duplication event that gave rise to proteins of 12 TMSs from primordial systems that presumably exhibited 6 TMSs (Saier & Tseng). Most dramatic is the ArsB protein where the degree of sequence similarity between the halves is sufficient to easily establish homology (Saier & Tseng, 1999 ; unpublished observation). Attempts to document an internal duplication in DctM led to recognition of sequence similarity between its two halves, but this similarity was insufficient to establish homology (unpublished observation). We suggest either that the two halves of DctM diverged in sequence more rapidly than did those in ArsB or that the duplication event leading to the full-length DctM homologues occurred earlier than the one that gave rise to ArsB homologues. The latter interpretation is in agreement with conclusions reached from studies of another large superfamily, the RND superfamily (Tseng et al., 1999
). Regardless of this possibility, however, we predict that all IT superfamily transporters will prove to have arisen by intragenic duplication.
In spite of the fact that an orphan DctP homologue appears to exist in Bacillus subtilis, an organism with a fully sequenced genome, and that a few extra receptors may be present in a few other bacteria, our phylogenetic analyses suggest that in bacteria, and probably in archaea as well, the three constituents of complete TRAP-T systems evolved in parallel from a single primordial three-component (or three-domain) transport system. If this contention is correct, it would imply that intact TRAP-T systems did not arise recently by the superimposition of accessory proteins on the putative DctM transporter, but that these three proteins have functioned together for billions of years with minimal shuffling of constituents between systems. This conclusion is in agreement with the observation that such shuffling has seldom occurred during the evolution of the ubiquitous ABC superfamily (TC no. 3.1; Kuan et al., 1995 ), the bacterial phosphotransferase functional superfamily (TC no. 6.16.6; Postma et al., 1993
) and the integral membrane constituents of F-type ATPases (TC no. 3.2; Blair et al., 1996
). Moreover, the demonstrable sequence similarity between the putative DctP homologues in Archaeoglobus fulgidus and the glutamine/glutamate-binding proteins of bacteria leads further to the suggestion that all DctP homologues are part of the binding protein superfamily (Quiocho & Ledvina, 1996
). The general picture that seems to be emerging is that once a multicomponent transport system has appeared, it undergoes diversification following gene duplication and during speciation with minimal shuffling of constituents between functional units. Both ABC and TRAP-T uptake systems must have arisen during molecular antiquity but have retained their independent lineages. The molecular, physiological and regulatory constraints imposed upon transporters that prevent shuffling have yet to be elucidated.
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ACKNOWLEDGEMENTS |
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Received 8 April 1999;
revised 9 August 1999;
accepted 16 August 1999.