The gene ginA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sudfolobus solfataricus ginA gene as the probe. An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp. The encoded protein was 49%o identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family. The P. woesei GSI was aligned with available homologs from other archaea (S. soifataricus, M. voitae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria. Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments. In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny. On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles. One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G+C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G+C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation). The GSIs of the Thermotoga and the Bacilus-Clostridium lineages shared a direct common ancestor with that of P. woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S. coelicolor.The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism. However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained.Glutamine synthetase (GS) catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonium (NH4+). The enzyme is a multimer found in at least two forms that are only distantly related to one another (15 to 19% sequence identity) (43, 56). One of these forms (GSI), composed of 12 identical subunits (443 to 474 amino acids each), occurs in bacteria and archaea. A second form (GSII), composed of eight identical subunits (332 to 378 amino acids each), occurs in eukarya but also in members of the family Rhizobiaceae (12, 23, 53) and in certain actinomycetes (4, 18, 34, 49); these last two groups harbor both a GSI and a GSII-like enzyme. A third form of the enzyme (GSIII), composed of six identical subunits (729 amino acids each), whose sequence is unrelated to both GSI and GSII, is harbored by Bacteroides fragilis (29).Because GSI is a relatively long polypeptide chain containing both semiconserved and highly conserved regions, and because it is ubiquitous in prokaryotes, GSI subunit sequences are suited, in principle, to trace the evolution of the bacterial and the archaeal lineages.A large inventory of (eu)bacterial GSI sequ...
The EF-2 coding genes of the Archaea Pyrococcus woesei and Desulfuoccus mobilis were cloned and sequenced. Global phylogenies were inferred by alternative tree-makig methods from available EF-2(G) sequence data and contrasted with phylogenies constructed from the more conserved but shorter EF-la(Tu) sequences. Both the monophyly (sensu Henig) of Archaea and their subdivision into the kingdoms Crenarchaeota and Euryarchaeota are cnsendy inferred by analysis of EF-2(G) sequences, usually at a high bootstrap confidence level. In contras, EF-la(Tu) phylogenies tend to be inconsistent with one another and show low bootstrap confidence levels. While evolutionary distance and DNA maximum parsimony analyses of EF-la(Tu) sequences do show archaeal monophyly, protein p ony and DNA maximum-likelihood analyses of these data do not. In no case, however, do any of the tree topologies inferred from EFla(Tu) sequence analyses receive si nt bootstrap support.Phylogenies spanning extant life-forms have been reconstructed from molecular sequence data by using small-and large-subunit rRNAs (1, 2), RNA polymerase core subunits (3), H+-ATPase a and (3 subunits (4, 5), and the two elongation factors EF-la (and its eubacterial homolog EF-Tu), which is involved in aminoacyl-tRNA binding (6, 7), and EF-2 (and its eubacterial homolog EF-G), which is involved in peptidyl-tRNA translocation (8).In that the stem leading to the archaeal branch in a global tree is always significantly shorter than those that lead to the Bacteria or the Eucarya, it is not surprising that the various tree-making methods applied to various molecular types do not always yield a (statistically significant) monophyletic grouping for the Archaea. This has led rightly or wrongly to the conclusion by some that the Archaea are not a monophyletic grouping (sensu Hennig) (9,10). Phylogenies based upon EF-la(Tu) are a good case in this point: Although most analyses (6, 7) yield a monophyletic archaeal grouping, the archaeal stem (joining the Archaea to the other lineages) is more than an order of magnitude shorter than its bacterial or eucaryal counterparts (6, 7). And some EF-la(Tu) analyses indeed have given paraphyletic archaeal groupings (11). Given the far-reaching implications of the topology of the global phylogenetic tree, it is important to understand and resolve these differences.Here we compare phylogenies inferred by various methods from the relatively short and conserved sequences of the EF-la(Tu) type (about 400 aa) to those inferred from its longer, less conserved counterpart, of the EF-2(G) type (about 700 aa). To increase the rather meager collection of archaeal EF-2 sequences we have cloned and sequenced the EF-2 coding genes from the sulfur-dependent Archaea Pyrococcus woesei and Desulfurococcus mobilis. § Phylogenetic trees have been inferred by using evolutionary (sequence) distance, parsimony, and maximum-likelihood methods, based either upon the first and second codon positions in the alignments or upon the second positions only. It is clear ...
Phylogenies were inferred from both the gene and the protein sequences of the translational elongation factor termed EF-2 (for Archaea and Eukarya) and EF-G (for Bacteria). All treeing methods used (distance-matrix, maximum likelihood, and parsimony), including evolutionary parsimony, support the archaeal tree and disprove the "eocyte tree" (i.e., the polyphyly and paraphyly of the Archaea). Distance-matrix trees derived from both the amino acid and the DNA sequence alignments (first and second codon positions) showed the Archaea to be a monophyletic-holophyletic grouping whose deepest bifurcation divides a Sulfolobus branch from a branch comprising Methanococcus, Halobacterium, and Thermoplasma. Bootstrapped distance-matrix treeing confirmed the monophyly-holophyly of Archaea in 100% of the samples and supported the bifurcation of Archaea into a Sulfolobus branch and a methanogen-halophile branch in 97% of the samples. Similar phylogenies were inferred by maximum likelihood and by maximum (protein and DNA) parsimony. DNA parsimony trees essentially identical to those inferred from first and second codon positions were derived from alternative DNA data sets comprising either the first or the second position of each codon. Bootstrapped DNA parsimony supported the monophyly-holophyly of Archaea in 100% of the bootstrap samples and confirmed the division of Archaea into a Sulfolobus branch and a methanogen-halophile branch in 93% of the bootstrap samples. Distance-matrix and maximum likelihood treeing under the constraint that branch lengths must be consistent with a molecular clock placed the root of the universal tree between the Bacteria and the bifurcation of Archaea and Eukarya. The results support the division of Archaea into the kingdoms Crenarchaeota (corresponding to the Sulfolobus branch and Euryarchaeota). This division was not confirmed by evolutionary parsimony, which identified Halobacterium rather than Sulfolobus as the deepest offspring within the Archaea.
The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacterium Thermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). The rpsG, fus, and tuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in the str operon of Escherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrograms, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies between Thermotoga and the other bacterial lineages.
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