Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales

Abstract

Background

The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry.

Results

We demonstrate that the two maltose ATP binding cassette (ABC) transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively) are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to have been horizontally acquired by a Thermotoga species that had only mal1.

Conclusion

These data demonstrate that the Tc. litoralis and P. furiosus mdx maltodextrin transporter operons arose in the Archaea while their mal maltose transporter operons arose in a bacterial lineage, but not the same lineage as the two maltose transporter operons found in the published Tt. maritima genome sequence. These Tt. maritima maltose transporters are phylogenetically and structurally similar to those found in enteric bacteria and the mal2 operon was horizontally transferred within the Thermotoga lineage. Other Thermotogales species have a third mal operon that is more closely related to the bacterial Thermococcales mal operons, but the data do not support a recent horizontal sharing of that operon between these groups.

Background

The genome sequence of the bacterial hyperthermophile Thermotoga maritima revealed evidence of extensive horizontal gene transfer (HGT) with archaea [1]. Subsequent analyses of its genome sequence along with analyses of large tracts of sequences from other members of the Thermotogales have supported and extended this observation [2, 3]. Many of the genes that have been shared with archaea encode ATP binding cassette (ABC) transporters. Although originally characterized as oligopeptide transporter genes, analysis of the substrate binding proteins encoded by these operons showed that oligosaccharides are their likely substrates [4]. The Tt. maritima genome encodes many other ABC transporters. Indeed, its genome encodes the second highest proportion of ATP-dependent transporter genes (including ABC transporters) among currently sequenced bacterial and archaeal genomes [5]. Some of the Tt. maritima transporters have been experimentally shown to encode sugar binding proteins [4] including two proteins that bind maltose [6].

Horizontal acquisition of transporter genes also occurred among the hyperthermophilic archaea. A striking example of this is the complex evolutionary history of the mal operons in the Thermococcales as depicted in Figure 1. While the genomes of Pyrococcus woesei, P. abyssi, P. horikoshii, and Thermococcus kodakarensis all encode one mal operon, the genomes of P. furiosus and Tc. litoralis each encode two [7, 8]. The second mal genes found in each of the latter two organisms (previously designated as the mal2 genes, but called the mdx genes here, as in [9]) are orthologous to the genes in the single operons of the former organisms. The mdx operon in P. furiosus (PF1938-1936 and PF1933) encodes a maltodextrin binding protein (MdxE _Pf , PF1938) and is upregulated in response to growth on maltose and starch [10, 11]. In P. furiosus, the mal operon (PF1739-1741 and PF1744) encodes MalE _Pf , a protein that binds maltose and trehalose, but it does not appear to function as its major maltose transporter [10–13]. The orthologous mal products in Tc. litoralis have been extensively studied [13–17]. The region encoding the mal genes are flanked by insertion sequences in both P. furiosus and Tc. litoralis suggesting these genes were shared between these organisms by HGT [8]. The similarity of the two MalK homologs (the ATP-binding proteins) in Tc. litoralis (MalK _Tcl and MdxK _Tcl ) suggests that one of them arose by a duplication of the other [18]. Subsequently, the mal-linked malK transferred to the ancestor of P. furiosus along with the entire mal operon [18] (Fig. 1).

Figure 1

The evolutionary history of the mal genes in the Thermococcales. mal E/mdx E encode the substrate binding proteins (SBPs), mal F/mdx F and mal G/mdx G encode membrane spanning proteins (MSPs) also known as permease subunits, and mal K/mdx K encode ATP binding proteins (ABP) containing an ATP binding cassette (ABC). Subscript An denotes ancestral genes.

This history of horizontal transfer of archaeal mal genes raises the possibility that bacterial thermophiles that live among these archaea may also have shared these maltose operons. Tt. maritima was originally isolated from sediments on Vulcano Island, Italy, the same area from which P. furiosus and Tc. litoralis were isolated [19–21]. Hamilton-Brehm, et al. speculated that because these two archaea lived together, they were more likely to exchange mal genes [7], so it is conceivable that bacteria living among them, like the ancestor of Tt. maritima, might have acquired those genes, too. There is evidence of transfer of whole operons to the Thermotoga lineage from the Thermococcales. Most striking, perhaps, is the horizontal transfer of the twelve-gene mbx operon encoding an NADH:ubiquinone oxidoreductase complex that is now found adjacent to the mal1 operon in Tt. maritima [22].

A simple BLAST analysis of the Tt. maritima Mal amino acid sequences suggested they are of bacterial origin [1]. The two sets of Tt. maritima mal genes are their closest homologs indicating a duplication event or HGT within the Thermotogales lineage gave rise to one of them. Other BLAST hits are mainly to bacterial sequences, though some archaeal sequences are also retrieved. Although these data suggest the Tt. maritima mal genes are of bacterial origin, no rigorous phylogenetic analysis has been published that rules out the possibility that these apparently bacterial genes originated from archaeal genes and perhaps were a part of the archaeal HGT events described above. This report provides a detailed examination of the evolutionary history of these mal genes.

The Tt. maritima mal genes are in two operons each containing three, collinearly transcribed genes encoding a substrate binding protein (SBP, MalE) and two membrane-spanning proteins (MSPs, MalF and MalG). However, the malF2 _Ttm ORF is truncated and contains an authentic frameshift mutation. A short ORF (TM1838) precedes it. Neither operon contains a gene encoding the necessary ATP-binding protein (ABP) that provides the energy for substrate transport. The mal1 _Ttm operon (TM1204-2) encodes an apparent mannooligosaccharide transporter that also can bind maltose and maltotriose [6]. The mal2 _Ttm operon (TM1839-36) encodes a maltose and trehalose transporter [6].

Recently, genome sequence data of several other members of the Thermotogales have become available. Sequence data from the genomes of Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae and others are publicly available and a partial genome sequence of Thermotoga neapolitana is available from The Institute for Genomic Research. This information from other Thermotogales species provides data to examine how the mal operons have evolved within this lineage and perhaps can provide evidence about the duplication event that gave rise to the two mal operons now found in Tt. maritima. In this examination of the deep phylogenetic relationships among the hyperthermophiles' mal genes, we also uncovered unreported features of the Thermotogales Mal protein sequences that not only enlighten our understanding of their evolutionary histories, but also suggest novel structural or functional features of the transporter proteins.

Results

Evolution of the mal and mdx genes in the Thermococcales

Previous sequence comparisons using BLAST analyses indicated that one mal operon (the ancestral mal1 operon, mal1_An, along with the ancestral malK2_An gene in Figure 1) transferred from an ancestor of Tc. litoralis to an ancestor of P. furiosus, likely involving a transposition mediated by the insertion sequences now found in P. furiosus [8]. Based on a simple comparison of the genes encoding ABPs in the mal and mdx operons in Tc. litoralis with their homologs in P. furiosus, a copy of an ancestral mdxK gene (malK2_An) was postulated to have recombined downstream of the mal1_An operon in an ancestor of Tc. litoralis (Fig. 1), where it now encodes the ABP for the mal-encoded transporter [18]. This ancestral mdxK could have originated from gene duplication or horizontal acquisition. We performed phylogenetic analysis of all the proteins encoded by the mal and mdx operons to test these hypotheses and to determine the origins of these evolutionarily mobile genes.

The MdxE _Pf (PF1938) protein sequence was used as a query for a BLAST search of the non-redundant protein database at NCBI. The top 250 hits were aligned using ClustalX and then used to construct neighbor joining trees. Using these preliminary relationships as a guide, sequences were chosen from archaea, Thermotogales species, and bacteria closely related to the archaeal sequences to represent a broad spectrum of taxa. These selected sequences were then placed in a new database (approximately 60 sequences for each gene). Associated MalF and MalG protein sequences were concatenated with each MalE-like sequence only if these three ORFs were the only ORFs in an apparent ABC transporter operon. These concatenated sequences were aligned and their phylogenetic relationships were examined by maximum likelihood (ML) and Bayesian analyses. The tree constructed from this ML analysis is shown in Figure 2 with ML bootstrap values and Bayesian posterior probabilities for each analysis placed thereon.

Figure 2

Relationships among families of apparent maltose ABC transporter operons in archaea and bacteria. Homologs of the P. furiosus MdxE (PF1938) were gathered using that sequence as query in BLASTP. Concatenated sequences of these SBP homologs and their cognate MSP and ABP from each operon were prepared, aligned and analyzed. A ML tree generated in PHYML is shown. Values shown are Bayesian posterior probabilities followed by bootstrap support values calculated with PHYML. Bootstrap support values below 800 and posterior probabilities below 0.8 are not shown. For reference, the gi numbers of the MalE (SBP) sequence for each operon are provided. Archaeal sequences are shown in red, those from Thermotogales in blue, and from other bacteria in black. The tree depicted here should be considered unrooted.

The most obvious feature of this tree in regard to the Thermococcales sequences is the independent evolution of the archaeal mal and mdx operons. The distant evolutionary relationship of these two operons is well supported. The Mdx sequences cluster with strong support with those from other archaea while the Mal sequences cluster separate from other archaeal sequences. The latter cluster with sequences from a variety of bacteria including cyanobacteria and members of the Thermotogales. These data support the independent evolution of these two sets of genes and suggest that the mal genes found in Tc. litoralis and P. furiosus were acquired from a bacterium by an ancestral member of the Thermococcales (Fig. 1). We shall discuss the relationship of these Thermotogales operons to the Thermococcales mal operons after considering the history of the Thermococcales malK genes.

Evolution of the malK/mdxK genes and their Thermotogales homologs

The archaeal malK genes have undergone a very different evolutionary history than those in their adjoining mal operons. We examined the relationships among malK homologs using both MrBayes and PHYML and both gave similar, though not identical trees, each with overall relatively weak support. However, within those trees, clusters of strong support were found and these provide reliable information about the evolutionary relationships within those clusters. As shown in the PHYML-derived phylogeny in Figure 3, all the Thermococcales malK and mdxK homologs group together in a well-supported clade. This supports the hypothesis that an ancestral malK2_An homolog either was duplicated in an ancestral Thermococcales or was horizontally acquired by that ancestor from another Thermococcales. The malK2_An was later transferred along with the mal1 _An operon to a P. furiosus ancestor (Fig. 1) [18]. In these genomes, these malK genes are near, but not immediately adjacent to, the three-gene mal/mdx ABC operons, each encoding two MSPs and one SBP. An amylopullulanase gene typically separates the malK homolog from the other genes, but some of these clusters have more genes in the intervening region, perhaps indicative of other HGT or deletion events in some species.

Figure 3

Maximum likelihood analysis of P. furiosus MalK homologs (ATP binding proteins). Homologs of the P. furiosus MdxK (PF1933) were gathered using that sequence as query in BLASTP. A ML tree using an alignment of these sequences is shown here. Only bootstrap support values over 800 are shown. For reference, the gi numbers of the MalK homologs are provided. Archaeal sequences are shown in red, those from Thermotogales in blue, and from other bacteria in black. The tree depicted here should be considered unrooted.

Since the Tt. maritima mal operons do not contain adjacent malK homologs, we sought in this tree any indication of the identity of a possible Thermotoga malK homolog, perhaps derived from the archaeal malK or mdxK genes. Unfortunately, only two Tt. maritima ORFs appear in Fig. 3 among archaeal sequences and all are distantly related to the Thermococcales malK/mdxK cluster. The functions of these archaeal transporters are unknown and since none of their ABP or MSP homologs appeared in the BLAST results using the P. furiosus MalE, MalF or MalG sequences as queries, they unlikely to be mal-related ABPs. A function of TM0421, one of the Tt. maritima ORFs found in the vicinity of these archaeal sequences (though with low bootstrap support), has been suggested. Data has indicated that it is a myo-inositol transporter's ABP (InoK) [4]. The function of the other ORF, TM1232, is unknown, though its expression was reported to be upregulated when Tt. maritima was grown in co-culture with Methanocaldococcus jannaschii [23].

A relatively weakly supported cluster of Thermotogales ORFs apparent in Fig. 3 is unrelated to any archaeal ORFs. This cluster includes the Tt. maritima ORF TM1276 which, based upon gene expression data, has been suggested to encode a maltose transporter ABP [24, 25]. Unfortunately this analysis does not provide additional support for that observation. It does not appear that any archaeal malK/mdxK homolog was acquired by ancestors of the currently sequenced species of Thermotogales.

Discovery of a third mal operon in some members of the Thermotogales

Using the P. furiosus MalE sequence as a query, homologs were revealed in the genome sequences from F. nodosum, Ts. melanesiensis, Tt. petrophila, and Tt. neapolitana but not Tt. maritima (Fig. 2). Each of these genomes contains complete malF and malG genes downstream from this malE homolog in the order malEFG. For Tt. petrophila and Tt. neapolitana this reveals a third apparent mal operon. Ts. melanesiensis and F. nodosum have only one other mal operon (see below), so this is a second for them. This third operon is closely more related to the mal operons of Tc. litoralis and P. furiosus than are the mal1 and mal2 operons (Fig. 2). Although in that figure they may appear to be specific relatives of one another, that relationship is not strongly supported by either maximum likelihood or Bayesian analyses.

To examine the possibility of HGT of these mal3 genes between the Archaea and the Thermotogales, we aligned and analyzed sequences from several additional bacteria in the clade that includes mal3. That analysis, shown in Figure 4, does not show a specific association of the Thermococcales Mal sequences with these Thermotogales sequences, discounting the possibility of HGT of these genes between the Thermococcales and Thermotogales.

Figure 4

Relationship among apparent maltose ABC transporters related to the Mal3 homologs found in some members of the Thermotogales. Homologs of the Tc. litoralis MalE were gathered using that sequence as query in BLASTP. Concatenated sequences of these SBP homologs and their cognate MSP and ABP from each operon were prepared, aligned and analyzed. A ML tree generated in PHYML is shown. Values shown are Bayesian posterior probabilities followed by bootstrap support values calculated with PHYML. Bootstrap support values below 800 and posterior probabilities below 0.8 are not shown. For reference, the gi numbers of the MalE (SBP) sequence for each operon are provided. Archaeal sequences are shown in red, those from Thermotogales in blue, and from other bacteria in black. The tree depicted here should be considered unrooted.

Interestingly, the additional mal operon in Tt. lettingae (depicted here as "Mal3 _Ttl " though it only has two mal operons) is not closely related to its homologs in the other members of the Thermotogales. It is also not a specific relative of the archaeal mal genes. It was likely acquired from a bacterial group different than the donor to the other Thermotogales.

Phylogenetic history of the Thermotogales mal1 and mal2 genes

Although the Tt. maritima Mal paralogs are associated with sequences from enteric bacteria in the P. furiosus mdxE-derived phylogeny (Fig. 2), the resolution of that analysis was too low to determine the detailed relationships among the bacterial orthologs. To examine the Tt. maritima mal1/mal2 evolutionary history in detail and to determine the relationships among the mal1 and mal2 genes in the Thermotogales, we used the MalE1 _Ttm (TM1204) sequence as a query and selected from that dataset sequences from those genes that are arranged in three-gene operons as described previously. Those sequences were concatenated with their MalF and MalG partners. The concatenated sequences from the Mdx _Pf operon was included as an outgroup and all these concatenates were aligned using ClustalX. Trees were constructed using ML and Bayesian analyses as above.

The resulting phylogenetic tree (Fig. 5) grouped Thermotogales sequences together and the relative arrangement of species in this group reflects their branching order as derived using 16S rRNA gene sequence comparisons [3, 26]. Tt. lettingae, F. nodosum, and Ts. melanesiensis have only one of this type of mal operon while the remaining three Thermotoga species each have two of this kind (Fig. 5). These mal operons in F. nodosum and Ts. melanesiensis are related to one another to the exclusion of those from the Thermotoga species. The single Tt. lettingae mal operon is most closely related to the mal1 orthologs. The mal2 operon appears to have arisen within the Thermotoga lineage either through a duplication of an ancestral mal1 or through a horizontal acquisition from an unknown close relative.

Figure 5

Relationship among apparent maltose ABC transporters related to the Mal1 and Mal2 homologs found in some members of the Thermotogales. Homologs of the Tt. maritima MalE1 (TM1204) were gathered using that sequence as query in BLASTP. Concatenated sequences of these SBP homologs and their cognate MSP and ABP from each operon were prepared, aligned and analyzed. A ML tree generated in PHYML is shown. Values shown are Bayesian posterior probabilities followed by bootstrap support values calculated with PHYML. Bootstrap support values below 800 and posterior probabilities below 0.8 are not shown. For reference, the gi numbers of the MalE (SBP) sequence for each operon are provided. The tree is rooted using the Mdx sequences from P. furiosus as outgroup, compare with Fig. 2. Archaeal sequences are shown in red, those from Thermotogales in blue, and from other bacteria in black.

An examination of the genomic contexts of these mal operons suggests that a simple loss of one of the mal operons does not best explain this evolutionary history. As shown in Table 1, the mal operons in F. nodosum and Ts. melanesiensis are not located in an area of the genome that has synteny with any of the other species considered here. This genomic context analysis also supports the observation that this mal operon in Tt. lettingae belongs to the mal1 cluster and that mal1 was likely found in the ancestor of all these Thermotoga species. We found no region in the Tt. lettingae genome sequence that contains a cluster of homologs of the genes surrounding the mal2 operon of the other species. Consequently, a simple loss of mal2 from the Tt. lettingae lineage is not evident. A duplication of mal1 after the divergence of Tt. lettingae would cause the Mal1 and Mal2 lineages to be exclusively related to one another, but that is not apparent in Fig. 5. Consequently, mal2 does not appear to have arisen by a duplication of mal1. Rather the data indicate that mal2 was acquired by an ancestral Thermotoga species from an unknown the Thermotoga species after the divergence of Tt. lettingae.

Table 1 Arrangements of ORFs in the vicinities of the mal1 and mal2 gene clusters in selected species of the Thermotogales. ORFs from different genome sequences are shown here as their best BLASTP hit in the genome sequence of Tt. maritima¹.

With the exception of Tt. maritima all species examined here have intact malF2 genes. Tt. maritima has a malF2 pseudogene. Our analyses cannot determine whether the mutations in malF2 _Ttm occurred during laboratory cultivation of this strain or prior to its isolation from nature.

MalF transmembrane topologies in the Thermotogales and enteric bacteria are similar

Sequence comparisons showed that the Thermotogales cluster of Mal1 and Mal2 proteins are specifically related to those from enteric bacteria, so we sought other evidence to support this unusual evolutionary association of proteins from such different kinds of bacteria. We examined putative structural features of the MalF and MalG membrane proteins by hydropathy analyses using the TMpred program [27].

The MalF _Ec is a member of the CUT1 (carbohydrate uptake) family [28] and is one of the most extensively studied membrane permeases [29]. Unlike other membrane permeases, MalF _Ec is unusual in that it consists of eight transmembrane helices instead of six and has a large periplasmic domain of 180 amino acids between transmembrane helices 3 and 4 (Fig. 6) [30]. This peculiar membrane topology is reportedly conserved in all MalF homologs from bacteria closely related to E. coli [30]. The function of this domain is unknown, but mutational alterations in this loop affect the localization of MalK _Ec to the membrane bound MalF _Ec [29].

Figure 6

Transmembrane helices of Tt. maritima MalF1 and Tt. petrophila MalF3 homologs as predicted by TMpred. Plots derived from TMpred [27] are shown. In selected proteins the locations of the proposed hydrophilic periplasmic domains and the EEA motifs (circled) are shown. The MalF _Ec is thought to interact with the MalG _Ec motif to promote MalK association with the permease complex [42].

To determine if the MalF topology found in E. coli is conserved in the MalF homologs from its close relatives, including members of the Thermotogales, transmembrane topological analyses of these sequences were performed. Representative data from those TMpred analyses are shown in Figure 6 and Additional file 1. Both MalF1 and MalF2 homologs from all members of the Thermotogales show the eight transmembrane helices, large transmembrane loop, and ABP-interaction motif found in MalF _Ec . These unusual features support the close relationship between the Thermotogales MalF1 and MalF2 homologs and those from E. coli relatives. The phylogenetic analyses reported above are not influenced by the presence of this large loop since tree topologies are not changed when these loops were removed from the sequences prior to alignment (data not shown).

The MalF homolog from Propionibacterium acnes, also related to the E. coli MalF, has a smaller, but still evident, loop (see Additional file 1). The conservation of this unique large loop in the MalF permeases in these distantly related organisms is consistent with their shared evolutionary history. Interestingly the MalF from Deinococcus radiodurans (and Thermus thermophilus, not shown) has a smaller loop while Thermoanaerobacter tengcongensis MalF (and those closely related to it) show no evidence of a loop (see Additional file 1). Whatever its function, this loop is more pronounced in close relatives of the enteric form of MalF and was apparently lost from the largely gram positive clade of organisms.

The MalF3 homologs found in some members of the Thermotogales and in P. furiosus and Tc. litoralis do not contain this loop feature (Fig. 6). This observation supports the large evolutionary distance between the mal3 genes and the mal1-mal2 genes.

Unusual domain architectures of MalG in the Thermotogales

The MalG1 and MalG2 homologs in the Thermotogales are unusually large as compared to their homologs from other bacteria including E. coli (Fig. 7). The alignments of MalG homologs showed that the C-terminal 300 amino acids of MalG1 _Ttm and MalG2 _Ttm are similar to the MalG homologs from E. coli and related bacteria, but the N-terminal 525 amino acids showed no significant sequence similarity to any known proteins. TMpred analyses revealed a large hydrophilic region of about 500 amino acids between transmembrane helices one and two of these MalG1 and MalG2 sequences (Fig. 7). The MalG3 homologs lack this region, supporting their phylogenetic placement relative to Mal1/Mal2 (Fig. 7).

Figure 7

Transmembrane helices of Tt. maritima MalG1 homologs as predicted by TMpred. Plots derived from TMpred [27] are shown. Solid and dashed lines depict inside to outside and outside to inside orientations of the helices predicted by TMpred, respectively. The location of the proposed hydrophilic region in the Tt. maritima MalG1 and the EEA motifs (circled) are shown. This motif is involved in MalK binding in the E. coli maltose transporter [42].

Discussion

Members of the archaeal Order Thermococcales have participated in intradomain lateral transmission of ABC transporter genes. This is most clearly seen in the sharing of maltose/trehalose transporter genes [8, 18]. The genome of the bacterium Tt. maritima has a disproportionate representation of ABC transporters, many of which appear to have been acquired from archaea [3, 31]. Since Tt. maritima has two mal operons and lives among species of the Thermococcales, we examined the evolutionary history of its mal genes to look for evidence of possible interdomain HGT.

Our analyses show that the mal operons found in the currently sequenced Thermococcales genomes have undergone a complex evolutionary history. We confirmed the earlier suggestions that P. furiosus acquired its mal operon from an ancestor of Tc. litoralis and that that operon acquired its malK homolog in an ancestor of Tc. litoralis from an ancestral mal2 operon. There is insufficient information to determine whether malK2_An was acquired by the ancestral mal1 operon from another organism via HGT or by gene duplication from the same chromosome.

We found that the ancestor of the Thermococcales mal genes was clearly in the bacterial lineage. Analysis of concatenations of MalE, MalF, and MalG sequences show the Thermococcales Mal proteins are most closely related to bacterial homologs. In contrast, the Mdx sequences cluster separately in a distant archaeal lineage. Since P. abyssi and Tc. kodakarensis do not contain a second mal operon, the mal1_An genes must have been acquired in the Tc. litoralis lineage.

The ancestors of the Thermococcales mal genes were acquired from a bacterial lineage relatively distant from that that gave rise to the two mal operons in Tt. maritima. However, several other members of the Thermotogales have a mal operon (in some of these it is a third mal operon) that is from the same bacterial lineage as the Thermococcales mal operons. We found no evidence that this operon was directly transferred between the Thermococcales and Thermotogales lineages, though. Sequence comparisons cannot demonstrate the functions of these newly revealed mal operons. Investigations into the binding properties of their SBPs are underway to elucidate their potential physiological roles.

The genome sequence of Tt. maritima strain MSB8 published by The Institute for Genome Research in 1999 did not contain a third mal operon. Prior to that publication, in 1993, W. Liebl deposited in GenBank the sequence of a Tt. maritima strain MSB8 gene encoding a β-glucosidase (bglA) that was contained on a cloned fragment that also contained a portion of an apparent malE gene upstream of this bglA [32]. Neither this bglA gene nor the adjacent malE appeared in the subsequent TIGR genome sequence. This suggests that after the strain was deposited in the DSMZ strain collection, it may have suffered a deletion of its mal3-bglA region. We are investigating this possibility using cultures of the type strain and the strain used for genome sequencing.

The modern Thermococcales MalK homologs are descended from an ancestral MalK2. Since all the examined archaeal mal/mdx operons have a nearby malK/mdxK gene, all of which are closely related to one another, we cannot know if the ancestral mal1 operon inherited from the bacteria contained a malK gene or not. Despite the relatively close relationship of the Thermococcales Mal sequences to those of the Thermotogales Mal3 sequences, there are no obvious orthologs of the archaeal MalK/MdxK in any of the Thermotogales. The membrane components of ABC transporters are known to recruit different ABPs to effect transport, so the lack of a malK near these Thermotogales operons is not unusual. Two Tt. maritima ORFs, TM1276 and TM1232, were identified here as potential MalK homologs by phylogenetic analyses, but we did not observe up regulation of either ORF in response to growth on maltose [33] though there is a report that TM1276 is expressed in response to growth on maltose [25].

Sequence comparisons of the Thermotogales Mal1 and Mal2 concatenates demonstrated their close relationship to one another. Three of the examined members of the Thermotogales have only one member of this family of mal operons. Gene synteny comparisons and phylogenetic analyses suggest that mal1 was present in the ancestor of Tt. maritima, Tt. petrophila, and Tt. neapolitana and that mal2 entered that ancestor by HGT from an unknown Thermotoga species. F. nodosum and Ts. melanesiensis both have a single mal operon of the mal1/mal2 type, but the genetic contexts of those operons are unlike those found in any of the other organisms. Their sequences do not place them uniquely with either the mal1- or mal2-type genes, a situation one might expect for ancestral-type sequences. It will be very interesting to determine the functions of these transporters from these two species to compare them with the evolved Mal1 and Mal2 transporters.

The Thermotogales Mal1 and Mal2 sequences consistently clustered in a lineage that included the Mal sequences from the gamma proteobacteria. The relationships revealed in the Mal1/Mal2 phylogeny are supported by the phylogenetic distribution of a unique secondary structure of the MalF homologs. All the MalF homologs with eight transmembrane helices and a large periplasmic loop between transmembrane helices three and four are clustered in our phylogenetic analysis (Fig. 5). A large unique hydrophilic region in the Thermotogales MalG1 and MalG2 proteins confirms their close evolutionary relationship with one another and indicates a relatively recent acquisition of this domain. This region is not found in any other MalG protein and its function is as yet unknown.

Conclusion

We confirmed earlier suggestions that P. furiosus acquired its mal operon from an ancestor of Tc. litoralis and that that operon acquired its malK homolog in an ancestor of Tc. litoralis from an ancestral mal2 operon. Our analyses show that the ancestor of the Thermococcales mal genes was in the bacterial lineage while the ancestor of the mdx genes was from an archaeal lineage. The bacterial lineage from which came the Tc. litoralis mal operon also gave rise to a newly discovered Thermotogales mal operon, the third in some extant Thermotoga species. We find no evidence that the archaeal mal genes or the Thermotogales mal3 genes were shared between these groups.

The Thermotogales Mal1/Mal2 sequences consistently clustered in a lineage that included the Mal sequences from the gamma proteobacteria. The appearance of paralogous mal operons in some Thermotoga species took place by acquisition of an orthologous mal2 operon by HGT.

The relationships among bacterial mal genes revealed in the Mal1/Mal2 phylogeny are supported by the phylogenetic distributions of unique secondary structures in the MalF proteins. The enteric bacteria and the Thermotogales contain MalF homologs with eight transmembrane helices and a large periplasmic loop between transmembrane helices three and four cluster. A unique large hydrophilic region in the MalG proteins from the members of the Thermotogales confirms their close evolutionary relationship to one another and indicates a relatively recent acquisition of this region.

Methods

Data acquisition

Protein sequences were retrieved by BLASTP searches of the nonredundant protein database at NCBI [34] using as queries amino acid sequences of Tt. maritima, P. furiosus, or Tc. litoralis MalE, MalF, or MalG. The P. furiosus MalK2 (PF1933) was used as the query to search for MalK homologs among bacteria and archaea. The top 250 sequences retrieved by BLASTP searches were assembled into datasets and repeated sequence entries and closely related sequences (determined using neighbor-joining trees of aligned sequences) were removed. Those sequences from three-gene operons (malE, malF and malG) were retained and the cognate genes were concatenated. Alignments of the amino acid sequences of these concatenates were prepared using ClustalX v1.83.1 [35].

TM1837, the malF2 _Ttm ORF, contains an authentic frameshift mutation. To make comparisons between MalF homologs, the pseudogene was corrected by removing nucleotides at positions 254 (A) and 298 (G) (identified using GENIO/frame [36]). Corrections at these positions restored the reading frame without introducing new stop codons. malF2 _Tm is preceded by a small ORF, TM1838. The putative 54 amino acid peptide product of TM1838 is 53% similar to the amino terminus of MalF1 _Ttm . By adding a nucleotide between positions 138 and 139 in the TM1838 sequence and combining it with the corrected malF2 _Ttm , a single polypeptide is obtained that has a high similarity to malF1 _Tm as measured by PRSS (P value 1.6 e-21) [37]. It appears that a series of point mutations in an ancestral malF2 created TM1838 and the translationally truncated malF2 _Ttm . Consequently, there is no functional MalF2 _Ttm protein produced by Tt. maritima.

Phylogenetic analyses

Aligned sequences from the concatenate datasets were used to construct consensus trees using MrBayes v3.1 [38] and PHYML v2.4.4. The MrBayes analyses were performed using the default GTR model and gamma distributed rate variation with two runs, each with four chains, for 1,000,000 generations (780,000 for the tree shown in Fig. 2) and taking a consensus tree after a burn-in of either 100,000 or 65,000 generations. These analyses were performed at the Bioinformatics Facility of the University of Connecticut Biotechnology Bioservices Center. Maximum likelihood analyses of these alignments were performed in PHYML with 1000 bootstrap resamplings, the JTT substitution model, a fixed proportion of invariable sites, one category of substitution rate, and the BIONJ input tree.

Other sequence analyses

The analyses above were confirmed with alternative alignments using MUSCLE [39] and T-COFFEE [40] each using their default parameters. These analyses produced phylogenies nearly identical to those shown here when used with PHYML (WAG model, estimated pinvar, estimated gamma distribution) (not shown). Transmembrane topological analyses were performed using TMpred [41, 27].