Phylogenetic Analyses of Two "Archaeal" Genes in Thermotoga maritima Reveal Multiple Transfers Between Archaea and Bacteria

Camilla L. Nesbo*, Stéphane L'Haridon, Karl O. Stetter and W. Ford Doolittle*

*Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada;
{dagger}Sciences de l'Environnement Marin, Centre National de la Recherche Scientifique and Université de Bretagne Occidentale, Plouzané, France;
{ddagger}Lehrsthul fuer Mikrobiologie und Archaeenzentrum, Universitaet Regensburg, Germany


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The genome sequence of Thermotoga maritima revealed that 24% of its open reading frames (ORFs) showed the highest similarity scores to archaeal genes in BLAST analyses. Here we screened 16 strains from the genus Thermotoga and other related Thermotogales for the occurrence of two of these "archaeal" genes: the gene encoding the large subunit of glutamate synthase (gltB) and the myo-inositol 1P synthase gene (ino1). Both genes were restricted to the Thermotoga species within the Thermotogales. The distribution of the two genes, along with results from phylogenetic analyses, showed that they were acquired from Archaea during the divergence of the Thermotogales. Database searches revealed that three other bacteria—Dehalococcoides ethenogenes, Sinorhizobium meliloti, and Clostridium difficile—possess archaeal-type gltBs, and the phylogenetic analyses confirmed at least two lateral gene transfer (LGT) events between Bacteria and Archaea. These LGT events were also strongly supported by gene structure data, as the three domains in bacterial-type gltB are homologous to three independent ORFs in Archaea and Bacteria with archaeal-type gltBs. The ino1 gene has a scattered distribution among Bacteria, and apart from the Thermotoga strains it is found only in Aquifex aeolicus, D. ethenogenes, and some high-G+C Gram-positive bacteria. Phylogenetic analysis of the ino1 sequences revealed three highly supported prokaryotic clades, all containing a mixture of archaeal and bacterial sequences, and suggested that all bacterial ino1 genes had been recruited from archaeal donors. The Thermotoga strains and A. aeolicus acquired this gene independently from different archaeal species. Although transfer of genes from hyperthermophilic Archaea may have facilitated the evolution of bacterial hyperthermophily, between-domain transfers also affect mesophilic species. For hyperthermophiles, we hypothesize that LGT may be as much a consequence as the cause of adaptation to hyperthermophily.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Lateral (or horizontal) gene transfer (LGT) is now recognized as an important evolutionary force. Comparisons of gene composition between completely sequenced genomes, comparative base composition and codon analyses within genomes, and phylogenetic trees based on single gene families leave no doubt that LGT, especially among and between prokaryotic domains (Archaea and Bacteria), can be the basis of specific biochemical and environmental adaptations (Lawrence and Ochman 1998Citation ; Doolittle 1999a, 1999bCitation ; Jain, Rivera, and Lake 1999Citation ; Lawrence 1999Citation ; Martin 1999Citation ). However, it is not simple to assess the relative contributions of LGT and vertical inheritance in establishing and maintaining phylogenetic patterns (Doolittle 1999aCitation ; Fitz-Gibbon and House 1999Citation ; Logsdon and Faguy 1999Citation ).

Several authors have claimed that hyperthermophilic Archaea and Bacteria have exchanged genes particularly often. For instance, Nelson et al. (1999)Citation reported that 24% of all open reading frames (ORFs) in the genome of the hyperthermophilic bacterium Thermotoga maritima showed the highest BLAST scores to genes found in Archaea. A similar situation was observed with the other completed hyperthermophilic bacterial genome sequence, that of Aquifex aeolicus. Aravind et al. (1998)Citation found that 16.2% of the ORFs in this genome exhibited the highest similarity to archaeal ORFs in BLAST analyses.

Such observations could be taken to indicate that extensive LGT has occurred between the hyperthermophilic Bacteria and Archaea and support the suggestion that this process has facilitated the acquisition of thermophily by one or both groups (Aravind et al. 1998Citation ; Nelson et al. 1999Citation ). However, Thermotoga and Aquifex are also members of the two lineages (Thermotogales and Aquificales) believed (primarily from 16S small-subunit [SSU] rRNA phylogenetic analyses) to have diverged the earliest from the rest of the Bacteria (Tiboni et al. 1991Citation ; Barns et al. 1996Citation ; Fitz-Gibbon and House 1999Citation ; Klenk et al. 1999Citation ; Bocchetta et al. 2000Citation ). Thus, Kyrpides and Olsen (1999)Citation and Logsdon and Faguy (1999)Citation have argued that if Thermotoga and Aquifex are truly deep-branching, it is equally parsimonious to suppose that some of the genes these hyperthermophilic Bacteria share with Archaea are primitive features, retained ancestral genes which have since been lost in most Bacteria as they adapted to mesophilic environments.

These alternatives can be distinguished only on a gene-by-gene basis. If phylogenetic analyses rigorously show that a gene in a bacterial genome occupies a specific branch within an otherwise archaeal gene tree (that is, is more closely related to some archaeal genes than to others) and/or that multiple bacterial outgroups (e.g., other members of the Thermotogales or the Aquificales) lack the gene, then LGT becomes the most parsimonious interpretation. We searched the Thermotoga genomes for genes with strong similarity to genes within the Archaea (BLAST cutoff at 1e-100). Of 65 candidate cases suitable for phylogenetic analysis (i.e., ORF found in both Archaea and Bacteria), we focused on two, encoding myo-inositol synthase and the large subunit of glutamate synthase, for more rigorous examination.

The T. maritima myo-inositol 1P synthase gene (ino1, TM1419), encoding the first enzyme in the de novo biosynthetic pathway to inositol, was most similar to its Pyrococcus abyssi and Pyrococcus horikoshii orthologs (AB1989, PH1605). The gene encoding the large subunit of glutamate synthase subunit (TM0397, incorrectly annotated gltA in the genome, but here more conventionally referred to as gltB) showed the highest similarity to the Archaeoglobus fulgidus gene (AF0953). Based on these data alone, Thermotoga has apparently exchanged genes with more than one archaeon. The two genes also display highly divergent patterns of phylogenetic distribution, suggesting that the Thermotoga transfer is not the only one in which these genes have participated.

Glutamate synthase (GltS) is one of the key enzymes in the nitrogen assimilation pathway, and it is widely distributed among all organisms (Vanoni and Curti 1999Citation ). In Bacteria, the L-glutamate produced not only is the precursor of other amino acids, but is also involved in osmoregulation (Martins et al. 1997Citation ; Martin, Ciulla, and Roberts 1999Citation ; Vanoni and Curti 1999Citation ). Outside the Archaea, three different classes of GltS have been described: a bacterial form (NADPH-GltS), an Fd-dependent form in cyanobacteria and plants (Fd-GltS), and a pyridine-linked form in both photosynthetic and nonphotosynthetic eukaryotes (NADH-GltS) (Vanoni and Curti 1999Citation ). In most bacteria, NADPH-GltS is composed of a large subunit (the gltB gene) and a small subunit (encoded by the gltD gene). In eukaryotes, glutamate synthase displays a single-subunit structure, corresponding either to the large bacterial subunit or to both the small and the large bacterial subunits (Jongsareejit et al. 1997Citation ; Temple, Vance, and Gnatt 1998Citation ; Vanoni and Curti 1999Citation ).

In the archaeon Pyrococcus sp. KOD1, a homolog of the bacterial small subunit (gltD gene product) has been shown to function alone (Jongsareejit et al. 1997Citation ), and the genomes of P. abyssi and P. horikoshii contain only gltD genes (Kawarabayasi et al. 1998Citation ). The genomes of A. fulgidus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum, on the other hand, encode only homologs of the large subunit (Bult et al. 1996Citation ; Klenk et al. 1997Citation ; Smith et al. 1997Citation ). These archaeal gltBs are much shorter than the bacterial polypeptides (~500 amino acids in Archaea, compared with 1,500 amino acids in Bacteria) and show homology to the central part of the bacterial gene (Vanoni and Curti 1999Citation ). This has lead to speculations that the archaeal gltBs are ancestral minimum forms of the enzyme (Temple, Vance, and Gnatt 1998Citation ). The question of how the enzymes can function with important domains missing is, however, unresolved, and our analysis showed that the entire gltB sequence is represented by three separate ORFs in all Archaea (where the short archaeal gltB has been found). Only the ORF encoding the central domain has been annotated gltB in these archaeal genome projects.

The ino1 gene, although found in most eukaryotes, has a scattered distribution among prokaryotes (Bachhawat and Mande 2000Citation ). The encoded protein can be used to produce the unusual osmolyte di-myo-inositol 1,1' phosphate (DIP). Among prokaryotes, this osmolyte appears to track hyperthermophily, as it has only been found in hyperthermophilic Archaea, Aquificales (Aquifex pyrophilus), and Thermotogales (T. maritima, Thermotoga neapolitana), where it is thought to be involved in protection from heat and salt (Martins et al. 1996Citation ; Chen, Spiliotis, and Roberts 1998Citation ; Martin, Ciulla, and Roberts 1999Citation ). Apart from the hyperthermophilic lineages, the ino1 gene has so far been found only in the high-G+C Gram-positive phyla Mycobacteria and Streptomyces (Bachhawat and Mande 1999, 2000Citation ). In mycobacteria, the cellular envelope contains large amounts of inositol compounds that facilitate receptor-mediated phagocytosis of Mycobacterium tuberculosis by host cells (Besra and Brennan 1997Citation ). In Streptomyces, myo-inositol is used in the synthesis of antibiotics, such as spectinomycin (Walker 1995Citation ).

To establish when within the divergence of Thermotogales the inferred LGT events occurred, we screened 16 strains belonging to the genus Thermotoga and other related Thermotogales for the occurrence of the ino1 and gltB genes and successfully amplified and sequenced portions of the genes from 10 different species of Thermotoga. We performed detailed phylogenetic analyses on these genes and homologous sequences available in databases. Since SSU rRNA sequences were not available for most of these isolates, we also amplified and sequenced these genes. Overall, LGT seems less likely to affect SSU rRNA genes than to affect genes for dispensable metabolic functions (e.g. Jain, Rivera, and Lake 1999Citation ), and we took the resulting SSU rRNA phylogeny as a backbone against which to assess LGT.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
DNA Extraction, PCR, and Sequencing
The bacterial strains used are listed in table 1 . DNA was extracted using the protocol of Charbonnier and Forterre (1994)Citation . Amplification of 935–947 bp of the gltB gene was carried out using the primers gltBFOR (ATC ATG AAG CAG GCN GAR AC) and gltBREV (GTT CCT ATG TAA CAN GCR TC). Primers used for amplifying 1,046 bp of the myo-inositol 1P synthase (myo1P) were ino1FOR (CTG GGT CAG GGC TAY GTN GC) and ino1REV (CCC GGG TTC TTC ATR TAR AA). Primers used for amplifying the SSU rRNA DNA were TSSU.F-3 (TAT GGA GGG TTT GAT CCT GG), TSSU.F-497 (AGA AAG CCC CGG CTA ACT AC), TSSU.R-1260 (ATT GTA CCG CCC ATT GTA GC), and TSSU.R-1528 (CCG GTA CGG CTA CCT TGT TA). The gltB primers were designed from the alignment of the T. maritima sequence along with the sequences from A. fulgidus and M. jannaschii. The ino1 primers were constructed from the alignment of the T. maritima and the P. abyssi and P. horikoshii sequences. The SSU primers were constructed from the alignment of all available Thermotogales sequences. Amplifications were carried out in a 50-µl final volume containing 1–5 ng template DNA, 1 x PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTP, 1 mM of each primer, and 0.5–1 U Taq polymerase (Eppendorf). The reactions were submitted to an initial denaturation at 96°C for 5 min, followed by 35 cycles with denaturation at 96°C for 1 min, primer annealing at 40–55°C for 1 min, and primer extension at 72°C for 2 min. The resulting PCR products were cloned using the TOPO TA cloning kit (Invitrogen), and individual clones were checked for inserts within the correct size range by PCR screening. PCR products of the correct size were sequenced manually with the Sequenase* PCR product sequencing kit (Amersham Pharmacia Biotech) using the m13FOR and m13REV sequencing primers. One or two clones containing the target gene were finally sequenced from both strands using either an ABI 373A or a LiCor 4000L automated sequencer. For the Thermotoga sp. RQ2 ino1 sequence, four clones from two different PCR reactions were sequenced (one was sequenced from both strands, and three were sequenced from one strand).


View this table:
[in this window]
[in a new window]
 
Table 1 Bacterial Strains Used in the Study, Along with the Genes Analyzed

 
Data Analysis
Thermotoga maritima sequences were obtained from the Thermotoga maritima Genome Database at http://www.tigr.org/. Preliminary sequence data from the unfinished genomes of Chlorobium tepidum, Mycobacterium avium, Vibrio cholerae, and Dehalococcoides ethenogenes were obtained from the Institute for Genomic Research web site at http://www.tigr.org. The Bordetella pertussis, Clostridium difficile, Corynebacterium diphtheriae, Mycobacterium bovis, Mycobacterium leprae, Streptomyces coelicolor, and Yersinia pestis sequence data were produced by the Microbial Genome Sequencing Group at the Sanger Center and were obtained from http://www.sanger.ac.uk/. Sulfolobus solfataricus sequences were obtained from http://niji.imb.nrc.ca/, Pyrobaculum aerophilum sequences were obtained from http://genome.caltech.edu/, Pyrococcus furiosus sequences were obtained from http://combdna.umbi.umd.edu/, Sinorhizobium meliloti sequences were obtained from http://sequence.toulouse.inra.fr/, and Methanosarcina mazeii sequences were obtained from http://www.g2l.bio.uni-goettingen.de/.

For gltB, a group of distantly related homologs were found in some species, including T. maritima (TM1058). However, preliminary phylogenetic analyses including these genes showed that they were very distantly related to all other gltB genes. These presumably homologous sequences were therefore excluded in all subsequent analyses. For bacterial sequences, we also only included one copy when multiple copies of gltB were submitted from the same organism and BLAST searches and preliminary phylogenetic analyses showed them to be most similar to each other (i.e., recent within-genome duplications).

Amino acid sequences were first aligned in CLUSTAL X (Thompson et al. 1997Citation ) and then checked and edited by eye in Se-Al, version 1.0 (Rambaut 1995Citation ). An alignment of available Thermotogales SSU sequences, along with the sequences from A. aeolicus and A. pyrophilus, was obtained from the European Small Subunit Ribosomal RNA database (Van de Peer et al. 2000Citation ). The new sequences generated in this study were manually added to this alignment.

The chi-square test in PAUP*, version 4.04b (Swofford 2000), revealed significant differences in G+C content among the Thermotogales SSU genes (P > 0.0000001). Such biases can have strong influences on phylogenetic reconstruction (Galtier and Gouy 1995Citation ). Hence, the SSU phylogeny including all the Thermotogales strains studied was built using logdet distances (Lockhart et al. 1994Citation ), the heuristic search option with tree bisection-reconnection (TBR) branch swapping, and 10 replicates with starting trees made by random stepwise additions, and the minimum-evolution objective function in PAUP*, version 4.04b. The SSU, gltB, and ino1 phylogenies including only the strains closely related to T. maritima were built using parsimony, applying the branch-and-bound option in PAUP*, version 4.04b, and rooted by midpoint rooting.

Phylogenetic analyses including sequences retrieved from the public databases were performed applying amino acid sequences, as well as DNA sequences, for a subset of the gltB sequences (see fig. 4B ). DNA analyses and protein parsimony were done in PAUP*, version 4.04b, applying the heuristic-search option with TBR branch swapping and 10 replicates with starting trees made by random stepwise additions. For distance trees, the minimum-evolution objective function was applied. For maximum-likelihood (ML) and ML distance analyses of the gltB DNA alignment, the Model test program (Posada and Crandall 1998Citation ) suggested two models: the Hasegawa-Kishino-Yano model (model 1, log likelihood ratio test) with gamma correction ({alpha} = 0.6551), and the general-time reversible model (model 2, model selection using the Aikaike Information Criterion criterion) with gamma correction ({alpha} = 0.6673). Both models gave the same topology in ML analyses and distance analyses. Hence, only results from model 1 are presented in figure 4B. Protein neighbor-joining (NJ) trees of PAM-based distances were constructed using the PROTDIST and NEIGHBOR programs in PHYLIP, version 3.572 (Felsenstein 1995Citation ). Protein ML analyses were performed using PROTML in the MOLPHY, version 2.2, package (Adachi and Hasegawa 1996Citation ) and PUZZLE, version 4.0 (Strimmer and von Haeseler 1996Citation ). Parameters used were the JTT model of substitutions (PROTML and PUZZLE), gamma-distributed rates over eight categories and the {alpha} parameter estimated from the data (PUZZLE), and the quick-add search with 1,000 replications (PROTML). The branch lengths of the PROTML trees shown in figure 4A and figure 5 were estimated in PUZZLE, applying the JTT model of substitutions with no gamma correction. Confidence of branch points was estimated by 1,000 bootstrap replications (distance and parsimony trees), by 1,000 puzzling quartets (ML trees estimated by PAUP and PUZZLE), and by calculating resampling estimated log likelihood (RELL) values using the 1,000 ML trees estimated by PROTML, applying the settings described above. The trees were arbitrarily rooted with the eukaryotic sequences.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.—A, Maximum-likelihood tree of all available archaeal gltB sequences, as well as a selection of sequences from bacteria and eukaryotes, estimated from the amino acid alignment using PROTML. Branches inferred to be involved in LGT are shaded gray. The same overall topology was obtained by quartet puzzling, neighbor-joining of PAM-based distances, and parsimony analyses. B, Maximum-likelihood tree of gltB DNA sequences. Bacterial species are given in plain text, archaeal species in italic text, and eukaryotes in bold text. Numbers at nodes represent the percentages of occurrence of nodes in 1,000 bootstrap resamplings (distance in bold type and parsimony in italic type), 1,000 puzzling steps (plain), or RELL values in percentages obtained using the 1,000 maximum-likelihood trees estimated with PROTML (indicated by R; A only). Black and gray circles at the nodes indicate that both the parsimony and the distance bootstrap values were above 90% and 70%, respectively

 


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.—Maximum-likelihood tree of all available prokaryotic ino1 sequences, as well as a selection of eukaryotes, estimated from the amino acid alignment using PROTML. Bacterial species are given in plain text, archaeal species in italic text, and eukaryotes in bold text. The same topology was obtained by quartet puzzling, neighbor-joining of PAM-based distances, and parsimony analyses. Numbers at nodes represent the percentages of occurrence of nodes in 1,000 bootstrap resamplings (distance in bold text and parsimony in italic text), 1,000 puzzling steps (plain), or RELL values in percentages obtained using the 1,000 ML trees estimated by PROTML (indicated by R). Black and gray circles at the nodes indicate that both the parsimony and the distance bootstrap values were above 90% and 70%, respectively.

 
Sequences have been submitted to the EMBL Nucleotide Sequence Database (accession numbers AJ00998–AJ401025).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
SSU rRNA Phylogeny of Thermotogales
The complete SSU RNA gene was amplified from strains for which no sequence had been submitted to GenBank. A phylogenetic tree estimated from these sequences, along with previously determined Thermotogales sequences, is shown in figure 1 . All strains sequenced herein clustered as very close relatives of T. maritima and fell into three groups: (1) the Thermotoga sp. KOL6 sequence, (2) T. maritima and the Thermotoga sp. RQ2 sequences, and (3) T. neapolitana and the remaining Thermotoga sp. sequences (fig. 1 ). The close relationship between T. maritima and T. neapolitana supported previous findings from DNA-DNA hybridization, morphology, and physiology (Belkin, Wirsen, and Jannasch 1986Citation ; Jannasch et al. 1988Citation ; Huber and Stetter 2000Citation ). Unexpectedly, two of the T. maritima strains, T. maritima SL7 and T. maritima FjSS3.B1, clustered with the T. neapolitana strain, not the T. maritima type strain. This does not, however, conflict with previous observations, as T. maritima SL7 was classified according to restriction maps of the SSU gene, using enzymes with which T. neapolitana and T. maritima display identical profiles (C. Jeanthon, personal communication). For T. maritima FjSS3.B1, the SSU phylogeny agreed with the distribution and similarity of xyn genes among T. neapolitana, T. maritima, and T. maritima FjSS3.B1 (Reeves et al. 2000).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.—Minimum-evolution tree of small-subunit sequences based on logdet distances of the Thermotogales strains included in the study, rooted by the Aquifex aeolicus and Aquifex pyrohilus sequences. Inclusion of additional Thermotogales sequences available in GenBank (Caldotoga, Geotoga, uncharacterized) did not alter the topology. Excluding or including invariable sites gave the same topology.

 
Archaeal-Type gltB and ino1 Distribution Among the Thermotogales: Both Genes Are Restricted to the Thermotoga Lineage
We successfully amplified gltB and ino1 genes from nine of the Thermotogales strains listed in table 1 , all corresponding to members of the Thermotoga lineage. For both genes, the amplified sequences were highly similar to the T. maritima and A. fulgidus (gltB) or Pyrococcus sp. (ino1) sequences. The distribution of the two genes did not completely overlap, however, as no gltB was obtained from Thermotoga sp. KOL6 and no ino1 could be amplified from Thermotoga thermarum.

In addition, no ino1 or archaeal-type gltB could be amplified from the other Thermotogales (Petrotoga, Fervidobacterium, Thermosipho). However, a bacterial-type gltB sequence was obtained from Petrotoga miotherma (see below). Although the absence of a gene cannot be asserted on the basis of negative PCR results, the PCR primers used did amplify the ino1 gene from P. furiosus and the gltB gene from A. fulgidus (see table 1 ), suggesting that if these genes are present in the other Thermotogales, they differ considerably from the sequences amplified here. Moreover, the presence/absence of ino1 PCR products matches the presence/absence of the inositol-derived osmolyte DIP in representatives of the Thermotogales lineages, analyzed by Martins et al. (1996Citation ).

Phylogenetic trees of the Thermotoga ino1 and gltB sequences are shown in figure 2A and B. The topologies of the SSU, gltB, and ino1 trees agreed, except for the placement of Thermotoga sp. RQ2, which clustered with T. maritima for SSU and gltB but branched between T. maritima and the T. neapolitana strains in the ino1 tree. Inspection of the Thermotoga sp. RQ2 ino1 sequence showed that this molecule was a mosaic of segments most similar to the T. maritima sequence and the T. neapolitana sequence, likely resulting from recombination between Thermotoga sp. RQ2 and a T. neapolitana strain. An alignment of the variable positions, as well as the distribution of variable sites, in these sequences is shown in figure 3 . Such a mosaic gene structure is commonly taken as evidence for intra- and interspecies recombination in well-studied Bacteria (such as Neisseria, Escherichia, and Streptococcus; e.g., Fudyk et al. 1999Citation ; McGraw et al. 1999Citation ; Feil, Enright, and Spratt 2000Citation ); this is, to our knowledge, the first evidence for interspecies intragenic recombination in hyperthermophiles.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.—A, Phylogenetic tree of the Thermotoga gltB sequences. B, Phylogenetic tree of the Thermotoga ino1 sequences. Both trees were constructed applying parsimony. Numbers at nodes indicate bootstrap support obtained from 1,000 branch-and-bound replicates. The Thermotoga sp. RQ2 sequence is indicated by . All trees were estimated from DNA sequences.

 


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 3.—A, Alignment of Thermotoga maritima, Thermotoga sp. RQ2, and Thermotoga neapolitana NS-ET sequences. Only variable positions are included. A dot denotes identity to the shaded nucleotide above. The arrows indicate the segments where the Thermotoga sp. RQ2 sequence is most similar to T. maritima. B, Distribution of the variable sites along the ino1 sequences.

 
gltB Phylogeny Suggests Multiple Transfers Between Archaea and Bacteria
Additional gltB homologs were obtained from the databases using BLAST searches with the T. maritima sequence as a probe. A phylogenetic tree of the gltB sequences is shown in figure 4A. All sequences except that of Plasmodium falciparum passed the chi-square tests in PUZZLE, in which the amino acid composition of each sequence is compared with the frequency distribution assumed in the ML model.

The deepest division in the tree separated two major groups of sequences with high confidence (>90% support in RELL, bootstrap, and quartet puzzling analyzes), one comprising most of the bacterial sequences (including A. aeolicus) along with the eukaryotes, and one harboring all archaeal sequences interspersed with the remaining bacterial sequences. The Thermotoga sequences clustered within the archaeal group, supporting a transfer from Archaea to the Thermotoga lineage. Three other bacterial sequences were found in the archaeal group: D. ethenogenes, a green nonsulfur bacterium; S. meliloti, an {alpha}-proteobacterium; and Clostridium difficile, a firmicute. Dehalococcoides ethenogenes was found in the same subcluster as the Thermotoga sequences, while S. meliloti and C. difficile clustered with two separate M. thermoautotrophicum genes. Altogether, this analysis identified at least two independent transfers of gltB from Archaea to Bacteria.

In the tree in figure 4A, the Thermotoga sequences appear polyphyletic, with the T. thermarum sequence branching with D. ethenogenes outside the Thermotoga clade. However, there was no support for this branching pattern. Hence, in order to better resolve the relationship among the Thermotoga sequences, a phylogeny for this clade was estimated from nucleotide rather than amino acid sequences. Including all codon positions, the Thermotoga strains were again polyphyletic in all analyses (including logdet), with the following branching pattern: (crenarchaeotes, P. furiosus, M. jannaschii, T. thermarum (D. ethenogenes (A. fulgidus (Thermotoga sp.))). However, significant differences in G+C content were observed for third positions among the Thermotoga sequences (T. maritima 0.52 G+C, T. thermarum 0.26 G+C; P < 0.0000001), while T. thermarum, P. furiosus, and M. jannaschii displayed similar base compositions (P = 0.99), suggesting that the polyphyly of the Thermotoga strains could be an artifact related to GC composition (Galtier and Gouy 1995Citation ). The phylogeny estimated when third positions were excluded is shown in figure 4B. The topology suggests monophyly of Thermotoga. Moreover, both the Thermotoga and the D. ethenogenes gltB appear to have been recruited from the Archaeoglobus lineage, and the two bacterial lineages are monophyletic. This would suggest a single transfer between Bacteria and Archaea, from A. fulgidus to the D. ethenogenes or Thermotoga lineage, followed by a second transfer between the bacterial lineages. Alternatively, the shared transfer in Thermotoga and D. ethenogenes could reflect common ancestry, as both lineages are considered to be "deep-branching". However, there is little support for the separation of A. fulgidus from D. ethenogenes and Thermotoga sp., so the possibility of two separate LGT events from the Archaeoglobus lineage to Bacteria cannot be excluded.

The phylogeny in figure 4A also suggests several LGT events of gltB within domains. For instance, within the bacterial part of the tree, several of the proteobacterial sequences cluster as sister taxa to other bacterial lineages—Bordtetella pertussis, Campylobacter jejuni, and one of the Vibrio cholerae genes—while the A. aeolicus gene clusters with cyanobacteria and chloroplasts (the Fd-gltBs; Vanoni and Curti 1999Citation ). Since two copies of the gltB gene are present in V. cholerae, as well as in cyanobacteria and plants, differential loss of paralogs can in part explain the mosaic pattern observed. However, since the proteobacteria cluster with different species, a minimum of four independent LGT events involving proteobacteria are necessary to explain the pattern observed for this bacterial group.

ino1 Phylogeny Suggests an Archaeal Origin of All Bacterial ino1 Homologs
Additional ino1 sequences were obtained from BLAST searches using the T. maritima, M. tuberculosis, and S. coelicolor genes as probes against the public databases. Apart from the sequences reported in Bachhawat and Mande (2000)Citation , we included the eight new Thermotoga sequences obtained by PCR amplification, along with homologs of ino1 found in databases for P. furiosus (identical to the gene amplified by us), S. solfataricus, C. diphtheriae, M. mazei, and D. ethenogenes.

A phylogenetic tree of the Thermotoga and retrieved ino1 sequences is shown in figure 5 . The S. coelicolor SC3F9.8 and SCE29.12c, Entamoeba histolytica, and Homo sapiens sequences failed the amino acid composition chi-square tests in PUZZLE. However, the same topology and level of support was observed when DNA sequences were used (third positions excluded) in logdet distance and parsimony analyses (data not shown). Also, the overall topology, with four divergent groups consisting of highly similar sequences, was the same as that recently reported by Bachhawat and Mande (2000)Citation . All eukaryote sequences cluster together as a monophyletic clade. The "prokaryotic" sequences form three groups, and, as noted in Bachhawat and Mande (2000)Citation , each cluster contains both archaeal and bacterial sequences, suggesting extensive transfer of the gene—in fact, all of the prokaryotic lineages containing this gene appear to have been involved in LGT. The alignment on which this tree was based contained several indels, particularly in the N-terminal end, mainly in comparisons between groups. However, deleting all positions with gaps did not change the overall topology or the support of the main groups (data not shown).

All three prokaryotic groups appeared to be rooted by archaeal sequences, while bacterial species occurred only at the tips of the tree. This strongly suggests that all bacterial ino1 homologs were derived from archaeal donors through LGT. In the first "prokaryote" group, the Thermotoga sequences clustered as a sister group to the Pyrococcus sequences, "rooted" by the Aeropyrum pernix sequence (prokaryote group 1). In the second group, the sequences of the Gram-positive bacteria were found along with D. ethenogenes, A. aeolicus, the two methanogens M. thermoautotrophicum and M. mazei, and the two sequences from S. solfataricus (prokaryote group 2). The bacterial and euryarchaeal sequences formed two monophyletic sister clades, rooted by the two S. solfataricus sequences. The third group contained the A. fulgidus sequence along with the last two S. coelicolor sequences (prokaryote group 3).

Taken together, therefore, we propose at least five instances of LGT, three of them between Archaea and Bacteria: a transfer involving Pyrococcus and Thermotoga, a transfer from Aeropyrum to the Pyrococcus or the Thermotoga lineage, at least one LGT from the Sulfolobus lineage to Bacteria (A. aeolicus, D. ethenogenes, and the Gram-positives) or the methanogens (M. thermoautotrophicum and M. mazei), a second LGT event involving the methanogens and Bacteria, and, finally, transfer of the gene from A. fulgidus to S. coelicolor. Notably, the two hyperthermophilic bacterial lineages, Thermotoga and Aquifex, were found in separate clades.

Gene Organization Data Support the Transfers Inferred from the Phylogenetic Trees
The transfers identified in the phylogenetic analysis were further investigated by assessing the affinity of genes flanking the gltB and ino1 gene using BLAST (PHI-BLAST searches). For the glutamate synthase gene, we found that a typical bacterial large-subunit gene was homologous to three separate ORFs in Archaea ("gltB-ORFs"—the ORF annotated gltB, which corresponds to the segments amplified in this study, and two additional ORFs). A three-ORF pattern also characterizes the Bacteria for which we infer an archaeal transfer—T. maritima, D. ethenogenes, and S. meliloti (fig. 6 ).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.—Archaeal "gltB gene clusters" compared with a standard bacterial gltB gene based on genomic data from Thermotoga maritima (Nelson et al. 1999Citation ), Archaeoglobus fulgidus (Klenk et al. 1997Citation ), Methanococcus jannaschii (Bult et al. 1996Citation ), and Methanobacterium thermoautotrophicum (Smith et al. 1997Citation ), the unfinished genome of Dehalococcoides ethenogenes (http://www.tigr.org), Pyrococcus furiosus (http://niji.imb.nrc.ca/sulfolobus), Sulfolobus solfataricus (http://niji.imb.nrc.ca/sulfolobus), Clostridium difficile (http://www.sanger.ac.uk/), and a 10,786-bp sequenced fragment of Sinorhizobium meliloti (accession number AF055582). Regions or open reading frames (ORFs) with similarity in BLAST searches are shaded with the same pattern. The ORF annotated gltB in archaea and T. maritima is shown as a gray box. For the ORF showing homology to the C-terminal domain in bacteria (black box), several homologs were observed in some genomes, but only the ORF linked to the two other gltB ORFs are included. In S. solfataricus, the two additional gltB domains formed one single ORF. ORFs interspersed in the cluster without homologs in the other "gltB gene clusters" are drawn as white boxes. In D. ethenogenes, the second ORF showed the highest similarity to an M. thermoautotrophicum gene (MTH1524), and ORF 5 showed highest similarity to an Aquifex aeolicus gene (AE000677). The sizes of the ORFs are only approximately drawn to scale

 
Archaeal gltB genes were not obtained from Petrotoga, Fervidobacterium, and Thermosipho, members of the Thermotogales which constitute an outgroup to Thermotoga (see fig. 1 ). Presumably, these organisms retained bacterial versions of gltB. Indeed, such a gene was fortuitously isolated from P. miotherma (data not shown). Our gltB primers amplified a 1,211-bp segment corresponding to a 149-aa fragment of the C-terminal part of gltB and 252 aa of the N-terminal part of the small subunit of glutamate synthase (gltD). For both gene fragments, the highest similarity scores in BLAST were to Cyanobacteria and low-G+C Gram-positives.

All T. maritima, D. ethenogenes, and S. meliloti gltB-ORFs included in figure 6 showed the highest similarity to their archaeal homologs in BLAST searches. The organizations of the gene clusters in A. fulgidus, T. maritima, and D. ethenogenes are very similar. In particular, a unique occurrence of the same two ORFs not related to the bacterial type gltB in T. maritima, D. ethenogenes, and A. fulgidus bolsters the close relationship inferred from the phylogenetic analyses (fig. 4 ) and suggests that the complete gene clusters were transferred in one event.

In sum, the difference in structure of the typical bacterial gltB genes, where all domains are included in one ORF, and the archaeal "gltB gene cluster" with three independent ORFs (except Sulfolobus), strongly support the findings from the phylogenetic analyses, where an archaeal origin of the gltB found in Thermotoga sp., D. ethenogenes, and S. meliloti appears most likely. Clostridium difficile also appears to have received an archaeal gltB gene, but no other homologs of the conserved gene cluster were found in the same contig. However, this gltB gene is flanked by two ORFs showing best match to M. jannaschii (MJ0448) and P. abyssi (PAB0085), supporting its archaeal origin. We note that the finding of additional ORFs homologous to the C- and N-terminal domains of the bacterial gltB may answer the question of how the archaeal glutamate synthase can function without the apparently missing domains (Vanoni and Curti 1999Citation ).

For the ino1 transfers identified in the phylogenetic analysis, corroboration was also found in flanking regions. The ino1 gene in T. maritima is flanked by three "archaeal" genes (TM1416–TM1418; best hit to Pyrococcus and Methanococcus), suggesting that these genes were transferred as one unit along with ino1 from a Pyrococcus ancestor. Linkage of the first two ORFs was conserved in A. pernix, P. abyssi, and P. horikoshii. Linkage of homologs of the last two genes (TM1418 and TM1419 = ino1), however, was only observed in A. pernix (APE1516, APE1517).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Both ino1 and gltB Have Been Acquired Recently by the Thermotoga Strains
The data reported here demonstrate that at least two of the "archaeal" T. maritima genes were recruited from Archaea by LGT and are not retained ancestral characters. Moreover, for 7 of 12 additional genes that were initially selected by us for preliminary phylogenetic analyses, LGT between T. maritima and Archaea was the most plausible explanation (data not shown).

BLAST analyses of the T. maritima genome suggested that the archaeal 24% of its genome resembled P. horikoshii (Nelson et al. 1999Citation ), indicating that the genes could have been acquired in one massive transfer event, involving a single archaeal lineage. The special relationship between Thermotoga and Pyrococcus is mirrored by BLAST analyses of the P. abyssi and P. horikoshii genomes; 8.2% and 6.4% (among a total of 16% and 20% "bacterial" genes), respectively, of the ORFs show the closest match to T. maritima, compared with 1.4%–3.5% in the other archaeal genomes (among a total 9%–20% "bacterial" genes) (data not shown). However, the results presented here, along with those of other studies of "archaeal" T. maritima genes for which phylogenetic analyses have been performed, such as reverse gyrase (Forterre et al. 2000Citation ), glutamate dehydrogenase (Kort et al. 1997Citation ), and glutamine synthase (Brown et al. 1994Citation ), suggest that the "archaeal" genes of T. maritima have been acquired from several different sources and that in some cases the transfers have been from Thermotoga to Archaea (Gribaldo et al. 1999Citation ). Moreover, our data also support different timing of the LGT events associated with ino1 and gltB (see below).

The distribution of the gltB and ino1 genes among the Thermotogales (table 1 and fig. 1 ), as well as the close phylogenetic relationship with Archaea (fig. 4 and 5 ), suggests that they were recruited from within the Archaea. Using the SSU phylogenies in figure 1 as a reference, the ino1 gene was probably recruited from the Pyrococcus lineage after the T. maritimaT. neapolitana group diverged from the remaining Thermotoga strains. Taking into account that myo-inositol 1P synthase catalyzes the first step in the production of the osmolyte DIP (Martins et al. 1996Citation ; Chen, Spiliotis, and Roberts 1998Citation ), the acquisition of ino1 could, in fact, be one of the important events leading to the divergence of these lineages, as one of the main characteristics that distinguishes the T. maritimaT. neapolitana group from the other Thermotogales is the ability to live in environments characterized by both high temperature and high salinity (Huber and Stetter 2000Citation ).

The archaeal gltB was probably recruited at an earlier stage, but nevertheless after the Thermotoga lineage diverged from the other Thermotogales (i.e., Petrotoga, Thermosipho, and Fervidobacterium), followed by loss of the gene in the lineage leading to T. subterranea and Thermotoga sp. KOL6. The finding of a bacterial-type gltB (and gltD) in P. miotherma might reflect that the archaeal-type gltB in the Thermotoga lineages replaced a bacterial homolog.

Does the High Number of Archaeal Genes in Hyperthermophiles Reflect a Unique Case of Adaptation to Life in Hot Biotopes or a General Evolutionary Mechanism?
The observation that the genomes of the two hyperthermophilic bacteria contain a larger number of archaeal genes (16%–24%) than have been detected in mesophilic bacteria (3%–5%) (Aravind et al. 1998Citation ; Nelson et al. 1999Citation ) has led some authors to suggest that this is mainly a reflection of adaptation to life in high-temperature biotopes and that hyperthermophily arose at least twice secondarily in eubacteria (Aravind et al. 1998Citation ; Forterre et al. 2000Citation ).

The phylogeny and distribution of the ino1 gene do in some ways support this hyperthermophilic-adaptation view. The presence of the osmolyte DIP, one of the products of the inositol pathway in hyperthermophiles, shows a strong correlation to growth in hyperthermophilic, high-salt environments (Ciulla et al. 1994Citation ; Martins et al. 1996, 1997Citation ). The phylogeny of ino1 indeed shows that it has been acquired independently from Archaea by the two bacterial hyperthermophilic lineages, in the same way as that observed for reverse gyrase (Forterre et al. 2000Citation ). However, the phylogeny also suggests that the gene has been transferred several times among the archaeal hyperthermophiles as well. Moreover, across-domain transfer of this gene is not restricted to hyperthermophilic lineages: several mesophilic bacteria have acquired it. The situation for S. coelicolor is particularly striking: this species has three copies of the gene, recruited from two different archaeal lineages. The selective advantage of multiple copies of ino1 in this species is probably related to the importance of myo-inositol in antibiotic production (Walker 1995Citation ).

There is, as well, no support for a thermophilic advantage driving the transfer of the archaeal-type gltB gene. First, A. aeolicus possesses a typical bacterial gltB. Second, archaeal gltBs are also found in three mesophilic bacteria: C. difficile, D. ethenogenes, and S. meliloti. In fact, T. maritima and D. ethenogenes have probably recruited the gltB gene from the same archaeal lineage (fig. 4 ). Notably, LGT involving proteobacteria accounts for four of the seven LGT events postulated for the gltB gene. This is the bacterial group for which the largest number of gltB-homologous genes has been submitted to GenBank. The LGTs of gltB reported here might therefore represent just the "tip of the iceberg."

It seems reasonable that LGT from hyperthermophiles to mesophiles would help the latter adapt to higher temperatures. This is, for instance, very likely the case for reverse gyrase (Forterre et al. 2000Citation ). However, hyperthermophiles might also be expected to share genes just because they share environments and have the opportunity to participate in LGT. The large number of archaeal genes in bacterial hyperthermophiles may be as much the consequence as the cause of their adaptation to hyperthermophily. The mesophilic anaerobic bacterium D. ethenogens (Maymo-Gatell et al. 1997Citation ) is noteworthy in that it possesses an "archaeal"-type gltB as well as the ino1 gene. Interestingly, SSU rRNA surveys of microbial communities dechlorinating polychlorinated biphenyls (PCBs) have shown that strains belonging to the Dehalococcoides and Thermotogales (as well as Clostridium and the {delta}-proteobacteria) SSU rRNA lineages co-occur in the same environment (mesophilic [30°C], anaerobic estuarine sediments), along with several methanogenic archaeal strains (Holoman et al. 1998Citation ). It will be exciting to see if the complete genome of D. ethenogenes shows large numbers of archaeal genes similar to those observed for T. maritima and A. aeolicus.

Analyses similar in rigor to those described here are required to verify how much of the 24% (451 genes) of the T. maritima genome identified as archaeal by BLAST analyses is indeed "archaeal." Our preliminary studies of an additional 12 gene families identified another 7 phylogenetically confirmable cases (data not shown). We anticipate that many of Thermotoga's archaeal genes will, like gltB and ino1, reveal themselves to have participated in many independent between- and within-domain transfers.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We are very grateful to Yan Boucher, John Archibald, Yuji Inagaki, and Jan Andersson for comments on earlier drafts of this work. We also acknowledge the members of W. F. Doolittle and A. Roger's lab for discussions and helpful comments. We are very grateful to Dr. H. Morgan (University of Waikato, New Zealand), Dr. N. Glansdorff (Universite Libre de Bruxelles, Belguim), Dr. M. Madsen and Dr. T. Lien (University of Bergen, Norway), and Dr. J. Brown (North Carolina State University, Raleigh, N.C.) for donating Thermotogales DNA. This work was supported by grant MT4467 to W.F.D. fellow of the Canadian Institute for Advanced Research from the Medical Research Council of Canada, an overseas fellowship (project 131945/410) from the Norwegian Research Council to C.L.N., and a grant from Fonds der Chemischen Industrie to K.O.S.


    Footnotes
 
Geoffrey McFadden, Reviewing Editor

1 Keywords: Thermotoga maritima, lateral gene transfer glutamate synthase myo-inositol 1P synthase phylogeny Back

2 Address for correspondence and reprints: Camilla L. Nesbø, Department of Biochemistry and Molecular Biology, Dalhousie University, 5859 University Avenue, Halifax, Nova Scotia, Canada B3H 4H7. E-mail: cnesbo{at}is.dal.ca Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 

    Adachi, J., and M. Hasegawa. 1996. MOLPHY version 2.3: programs for molecular phylogenetics based on maximum likelihood. Comput. Sci. Monogr. 28:1–150.

    Aravind, L., R. L. Tatusov, Y. I. Wolf, D. R. Walker, and E. V. Koonin. 1998. Evidence for massive gene exchange between archaeal and bacterial hyperthermophiles. Trends Genet. 14:442–444.[ISI][Medline]

    Bachhawat, N., and S. C. Mande. 1999. Identification of the INO1 gene of Mycobacterium tuberculosis H37Rv reveals a novel class of inositol-1-phosphate synthase enzyme. J. Mol. Biol. 291:531–536.[ISI][Medline]

    ———. 2000. Complex evolution of the inositol-1-phosphat synthase gene among archaea and eubacteria. Trends Genet. 16:111–113.[ISI][Medline]

    Barns, S. M., C. F. Delwiche, J. D. Palmer, and N. R. Pace. 1996. Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences. Proc. Natl. Acad. Sci. USA 93:9188–9193.

    Belkin, S., C. O. Wirsen, and H. W. Jannasch. 1986. A new sulfur-reducing, extremely thermophilic eubacterium from a submarine thermal vent. Appl. Environ. Microbiol. 51:1180–1185.[ISI]

    Besra, G. S., and P. J. Brennan. 1997. The mycobacterial cell wall: biosynthesis of arabinogalactan and lipoarabinomannan. Biochem. Soc. Trans. 25:845–850.[ISI][Medline]

    Bocchetta, M., S. Gribaldo, A. Sanangelantoni, and P. Cammarano. 2000. Phylogenetic depth of bacterial genera Aquifex and Thermotoga inferred from analysis of ribosomal protein, elongation factor and RNA polymerase subunit sequences. J. Mol. Evol. 50:366–380.[ISI][Medline]

    Brown, J. R., Y. Masuchi, F. T. Robb, and W. F. Doolittle. 1994. Evolutionary relationships of bacterial and archaeal glutamine synthetase genes. J. Mol. Evol. 38:566–576.[ISI][Medline]

    Bult, C. J., O. White, G. J. Olsen et al. (20 co-authors). 1996. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273:1058–1073.

    Charbonnier, F., and P. Forterre. 1994. Comparison of plasmid DNA topology among mesophilic and thermophilic eubacteria and archaebacteria. J. Bacteriol. 176:1251–1259.[Abstract]

    Chen, L., E. T. Spiliotis, and M. F. Roberts. 1998. Biosynthesis of Di-myo-inositol-1,1'-phosphate, a novel osmolyte in hyperthermophilic archaea. J. Bacteriol. 180:3785–3792.[Abstract/Free Full Text]

    Ciulla, R. A., S. Burggraf, O. Stetter, and M. Roberts. 1994. Occurrence and role of di-myo-inositol-1–1' phosphate in Methanococcus igneus. Appl. Environ. Microbiol. 60:3660–3664.

    Davey, M. E., W. A. Wood, R. Key, K. Nakamura, and D. Stahl. 1993. Isolation of three species of Geotoga and Petrotoga: two new genera, representing an new lineage in the bacterial line of descent distantly related to the "Thermotogales". Syst. Appl. Microbiol. 16:191–200.[ISI]

    Doolittle, W. F. 1999a. Lateral genomics. Trends Cell. Biol. 9:M5–M8.

    ———. 1999b. Phylogenetic classification and the universal tree. Science 284:2124–2129.

    Feil, E. J., M. C. Enright, and B. G. Spratt. 2000. Estimating the relative contributions of mutation and recombination to clonal diversification: a comparison between Neisseria meningitidis and Streptococcus pneumoniae. Res. Microbiol. 151:465–469.[ISI][Medline]

    Felsenstein, J. 1995. PHYLIP (phylogeny inference package). Version 3.572. Distributed by the author, Department of Genetics, University of Washington, Seattle.

    Fiala, G., and K. O. Stetter. 1986. Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 C. Arch. Microbiol. 145:56–61.

    Fitz-Gibbon, S. T., and C. H. House. 1999. Whole genome-based phylogenetic analysis of free-living microorganisms. Nucleic Acids Res. 27:4218–4222.[Abstract/Free Full Text]

    Forterre, P., C. Bouthier De La Tour, H. Philippe, and M. Duguet. 2000. Reverse gyrase from hyperthermophiles: probable transfer of a thermoadaptation trait from Archaea to Bacteria. Trends Genet. 16:152–154.[ISI][Medline]

    Fudyk, T. C., I. W. Maclean, J. N. Simonsen, E. N. Njagi, J. Kimuni, R. C. Brunham, and F. A. Plummer. 1999. Genetic diversity and mosaicism at the por locus of Neisseria gonorrhoeae. J. Bacteriol. 181:5591–5599.[Abstract/Free Full Text]

    Galtier, N., and M. Gouy. 1995. Inferring phylogenies from DNA sequences of unequal base compositions. Proc. Natl. Acad. Sci. USA 92:11317–11321.

    Gribaldo, S., V. Lumia, R. Creti, A. Sanangekintoni, and P. Cammarano. 1999. Discontinuous occurrence of the hsp70 (dnaK) gene among Archaea and sequence features of HSP70 suggest a novel outlook on phylogenies inferred from this protein. J. Bacteriol. 181:434–443.[Abstract/Free Full Text]

    Holoman, T. R., M. A. Elberson, L. A. Cutter, H. D. May, and K. R. Sowers. 1998. Characterization of a defined 2,3,5,6-tetrachlorobiphenyl-ortho-dechlorinating microbial community by comparative sequence analysis of genes coding for 16S rRNA. Appl. Environ. Microbiol. 64:3359–3367.[Abstract/Free Full Text]

    Huber, R., and K. O. Stetter. 2000. The order thermotogales. Pp. 1–11 in M. Dworkin, ed. The prokaryotes. An evolving electronic resource for the microboiological community (http://link.springer-ny.com/link/service/books/10125/index.html).

    Jain, R., M. C. Rivera, and J. A. Lake. 1999. Horizontal gene transfer among genomes: the complexity hypothesis. Proc. Natl. Acad. Sci. USA 96:3801–3806.

    Jannasch, H. W., R. Huber, S. Belkin, and K. O. Stetter. 1988. Thermotoga neapolitana sp. nov. of the extremely thermophilic, eubacterial genus Thermotoga. Arch. Microbiol. 150:103–104.

    Jeanthon, C., A. L. Reysenbach, S. L'Haridon, N. R. Pace, P. Glenat, and D. Prieur. 1995. Thermotoga subterranea sp. nov., a new thermophilic bacterium isolated from a continental oil reservoir. Arch. Microbiol. 164:91–97.[ISI][Medline]

    Jongsareejit, B., R. N. Rahman, S. Fujiwara, and T. Imanaka. 1997. Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1. Mol. Gen. Genet. 254:635–642.

    Kawarabayasi, Y., M. Sawada, H. Horikawa et al. (25 co-authors). 1998. Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res. 5:55–76.[Medline]

    Klenk, H. P., R. A. Clayton, J. F. Tomb et al. (25 co-authors). 1997. The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390:364–370.

    Klenk, H. P., T. D. Meier, P. Durovic, V. Schwass, F. Lottspeich, P. P. Dennis, and W. Zillg. 1999. RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria. J. Mol. Evol. 48:528–541.[ISI][Medline]

    Kort, R., W. Liebl, B. Labedan, P. Forterre, R. I. Eggen, and W. M. deVos. 1997. Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications. Extremophiles 1:52–60.

    Kyrpides, N. C., and G. J. Olsen. 1999. Archaeal and bacterial hyperthermophiles: horizontal gene exchange or common ancestry? Trends Genet. 15:298–299.

    Lawrence, J. G. 1999. Gene transfer, speciation, and the evolution of bacterial genomes. Curr. Opin. Microbiol. 2:519–523.[ISI][Medline]

    Lawrence, J. G., and H. Ochman. 1998. Molecular archaeology of the Escherichia coli genome. Proc. Natl. Acad. Sci. USA 95:9413–9417.

    Lien, T., M. Madsen, F. A. Rainey, and N. K. Birkeland. 1998. Petrotoga mobilis sp. nov., from a North Sea oil-production well. Int. J. Syst. Bacteriol. 48:1007–1013.

    Lockhart, P. J., M. A. Steel, M. D. Hendy, and D. Penny. 1994. Recovering evolutionary trees under a more realistic model of sequence evolution. Mol. Biol. Evol. 11:605–612.[Free Full Text]

    Logsdon, J. M., and D. M. Faguy. 1999. Thermotoga heats up lateral gene transfer. Curr. Biol. 9:R747–R751.

    McGraw, E. A., J. Li, R. K. Selander, and T. S. Whittam. 1999. Molecular evolution and mosaic structure of alpha, beta, and gamma intimins of pathogenic Escherichia coli. Mol. Biol. Evol. 16:12–22.[Abstract]

    Martin, D. D., R. A. Ciulla, and M. F. Roberts. 1999. Osmoadaptation in archaea. Appl. Environ. Microbiol. 65:1815–1825.[Free Full Text]

    Martin, W. 1999. Mosaic bacterial chromosomes: a challenge en route to a tree of genomes. Bioessays 21:99–104.

    Martins, L. O., L. S. Carreto, M. S. Da Costa, and H. Santos. 1996. New compatible solutes related to Di-myo-inositol-phosphate in members of the order Thermotogales. J. Bacteriol. 178:5644–5651.[Abstract/Free Full Text]

    Martins, L. O., R. Hubert, H. Hubert, K. O. Stetter, M. S. DaCosta, and H. Santos. 1997. Organic soultes in hyperthermophilic archaea. Appl. Environ. Microbiol. 63:896–902.[Abstract]

    Maymo-Gatell, X., Y. Chien, J. M. Gossett, and S. H. Zinder. 1997. Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 276:1568–1571.

    Nelson, K. E., R. A. Clayton, S. R. Gill et al. (29 co-authors). 1999. Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399:323–329.

    Patel, B. K. C., H. W. Morgan, and R. M. Daniel. 1985. Fervidobacterium nodosum gen. nov. and spec. nov., a new chemoorganotrophic, caldoactive, anaerobic bacterium. Arch. Microbiol. 141:63–69.

    Posada, D., and K. A. Crandall. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818.

    Rambaut, A. 1995. Se-Al Sequence alignment program. Version 1.d1. Department of Zoology, University of Oxford, Oxford, England.

    Reeves, R. A., M. D. Gibbs, D. D. Morris, K. R. Griffiths, D. J. Saul, and P. L. Bergquist. 2000. Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1. Appl. Environ. Microbiol. 66:1532–1537.[Abstract/Free Full Text]

    Smith, D. R., L. A. Doucette-Stamm, C. Deloughery et al. (25 co-authors). 1997. Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics. J. Bacteriol. 179:7135–7155.[Abstract]

    Strimmer, K., and A. von Haeseler. 1996. Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13:964–969.[Free Full Text]

    Swofford, D. L. 2000. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer, Sunderland, Mass.

    Temple, S. J., C. P. Vance, and J. S. Gnatt. 1998. Glutamate synthase and nitrogen assimilation. Trends Plant Sci. 3:51–56.[ISI]

    Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876–4882.

    Tiboni, O., R. Cantoni, R. Creti, P. Cammarano, and A. M. Sanangelantoni. 1991. Phylogenetic depth of Thermotoga maritima inferred from analysis of the fus gene: amino acid sequence of elongation factor G and organization of the Thermotoga str operon. J. Mol. Evol. 33:142–151.[ISI][Medline]

    Van de Peer, Y., P. De Rijk, J. Wuyts, T. Winkelmans, and R. De Wachter. 2000. The European Small Subunit Ribosomal RNA database. Nucleic Acids Res. 28:75–176.

    Vanoni, M. A., and B. Curti. 1999. Glutamate synthase: a complex iron-sulfur flavoprotein. Cell. Mol. Life Sci. 55:617–638.[ISI][Medline]

    Walker, J. B. 1995. Enzymatic synthesis of aminocyclitol moieties of aminoglycoside antibiotics from inositol by Streptomyces spp.: detection of glutamine-aminocyclitol aminotransferase and diaminocyclitol aminotransferase activities in a spectinomycin producer. J. Bacteriol. 177:818–822.[Abstract]

Accepted for publication October 31, 2000.