Anna M. Sanangelantoni

Early evolutionary relationships among known life forms inferred from elongation factor EF-2/EF-G sequences: Phylogenetic coherence and structure of the archaeal domain

SummaryPhylogenies 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 monophyletia-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.

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

SummaryThe gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacteriumThermotoga 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). TherpsG, fus, andtuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in thestr operon ofEscherichia 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 dendrogram, 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 betweenThermotoga and the other bacterial lineages.

Arrangement and nucleotide sequence of the gene (fus) encoding elongation factor G (EF-G) from the hyperthermophilic bacterium Aquifex pyrophilus: Phylogenetic depth of hyperthermophilic bacteria inferred from analysis of the EF-G/fus sequences

The gene fus (for EF-G) of the hyperthermophilic bacterium Aquifex pyrophilus was cloned and sequenced. Unlike the other bacteria, which display the streptomycin-operon arrangement of EF genes (5′-rps12-rps7 fus-tuf-3′), the Aquifex fus gene (700 codons) is not preceded by the two small ribosomal subunit genes although it is still followed by a tuf gene (for EF-Tu). The opposite strand upstream from the EF-G coding locus revealed an open reading frame (ORF) encoding a polypeptide having 52.5% identity with an E. coli protein (the pdxJ gene product) involved in pyridoxine condensation. The Aquifex EF-G was aligned with available homologs representative of Deinococci, high G + C Gram positives, Proteobacteria, cyanobacteria, and several Archaea. Outgroup-rooted phylogenies were constructed from both the amino acid and the DNA sequences using first and second codon positions in the alignments except sites containing synonymous changes. Both datasets and alternative tree-making methods gave a consistent topology, with Aquifex and Thermotoga maritima (a hyperthermophile) as the first and the second deepest offshoots, respectively. However, the robustness of the inferred phylogenies is not impressive. The branching of Aquifex more deeply than Thennotoga and the branching of Thermotoga more deeply than the other taxa examined are given at bootstrap values between 65 and 70% in the fus-based phylogenies, while the EF-G(2)-based phylogenies do not provide a statistically significant level of support (⩽ 50% bootstrap confirmation) for the emergence of Thermotoga between Aquifex and the successive offshoot (Thermus genus). At present, therefore, the placement of Aquifex at the root of the bacterial tree, albeit reproducible, can be asserted only with reservation, while the emergence of Thermotoga between the Aquificales and the Deinococci remains (statistically) indeterminate.

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

SummaryThe glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli.

The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb HindIII DNA fragment. Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50,088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolobus solfataricus). The T. maritima glnA gene was expressed in E. coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051. The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation. The molecular mass of the Thermotoga GS (590,000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type. Comparison of the amino acid sequence of T. maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability.

Expression in Escherichia coli of the tuf Gene from the Extremely Thermophilic Eubacterium Thermotoga maritima: Purification of the Thermotoga Elongation Factor Tu by Thermal Denaturation of the Mesophile Host Cell Proteins

Summary The gene for the elongation factor Tu (EF-Tu) of the extemely thermophilic eubacterium Thermotoga maritima was identified by cross-hybridization with the tufA gene of Escherichia coli and cloned into plasmid pBR322. The tuf gene of T. maritima was found to be present in a single copy in the Thermotoga chromosome and to lie only 11 nucleotides downstream of the terminating codon of the EF-G gene. The Thermotoga tuf gene was expressed in E. coli either following transcription from its own promoter or from the trp promoter of plasmid pDR720; the tuf gene expression was demonstrated by assaying the GDP-binding activity surviving heat-treatment (up to 90°C) of host-cell extracts. The formation of the thermostable EF-Tu in E. coli cells was also verified by the presence, in the cell extracts, of a protein specifically recognized by T. maritima anti-EF-Tu antibodies, and having the same relative molecular mass (Mr 49,000), and the same V8 protease cleavage pattern, as authentic Thermotoga EF-Tu. That protein was the predominant species found in the extracts following heating at 65°C and removal of precipitated proteins. The Thermotoga factor expressed in E. coli was unable to bind GDP at 37°C, a heat-treatment at high temperatures being required to overcome conformational barriers that hinder the proper folding of the polypeptide.

Chromosomal organization and nucleotide sequence of the genes for elongation factors EF-1α and EF-2 and ribosomal proteins S7 and S10 of the hyperthermophilic archaeum Desulfurococcus mobilis

The Desulfurococcus mobilis genes fus (encoding EF-2) and tuf (for EF-1α) were cloned and sequenced together with genes for ribosomal proteins S10 (rps10) and S7 (rps7). Unlike Methanococcus, which displays the bacterial-like fus and tuf gene context 5′-rps12-rps7-fus-tuf-3′, and similar to Sulfolobus and Pyrococcus, the Desulfurococcus fus gene (734 codons) has a distinct chromosomal location. Moreover, tuf (441 codons) is the promoter-proximal unit of a three-gene cluster comprising the genes rps10 (98 codons) and tRNASer; the arrangement of the cluster is 5′-tuf-91 bp spacer-rps10-138 bp spacer-tRNASer: 3′ and the tuf gene is preceded by a canonical archaeal promoter. The D. mobilis gene rps7 (198 codons) is located further upstream from tuf (535 bp ‘silent’ intergenic spacing) and no rps12 homolog occurs in its immediate vicinity. Also, judging from putative promoter and transcription termination sequences, rps7 appears to be separately transcribed. Analysis of the predicted fus and tuf gene products revealed the three consensus motifs characteristic of GTP-binding proteins, and the fus-encoded EF-2 protein also displayed the consensus sequence required for ADP-ribosylation by Diphtheria toxin. Both EF sequences were definitely crenarchaeal by comparison with available homologs from other Archaea. Outgroup-rooted phylogenies derived from the sequences of ribosomal proteins S10 and S7 yielded the Sulfolobus-Desulfurococcus association at a high bootstrap confidence level.

Immunochemical Cross-Reactivities of Protein Synthesis Elongation Factors (EF-Tu and EF-1α Proteins) Support the Phylogenetic Coherence of Archaebacteria

Summary Elongation factor Tu (EF-Tu) proteins have been purified by affinity chromatography on GDP-Sepharose columns, from the eubacterium Thermotoga maritima and from archaebacteria (Sulfolobus solfataricus, Thermoproteus tenax, Thermococcus celer, Pyrococcus wosei, Archaeoglobus fulgidus, Methanococcus thermolitotrophicus, Thermoplasma acidophilum) representative of all known divisions in the arachaebacterial tree except halophiles. Polyclonal antibodies raised against the purified Tu proteins were challenged with homologous and heterologous factors including eukaryotic EF-1α and cross-reactivities were quantified using 125 I labelled Protein A as the reporter molecule. The immunochemical relationships among factors both within and across kingdom boundaries demonstrated that (i) every archaebacterial EF-Tu is closer (immunochemically) to every other archaebacterial EF-Tu than to the analogous proteins of eubacteria and eukaryotes, (ii) within the archaebacteria the immunochemical cross-reactivities grossly correlate with the phylogenetic relatedness of the organisms inferred from similarities in rRNA sequences. The data support the notion that archaebacteria form a phylogenetically coherent taxon.

Construction of a cosmid library of Spirulina platensis as an approach to DNA physical mapping

A Spirulina platensis gene library has been constructed using cosmid vector pMMB34. The cosmid bank was controlled for its random gene distribution by colony hybridization. Genes were identified using either homologous or heterologous probes of genes involved in photosynthesis (large and small subunit of d-ribulose 1,5-bisphosphate carboxylase, 32 kDa thylakoid protein, α, β subunits of C-phycocyanin) and protein synthesis (elongation factors EF-Tu, EF-G).