Orsola Tiboni

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.

Characterization of the str operon genes from Spirulina platensis and their evolutionary relationship to those of other prokaryotes.

SummaryA 5.3 kb DNA segment containing the str operon (ca. 4.5 kb) of the cyanobacterium Spirulina platensis has been sequenced. The str operon includes the structural genes rpsL (ribosomal protein S12), rpsG (ribosomal protein S7), fus (translation elngation factor EF-G) and tuf (translation elongation factor EF-Tu). From the nucleotide sequence of this operon, the primary structures of the four gene products have been derived and compared with the available corresponding structures from eubacteria, archaebacteria and chloroplasts. Extensive homologies were found in almost all cases and in the order S12>EF-Tu>EF-G>S7; the largest homologies were generally found between the cyanobacterial proteins and the corresponding chloroplast gene products. Overall codon usage in S. platensis was found to be rather unbiased.

Nucleotide sequence of a DNA region comprising the gene for elongation factor 1α (EF-1α) from the ultrathermophilic archaeotePyrococcus woesei: Phylogenetic implications

SummaryThe gene encoding elongation factor 1α (EF-1α, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeotePyrococcus woesei was localized within aBglII fragment of chromosomal DNA. Sequence analysis showed that the EF-1α gene is the upstream unit of a three-gene cluster comprising the genes for ribosomal protein S10 (306 bp) and transfer RNAser (GGA). The three genes follow each other immediately in the order EF-1α·S10·tRNAser after a putative promoter located 55 bp upstream of the EF-1α gene. Alignment of the derived EF-1α sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed thatPyrococcus EF-1α is highly homologous (89% identity) toThermococcus celer EF-1α, both being strikingly more similar to eukaryotic EF-1α than to bacterial EF-Tu. Unrooted dendrograms computed from aligned sequences by distance matrix and DNA parsimony methods, including evolutionary parsimony, showed the Archaea to be a monophyletic-holophyletic cluster closer to Eukarya than to Bacteria. Both distance matrix and DNA parsimony-although not evolutionary parsimony-support the partition of the known archaeal lineages between the kingdoms Crenarchaeota and Euryarchaeota, and the affiliation of thePyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation ofPyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins.

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.

Presence of two sets of ribosome-specific transfer factors in the cell-free extracts from the non-photosynthetic alga Prototheca zopfii

Abstract Preparations of polymerizing enzymes from the non-photosynthetic alga Prototheca zopfii possess two transfer factors G, one active on the ribosomes of the 70 s type ( Escherichia coli ) and one active on those of the 80 s type ( Saccharomyces cerevisiae ). Evidence is presented in favour of the presence also of two ribosome-specific transfer factors T. The G-like activity for E. coli ribosomes present in the polymerizing enzymes from the yeast S. cerevisiae (Ciferri, Parisi, Perani & Grandi, 1968) has been demonstrated to be due to the presence of a transfer factor G, specific for ribosomes of the 70 s type that may be separated from that active on ribosomes of the 80 s type.

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.

Ribosomes and translation factors from isolated spinach chloroplasts

Abstract Chloroplasts isolated from spinach leaves carry on a light-driven, RNAase-insensitive incorporation of amino acids. From such chloroplasts an active S-20 fraction was prepared which was capable of incorporating amino acids under the direction of the endogenous mRNA and phenylalanine under that of polyuridylic acid (poly U). Initiation and elongation factors as well as ribosomes active in vitro were obtained from isolated chloroplasts.

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.

Cloning and expression of the genes for ribulose-1,5-bisphosphate carboxylase from Spirulina platensis

Abstract The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase have been cloned from the filamentous cyanobacterium Spirulina platensis. The two genes, located very closely on a 4.6 kbp DNA fragment, appear to be expressed although to a different extent in minicells of Escherichia coli. The amount of large subunit produced in the bacterial host represents at least 10% of the total protein.

Selective inhibition of the reactions catalyzed by ribosome-specific transfer factors G

It is well known that one of the transfer factors required for peptide chain elongation, transfer factor G (translocase) catalyzes the translocation of peptidyl-tRNA onto the ribosome [l] The translocation requires GTP and it has been calculated that one GTP is hydrolyzed to GDP and inorganic phosphate for every peptide bond formed [2]. In addition the factor, whether isolated from prokaryotic or eukaryotic organisms, is endowed with GTP-ase activity dependent on the presence of ribosomes [2-51. Albeit evident also under conditions in which there is no peptide chain elongation, it has been assumed that the hydrolytic activity is related to the synthetic activity [4,6] since the two activities are purified together and all the antibiotics so far tested that inhibit translocation also inhibit ribosome dependent GTP-ase. Indeed fusidic acid appears to inhibit both activities in the case of the factors G from prokaryotic and eukaryotic organisms [7, 81 , while siomycin exerts a similar effect on Escherichia coli transfer factor G and E. coli ribosomes [6] and diphtheria toxin and factors G (transfer factors II) * and ribosomes from mammalian cells and organs [9111. We have previously shown that the achloric alga Prototheca zopfii is endowed with two separated transfer factors G, one specific for 70 S ribosomes (such as are those present in prokaryotic organisms and in cellular organelles) and the other specific for ribosomes of the 80 S type as are those present in the cytoplasm of eukaryotic organisms [ 121. The availability of the two transfer factors G at a degree