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31.
A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA-DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov. 相似文献
32.
33.
By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their
function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less
archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of
approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein
microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an
MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation
by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of
T. acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of
structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL
− knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was charaterized by very brief openings
of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL
+ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL
in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or
modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids
having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels. 相似文献
34.
The presence of Archaea in the Great Barrier Reef marine sponge Rhopaloeides odorabile was investigated by 16S ribosomal RNA community analysis of total DNA extracted from the sponge tissue. The 16S rRNA gene
sequences corresponding to group I crenarchaeotes and group II euryarchaeotes were recovered from R. odorabile tissue. The location of archaeal cells within the sponge tissue was investigated using fluorescently labeled oligonucleotide
probes. The presence of Archaea was confirmed within all regions of the sponge tissue from R. odorabile, with a significantly higher number of archaeal cells located in the pinacoderm than the mesohyl region. This is the first
report of euryarchaeaotes associated with marine sponges.
Received April 16, 2001; accepted June 27, 2001 相似文献
35.
Genomic trees have been constructed based on the presence and absence of families of protein-encoding genes observed in 27
complete genomes, including genomes of 15 free-living organisms. This method does not rely on the identification of suspected
orthologs in each genome, nor the specific alignment used to compare gene sequences because the protein-encoding gene families
are formed by grouping any protein with a pairwise similarity score greater than a preset value. Because of this all inclusive
grouping, this method is resilient to some effects of lateral gene transfer because transfers of genes are masked when the
recipient genome already has a homolog (not necessarily an ortholog) of the incoming gene. Of 71 genes suspected to have been
laterally transferred to the genome of Aeropyrum pernix, only approximately 7 to 15 represent genes where a lateral gene transfer appears to have generated homoplasy in our character
dataset. The genomic tree of the 15 free-living taxa includes six different bacterial orders, six different archaeal orders,
and two different eukaryotic kingdoms. The results are remarkably similar to results obtained by analysis of rRNA. Inclusion
of the other 12 genomes resulted in a tree only broadly similar to that suggested by rRNA with at least some of the differences
due to artifacts caused by the small genome size of many of these species. Very small genomes, such as those of the two Mycoplasma genomes included, fall to the base of the Bacterial domain, a result expected due to the substantial gene loss inherent to
these lineages. Finally, artificial ``partial genomes' were generated by randomly selecting ORFs from the complete genomes
in order to test our ability to recover the tree generated by the whole genome sequences when only partial data are available.
The results indicated that partial genomic data, when sampled randomly, could robustly recover the tree generated by the whole
genome sequences.
Received: 30 May 2001 / Accepted: 10 October 2001 相似文献
36.
Jeon SJ Fujiwara S Takagi M Tanaka T Imanaka T 《Biochemical and biophysical research communications》2002,295(2):508-514
The Tk-ptp gene encoding a protein tyrosine phosphatase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned and biochemical characteristics of the recombinant protein (Tk-PTP) were examined. A series of mutants, D63A (replacing Asp-63 with Ala), C93S, C93A, R99K, and R99M, were also constructed and analyzed. Two unique features were found. First, the Tk-PTP showed the phosphatase activity not only toward phosphotyrosine but also toward phosphoserine. Second, the conserved Asp-63, which corresponds to a critical residue among other known PTPs, was not essential for catalysis. Cys-93 and Arg-99 residues played a crucial role in substrate binding and catalysis. To know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate proteins from cell extract of KOD1. Phenylalanyl-tRNA synthetase subunit beta-chain, one of the gene products of RNA terminal phosphate cyclase operon and phosphomannomutase, was identified, suggesting that they functioned for phosphate donation. 相似文献
37.
Ward DE Shockley KR Chang LS Levy RD Michel JK Conners SB Kelly RM 《Archaea (Vancouver, B.C.)》2002,1(1):63-74
Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus solfataricus, and the bacterium Thermotoga maritima. An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putative proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms. 相似文献
38.
Facchin S Sarno S Marin O Lopreiato R Sartori G Pinna LA 《Biochemical and biophysical research communications》2002,296(5):1366-1371
Yeast piD261/Bud32 and its homologues are present in eukaryotes and in archaea but not in bacteria and are believed to make up a primordial branch of the eukaryotic protein kinase superfamily. Here, we show that, at variance with the majority of Ser/Thr protein kinases which recognize phosphoacceptor sites specified by basic and/or proline residues, piD261 phosphorylates in vitro a number of acidic proteins and peptides, and it recognizes seryl residues specified by carboxylic side chains. These data suggest that recognition of acidic sites might have been a primordial trait of protein kinases, which was modified during evolution to cope with the increasing complexity of protein phosphorylation in eukaryotes. 相似文献
39.
Complete archaeal genomes were probed for the presence of long (> or = 25 bp) oligonucleotide repeats (words). We detected the presence of many words distributed in tandem with narrow ranges of periodicity (i.e., spacer length between repeats). Similar words were not identified in genomes of non-archaeal species, namely Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Mycoplasma genitalium and Mycoplasma pneumoniae. BLAST similarity searches against the GenBank nucleotide sequence database revealed that these words were archaeal species-specific, indicating that they are of a signature character. Sequence analysis and genome viewing tools showed these repeats to be restricted to non-coding regions. Thus, archaea appear to possess a non-coding genomic signature that is absent in bacterial species. The identification of a species-specific genomic signature would be of great value to archaeal genome mapping, evolutionary studies and analyses of genome complexity. 相似文献
40.
The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development
of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic
Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure
for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without
the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na2S) was replaced by sodium sulfite (Na2SO3), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace
of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that
were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H2S was simply added to the headspace of serum bottles by a syringe. The use of H2S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea
studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application
of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably.
Received: January 4, 2000 / Accepted: April 5, 2000 相似文献