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P Cammarano

Publications and source records attributed to P Cammarano.

At least 19 recordsLinked to original sources
PubMed1998

The root of the universal tree of life inferred from anciently duplicated genes encoding components of the protein-targeting machinery.

The key protein of the signal recognition particle (termed SRP54 for Eucarya and Ffh for Bacteria) and the protein (termed SRalpha for Eucarya and Ftsy for bacteria) involved in the recognition and binding of the ribosome SRP nascent polypeptide complex are the products of an ancient gene duplication that appears to predate the divergence of all extant taxa. The paralogy of the genes encoding the two proteins (both of which are GTP triphosphatases) is argued by obvious sequence similarities between the N-terminal half of SRP54(Ffh) and the C-terminal half of SRalpha(Ftsy). This enables a universal phylogeny based on either protein to be rooted using the second protein as an outgroup. Phylogenetic trees inferred by various methods from an alignment (220 amino acid positions) of the shared SRP54(Ffh) and SRalpha(Ftsy) regions generate two reciprocally rooted universal trees corresponding to the two genes. The root of both trees is firmly positioned between Bacteria and Archaea/Eucarya, thus providing strong support for the notion (Iwabe et al. 1989; Gogarten et al. 1989) that the first bifurcation in the tree of life separated the lineage leading to Bacteria from a common ancestor to Archaea and Eucarya. None of the gene trees inferred from the two paralogues support a paraphyletic Archaea with the crenarchaeota as a sister group to Eucarya.

Algorithms↗
PubMed1996

Manipulation of the tuf gene provides clues to the localization of sequence element(s) involved in the thermal stability of Thermotoga maritima elongation factor Tu.

Truncated versions of the tuf gene for elongation factor Tu (EF-Tu; 400 aa) from the hyperthermophilic bacterium Thermotoga maritima have been produced by progressive 3'-->5' trimming. The truncated genes have been expressed in Escherichia coli and the thermal stability of the gene products has been assayed by monitoring their GDP-binding capacity after preheating the cell-free extracts at various temperatures (65-95 degrees C). One of the truncated proteins, corresponding to the nucleotide-binding domain (G domain; aa 1-200) appears to be only slightly less stable than the full-length EF-Tu. Replacement of the first 90 N-terminal residues of both the full-length Thermotoga EF-Tu and the isolated G domain with the corresponding sequence of the mesophilic bacterium E. coli, drastically destabilizes both the complete and the truncated protein, indicating that sequence element(s) that are crucial for the attainment of a thermally stable conformation of the Thermotoga EF-Tu lie well within the initial portion of the G domain between residues 1 and 90. The relevant residues defy identification, however, as no amino acid preferences, or exclusive sequence element(s), appear to distinguish the N-terminal region of the thermophilic proteins from those of mesophilic counterparts. It is suggested that the thermal stability of Thermotoga EF-Tu is critically dependent upon unique tertiary structural interactions involving certain N-terminal residues of the molecule.

Amino Acid Sequence↗
PubMed1996

Sensitivity of ribosomes of the hyperthermophilic bacterium Aquifex pyrophilus to aminoglycoside antibiotics.

A poly(U)-programmed cell-free system from the hyperthermophilic bacterium Aquifex pyrophilus has been developed, and the susceptibility of Aquifex ribosomes to the miscoding-inducing and inhibitory actions of all known classes of aminoglycoside antibiotics has been assayed at temperatures (75 to 80 degrees C) close to the physiological optimum for cell growth. Unlike Thermotoga maritima ribosomes, which are systematically refractory to all known classes of aminoglycoside compounds (P. Londei, S. Altamura, R. Huber, K. O. Stetter, and P. Cammarano, J. offteriol. 170-4353-4360, 1988), Aquifex ribosomes are susceptible to all of the aminoglycosides tested (disubstituted 2-deoxystreptamines, monosubstituted 2-deoxystreptamines, sand streptidine compounds). The significance of this result in light of the Aquifex and Thermotoga placements in phylogenetic trees of molecular sequences is discussed.

Aminoglycosides↗
PubMed1995

Chromosomal organization and nucleotide sequence of the genes for elongation factors EF-1 alpha 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-1 alpha) 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 tRNA(Ser); the arrangement of the cluster is 5'-tuf-91 bp spacer -rps10-138 bp spacer -tRNA(Ser)-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.

Amino Acid Sequence↗
PubMed1995

Chromosomal organization and nucleotide sequence of the fus-gene encoding elongation factor 2 (EF-2) of the hyperthermophilic archaeum Pyrococcus woesei.

A Pyrococcus woesei EcoRI DNA fragment (3400 bp) harbouring the gene fus for elongation factor 2 (EF-2) was cloned and almost completely sequenced. Unlike Methanococcus vannielii (which displays the 'str operon'-like fus and tuf gene context, 5'-rps12-rps7-fus-tuf-3'), and similar to Sulfolobus acidocaldarius and Desulfurococcus mobilis, the Pyrococcus fus gene (732 codons) is unlinked to the rps and tuf genes, and is immediately followed (57 bp intergenic spacing) by an ORF of 106 codons. Both ORFs are preceded by potential archaeal promoters located 52 bp (for fus) and 37 bp (for ORF106) upstream of the putative start codons. The Pyrococcus EF-2(G) equivalent factor is somewhat closer to the eukaryal than to the bacterial homolog, and also shares with the former the C-terminal sequence required for ADP ribosylation of EF-2 by Diphtheria toxin.

Amino Acid Sequence↗
PubMed1995

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 Thermotoga 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 (< or = 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.

Amino Acid Sequence↗
PubMed1994

Evolution of translational elongation factor (EF) sequences: reliability of global phylogenies inferred from EF-1 alpha(Tu) and EF-2(G) proteins.

The EF-2 coding genes of the Archaea Pyrococcus woesei and Desulfurococcus mobilis were cloned and sequenced. Global phylogenies were inferred by alternative tree-making methods from available EF-2(G) sequence data and contrasted with phylogenies constructed from the more conserved but shorter EF-1 alpha(Tu) sequences. Both the monophyly (sensu Hennig) of Archaea and their subdivision into the kingdoms Crenarchaeota and Euryarchaeota are consistently inferred by analysis of EF-2(G) sequences, usually at a high bootstrap confidence level. In contrast, EF-1 alpha(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-1 alpha(Tu) sequences do show archaeal monophyly, protein parsimony and DNA maximum-likelihood analyses of these data do not. In no case, however, do any of the tree topologies inferred from EF-1 alpha(Tu) sequence analyses receive significant bootstrap support.

Amino Acid Sequence↗
PubMed1994

Phylogenetic depth of S10 and spc operons: cloning and sequencing of a ribosomal protein gene cluster from the extremely thermophilic bacterium Thermotoga maritima.

A segment of Thermotoga maritima DNA spanning 6,613 bp downstream from the gene tuf for elongation factor Tu was sequenced by use of a chromosome walking strategy. The sequenced region comprised a string of 14 tightly linked open reading frames (ORFs) starting 50 bp downstream from tuf. The first 11 ORFs were identified as homologs of ribosomal protein genes rps10, rpl3, rpl4, rpl23, rpl2, rps19, rpl22, rps3, rpl16, rpl29, and rps17 (which in Escherichia coli constitute the S10 operon, in that order); the last three ORFs were homologous to genes rpl14, rpl24, and rpl5 (which in E. coli constitute the three promoter-proximal genes of the spectinomycin operon). The 14-gene string was preceded by putative -35 and -10 promoter sequences situated 5' to gene rps10, within the 50-bp spacing between genes tuf and rps10; the same region exhibited a potential transcription termination signal for the upstream gene cluster (having tuf as the last gene) but displayed also the potential for formation of a hairpin loop hindering the terminator; this suggests that transcription of rps10 and downstream genes may start farther upstream. The similar organization of the sequenced rp genes in the deepest-branching bacterial phyla (T. maritima) and among Archaea has been interpreted as indicating that the S10-spc gene arrangement existed in the (last) common ancestor. The phylogenetic depth of the Thermotoga lineage was probed by use of r proteins as marker molecules: in all except one case (S3), Proteobacteria or the gram-positive bacteria, and not the genus Thermotoga, were the deepest-branching lineage; in only two cases, however, was the inferred branching order substantiated by bootstrap analysis.

Amino Acid Sequence↗
PubMed1993

Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences.

The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA 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% 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. solfataricus, M. voltae) 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 Bacillus-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.

Amino Acid Sequence↗
PubMed1992

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

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.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of a DNA region comprising the gene for elongation factor 1 alpha (EF-1 alpha) from the ultrathermophilic archaeote Pyrococcus woesei: phylogenetic implications.

The gene encoding elongation factor 1 alpha (EF-1 alpha, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeote Pyrococcus woesei was localized within a Bg/II fragment of chromosomal DNA. Sequence analysis showed that the EF-1 alpha 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-1 alpha.S10.tRNA(ser) after a putative promoter located 55 bp upstream of the EF-1 alpha gene. Alignment of the derived EF-1 alpha sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed that Pyrococcus EF-1 alpha is highly homologous (89% identity) to Thermococcus celer EF-1 alpha, both being strikingly more similar to eukaryotic EF-1 alpha 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 the Pyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation of Pyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins.

Amino Acid Sequence↗
PubMed1991

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.

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.

Amino Acid Sequence↗
PubMed1990

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

The 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.

Amino Acid Sequence↗
PubMed1989

Phylogenetic conservation of antigenic determinants in archaebacterial elongation factors (Tu proteins).

By using affinity chromatography methods, we have purified elongation factor Tu (EF-Tu) proteins from a host of archaebacteria covering all known divisions in the archaebacterial tree except halophiles, and from such distantly related eubacteria as Thermotoga maritima and Escherichia coli. Polyclonal antibodies were raised against the Tu proteins of Sulfolobus solfataricus, Thermoproteus tenax, Thermococcus celer, Pyrococcus wosei, Archaeoglobus fulgidus, Methanococcus thermolitotrophicus, Thermoplasma acidophilum, and Thermotoga and used to probe the immunochemical relatedness of elongation factors both within and across kingdom boundaries. A selection of the results, presented here, indicates that (i) every archaebacterial EF-Tu is closer (immunochemically) to every other archaebacterial EF-Tu than to the functionally analogous proteins of eubacteria and eukaryotes, with only one possible exception concerning the recognition of eukaryotic (EF-1 alpha) factors by Thermococcus EF-Tu antibodies, and (ii) within the archaebacteria there appears to be a correlation between EF-Tu immunochemical similarities and the phylogenetic relatedness of the organisms inferred from other (sequence) criteria. On the whole, immunochemical similarity data argue against the proposal that the archaebacterial taxon should be split and redistributed between two superkingdoms.

Antigens, Bacterial↗
PubMed1988

Ribosomes of the extremely thermophilic eubacterium Thermotoga maritima are uniquely insensitive to the miscoding-inducing action of aminoglycoside antibiotics.

Poly(U)- and poly(UG)-programmed cell-free systems were developed from the extreme thermophilic, anaerobic eubacterium Thermotoga maritima, and their susceptibility to aminoglycoside and other antibiotics was assayed at a temperature (75 degrees C) close to the physiological optimum (80 degrees C) for cell growth and in vitro polypeptide synthesis, using a Bacillus stearothermophilus system as the reference. The synthetic capacity of the Thermotoga assay mixture was abolished by the eubacterium-targeted drugs chloramphenicol, thiostrepton, and kirromycin. However, streptomycin, the disubstituted 2-deoxystreptamines (kanamycin, gentamicin, neomycin, and paromomycin), and the monosubstituted 2-deoxystreptamine (hygromycin) all failed to promote translational misreading of poly(U) on Thermotoga ribosomes; they also failed to block polyphenylalanine synthesis at a low (less than 10(-4) M) concentration and did not inhibit Thermotoga cell growth at a high (10 micrograms/ml) concentration even though Thermotoga ribosomes possess the 16S rRNA sequences required for aminoglycoside action. In contrast to the other eubacteria, Thermotoga elongation factor G was also refractory to the steroid inhibitor of peptidyl-tRNA translocation fusidic acid.

Aminoglycosides↗
PubMed1986

Archaebacterial and eukaryotic ribosomal subunits can form active hybrid ribosomes.

Purified ribosomal subunits from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus are able to recognize ribosomal subunits from the yeast Saccharomyces cerevisiae forming hybrid monosomes that can be revealed by sucrose gradient analysis and are active in peptide bond formation. Both reciprocal combinations (archaebacterial 30 S + eukaryotic 60 S and archaebacterial 50 S + eukaryotic 40 S) are functional. In contrast, no hybrid couples are formed between subunits of yeast and Escherichia coli ribosomes. These results indicate that ribosomes of at least one archaebacterial species share specific structural features with those of the lower eukaryote S. cerevisiae.

Archaea↗