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At least 991 records · Page 55Linked to original sources
PubMed2003

Characterization of aldehyde ferredoxin oxidoreductase gene defective mutant in Magnetospirillum magneticum AMB-1.

A non-magnetic mutant of Magnetospirillum magneticum AMB-1, designated as NMA21, was generated by mini-Tn5 transposon mutagenesis to identify genes involved in bacterial magnetic particle (BMP) synthesis. Alignment of the DNA sequences flanking the transposon allowed the isolation of an open reading frame (ORF2) within an operon consisting of five genes. The amino acid sequence of ORF2 showed homology with tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (48% identity and 64% similarity), which functions for aldehyde oxidation. AOR was found to be expressed under microaerobic conditions and localized in the cytoplasm of AMB-1. Iron uptake and growth of NMA21 were lower than wild type. Transmission electron microscopy (TEM) of NMA21 revealed that no BMPs were completely synthesized, but polyhydroxybutyrate (PHB)-like granules were persistently produced. These results indicate that AOR may contribute to ferric iron reduction during BMP synthesis in M. magneticum AMB-1 under microaerobic respiration.

Aldehyde Oxidoreductases↗
PubMed1997

Protein contributions to redox potentials of homologous rubredoxins: an energy minimization study.

The energetic contributions of the protein to the redox potential in an iron-sulfur protein are studied via energy minimization, comparing homologous rubredoxins from Clostridium pasteurianum, Desulfovibrio gigas, Desulfovibrio vulgaris, and Pyrococcus furiosus. The reduction reaction was divided into 1) the change in the redox site charge without allowing the protein to respond and 2) the relaxation of the protein in response to the new charge state, focusing on the latter. The energy minimizations predict structural relaxation near the redox site that agrees well with that in crystal structures of oxidized and reduced P. furiosus rubredoxin, but underpredicts it far from the redox site. However, the relaxation energies from the energy-minimized structures agree well with those from the crystal structures, because the polar groups near the redox site are the main determinants and the charged groups are all located at the surface and thus are screened dielectrically. Relaxation energies are necessary for good agreement with experimentally observed differences in reduction energies between C. pasteurianum and the other three rubredoxins. Overall, the relaxation energy is large (over 500 mV) from both the energy-minimized and the crystal structures. In addition, the range in the relaxation energy for the different rubredoxins is large (300 mV), because even though the structural perturbations of the polar groups are small, they are very near the redox site. Thus the relaxation energy is an important factor to consider in reduction energetics.

Amino Acid Sequence↗
PubMed2003

Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers.

Escherichia coli maltose-binding protein (MBP) is frequently used as an affinity tag to facilitate the purification of recombinant proteins. An important additional attribute of MBP is its remarkable ability to enhance the solubility of its fusion partners. MBPs are present in a wide variety of microorganisms including both mesophilic and thermophilic bacteria and archaea. In the present study, we compared the ability of MBPs from six diverse microorganisms (E. coli, Pyrococcus furiosus, Thermococcus litoralis, Vibrio cholerae, Thermotoga maritima, and Yersinia pestis) to promote the solubility of eight different aggregation-prone proteins in E. coli. In contrast to glutathione S-transferase (GST), all of these MBPs proved to be effective solubility enhancers and some of them were even more potent solubilizing agents than E. coli MBP.

Archaeal Proteins↗
PubMed1999

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis.

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum deltaH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl-tRNA synthetase (CysRS). The characterization of CysRS-encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysSts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.

Amino Acid Sequence↗
PubMed2004

Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus.

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.

Archaeal Proteins↗
PubMed1997

Physical characterization of a totally synthetic rubredoxin.

The entire polypeptide of hyperthermophilic Pyrococcus furiosus rubredoxin was synthesized in order to specifically probe structural determinants of protein thermostability. The uv-visible, circular dichroic, electron paramagnetic, and nuclear magnetic resonance spectra, and electrochemical properties, of the native and synthetic proteins were essentially identical. The synthetic protein had a half-life for denaturation of 24 hr at 80 degrees C. The synthetic protein is considerably more thermostable than nonhyperthermophilic rubredoxins, but not as stable as the native protein. Based on the spectroscopic evidence, it appears that the synthetic protein is incorporating iron properly to form holoprotein, but the peptide still may not be folded correctly.

Amino Acid Sequence↗
PubMed1998

The pyruvate dehydrogenase complex from thermophilic organisms: thermal stability and re-association from the enzyme components.

Examples of pyruvate dehydrogenase complexes, and of its probable precursors, the pyruvate ferredoxin oxidoreductases, both isolated from thermophilic organisms, are described. The pyruvate ferredoxin oxidoreductases are mostly characterized from thermophilic archaea like Sulfolobus solfataricus and Pyrococcus furiosus. They retain their catalytic activity up to 60 and 90 degreesC, respectively. Characteristic for the thermophilic nature is a biphasic temperature behavior, reflecting a more stable low temperature and a metastable high temperature form. Another feature is the strong binding of the cofactor thiamin diphosphate. Detailed analysis of thermostable pyruvate dehydrogenase complexes so far only exist for the enzymes from Bacillus stearothermophilus and Thermus flavus. In most respects, especially in the structural features, the enzyme complex from B. stearothermophilus resembles its mesophilic counterparts and only an elevated temperature maximum for the catalytic activity reveals the thermophilic nature. In contrast to this, the more thermostable enzyme complex from T. flavus shows a quite distinct behavior. One single protein chain (Mr=100 kDa) instead of an alpha2beta2 aggregate was found for the pyruvate dehydrogenase (E1) subunits of this enzyme complex. Its catalytic activity is controlled by allosteric regulation, while the enzyme complex from B. stearothermophilus shows no such regulation. Reversible phosphorylation as a regulatory principle of pyruvate dehydrogenase complexes from higher organisms does not take place in the thermophilic enzyme complexes. The overall activity of the enzyme complex from B. stearothermophilus remains stable at 60 degreesC for 50 min while that from T. flavus is active up to 83 degreesC. Thermophilic pyruvate dehydrogenase complexes do not spontaneously renature from their separated enzyme components. However, chaperonins from Thermus thermophilus stimulate the reactivation of the enzyme complex from T. flavus.

Archaea↗
PubMed1999

The NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum: cloning, sequencing, and expression of the enzyme gene(1).

The NAD-dependent glutamate dehydrogenase (GluDH) gene from the hyperthermophilic archaeon Pyrobaculum islandicum was cloned and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1266 bp encoding a protein of 421 amino acids with a molecular weight of 46,905. In the alignment of the amino acid sequence with those of mesophilic Clostridium symbiosum NAD-dependent GluDH and hyperthermophilic NADP-dependent enzymes from Thermococcus profundus and Pyrococcus furiosus, substitutions in the residues involved in dinucleotide binding were observed. On the other hand, the residues involved in glutamate binding were well conserved. This is the first description of the primary structure of NAD-dependent GluDH in hyperthermophilic archaea.

Amino Acid Sequence↗
PubMed2001

Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme.

DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp. KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, when long distance PCR (>5 kbp) was performed with KOD DNA polymerase, amplification efficiency (product yield) becomes lower because of its strong 3'-5' exonuclease activity for proof-reading. In order to improve a target length limitation in PCR, mutant DNA polymerases with decreased 3'-5' exonuclease activity were designed by substituting amino acid residues in conserved exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn210-Xaa-Xaa-Xaa-Phe-Asp), and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification fidelity (error rate) of the DNA polymerases were altered by mutagenesis. However, long and accurate PCR by a single-type of mutant DNA polymerase was very difficult. The wild-type DNA polymerase (WT) and its exonuclease deficient mutant (N210D) were mixed in different ratio and their characteristics in PCR were examined. When the mixed enzyme (WT and N210D) was made at the ratio of 1:40, long PCR (15 kbp) at lower mutation frequency could be efficiently achieved.

Amino Acid Sequence↗
PubMed2001

The form of chromosomal DNA molecules in bacterial cells.

The circular concept of the bacterial chromosome was based initially on experiments involving conjugation mapping and autoradiographic imaging of DNA. This view was then supported by DNA fragment mapping, genome sequencing, and the analysis of linear DNA produced by a single cleavage of chromosomal DNA. A circular chromosome is also indicated by the existence of a mechanism for segregating dimeric chromosomes produced by recombination and the replication of DNA on both sides of the replication terminus. The evidence for circularity is reviewed here and found to be compatible with either a circular or a linear chromosomal DNA molecule. Moving pictures of ethidium-stained DNA revealed most chromosomal DNA as a rosette form with loops emanating from a dense node or as a network of strands lacking a node. This description applies to Escherichia coli, Agrobacterium tumefaciens, Pyrococcus endeavorii, Vibrio cholerae, and both the linear-mapping chromosome of Streptomyces lividans and its circular-mapping derivative. Networks without nodes were found for two linear-mapping Borrelia species. For the E. coli chromosome, open-form circles of various sizes were found only at extremely low frequency. The node of the rosette was reduced in size or eliminated in recA mutants, as well as by treatment with either ribonuclease, topoisomerase IV, 1 M NaCl, or lysozyme. A model is presented for the bacterial chromosome in which the DNA is compacted by many points of strand association (including recombination junctions, tangles and knots) created during the repair of DNA damage that occurs many times in each chromosome replication cycle.

Bacteria↗
PubMed1997

Molecular physiology of carbamoylation under extreme conditions: what can we learn from extreme thermophilic microorganisms?

The importance of protein-protein interactions in the physiology of extreme thermophiles was investigated by analyzing the enzymes involved in biosynthetic carbamoylation in Thermus ZO5 and by comparing the results obtained with already available or as yet unpublished information concerning other thermophilic eu- and archaebacteria such as Thermotoga, Sulfolobus, and Pyrococcus. Salient observations were that (i) the highly thermolabile and reactive carbamoylphosphate molecule appears to be protected from thermodegradation by channelling towards the synthesis of citrulline and carbamoylaspartate, respectively precursors of arginine and the pyrimidines; (ii) Thermus ornithine carbamoyltransferase is clearly a thermophilic enzyme, intrinsically thermostable and showing a biphasic Arrhenius plot, whereas aspartate carbamoyltransferase is inherently unstable and is stabilized by its association with dihydroorotase, another enzyme encoded by the Thermus pyrimidine operon. Possible implications of these results are discussed.

Archaea↗
PubMed2002

Expression of long- and short-type FK506 binding proteins in hyperthermophilic archaea.

It has been reported that the hyperthermophilic archaeon, Methanococcus jannaschii, possesses two FKBP (FK506 binding protein) genes in the genome, one being 26 kDa FKBP (long-type FKBP) and the other, 18 kDa FKBP (short-type FKBP). FKBP is a family of peptidyl-prolyl cis-trans isomerases (PPIases). In order to clarify the difference between their roles in archaeal cells, they were expressed in Escherichia coli, and their PPIase and chaperone-like protein-folding activities were investigated. The catalytic efficiency of the PPIase activity of the long-type FKBP was significantly lower than that of short-type FKBP (less than 1/1000) which is comparable to that of human FKBP12. Both FKBPs showed chaperone-like protein-folding activity to enhance the refolding yield of an unfolded protein (Thermoplasma citrate synthase) in vitro. The chaperone-like protein-folding activity of the short type was higher than that of the long type. While the intracellular content of long-type FKBP in M. jannaschii tended to increase, that of short-type FKBP obviously decreased at growth temperatures higher than the optimum of 85 degrees C. In Pyrococcus horikoshii, another hyperthermophilic archaeon, the intracellular content of long-type FKBP did not change with temperature (80-102 degrees C). These results suggest that long-type FKBP functions at any temperature in the cells as a chaperone to maintain the folding states of intracellular proteins. On the other hand, short-type FKBP may be required at lower temperatures. Peptidyl-prolyl cis-trans isomerization is known to be a rate-limiting step in protein-folding and is slower at low temperature. Since the PPIase activity of short-type FKBP was much stronger than that of the long type, it may be required to accelerate the folding of intracellular proteins and for the hyperthermophilic cell to live at low growth temperatures.

Catalysis↗
PubMed1996

The world of archaea: genome analysis, evolution and thermostable enzymes.

Pyrococcus sp. KOD1 is a newly isolated hyperthermophilic archaeon from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. A physical map of the KOD1 chromosome was constructed using pulsed-field gel electrophoresis of restriction fragments generated by AscI, PacI and PmeI. The order of the AscI fragments was deduced from Southern hybridization using the AscI, PmeI and PacI fragments as a probe. The derived physical map indicates that KOD1 possesses a circular-form genome and its size was estimated to be 2036 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the physical map. Some genes involved in the central dogma were located on the restricted segment of the genome. Novel characteristics of KOD1 enzymes are also introduced in this article.

Archaea↗
PubMed1997

Cloning and identification of the Sulfolobus solfataricus lrp gene encoding an archaeal homologue of the eubacterial leucine-responsive global transcriptional regulator Lrp.

The lrp gene of the extreme thermophilic archaeon Sulfolofus solfataricus, encoding a homologue of the eubacterial global leucine-responsive regulatory protein, was identified by DNA sequencing and sequence comparisons on a 6.9-kb genomic fragment cloned into Escherichia coli. The S. solfataricus Lrp subunit is a 155-aa polypeptide that bears between 24.5 and 29% sequence identity with eubacterial regulatory proteins of the Lrp/AsnC family and 30.6% and 25.8% with the archaeal homologues of respectively Methanococcus jannaschii and Pyrococcus furiosus. Transcription initiation from the strong S. solfataricus lrp promoter was analyzed by primer extension mapping. The abundance of the S. solfataricus lrp messenger strongly suggests that this protein might function in archaea as a global transcriptional regulator and genome organizer, as proposed for E. coli Lrp, rather than as a local, specific regulatory protein. Our findings suggest the presence of a eubacterial type of regulatory mechanism in archaea, a situation that is noteworthy indeed, since the transcriptional machinery of archaea is more closely related to that of eukaryotes, whereas these latter apparently do not possess a homologue of Lrp.

Amino Acid Sequence↗
PubMed1999

Cloning and sequencing of an acetyl-CoA synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia.

A Giardia lamblia gene, Glacs, was cloned, sequenced and expressed in Escheria Coli. This gene codes for a 726 residue long acetyl-CoA synthetase (ADP-forming). This enzyme is responsible for the formation of acetate, a metabolic endproduct of G. lamblia. It is known from only two Type I amitochondriate eukaryotes, G. lamblia and Entamoeba histolytica and from the archaebacterium, Pyrococcus furiosus. With Glacs as query, homologous unidentified open reading frames were detected in the complete genomes of only a few archaebacteria and eubacteria. These form a new protein family present in all three domains of life, which probably plays a central role in the acyl-CoA metabolism but is of restricted taxonomic distribution.

Animals↗
PubMed1999

Studies of codon usage and tRNA genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: gene expression level and species-specific diversity of codon usage based on multivariate analysis.

We examined codon usage in Bacillus subtilis genes by multivariate analysis, quantified its cellular levels of individual tRNAs, and found a clear constraint of tRNA contents on synonymous codon choice. Individual tRNA levels were proportional to the copy number of the respective tRNA genes. This indicates that the tRNA gene copy number is an important factor to determine in cellular tRNA levels, which is common with Escherichia coli and yeast Saccharomyces cerevisiae. Codon usage in 18 unicellular organisms whose genomes have been sequenced completely was analyzed and compared with the composition of tRNA genes. The 18 organisms are as follows: yeast S. cerevisiae, Aquifex aeolicus, Archaeoglobus fulgidus, B. subtilis, Borrelia burgdorferi, Chlamydia trachomatis, E. coli, Haemophilus influenzae, Helicobacterpylori, Methanococcusjannaschii, Methanobacterium thermoautotrophicum, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma pneumoniae, Pyrococcus horikoshii, Rickettsia prowazekii, Synechocystis sp., and Treponema pallidum. Codons preferred in highly expressed genes were related to the codons optimal for the translation process, which were predicted by the composition of isoaccepting tRNA genes. Genes with specific codon usage are discussed in connection with their evolutionary origins and functions. The origin and terminus of replication could be predicted on the basis of codon usage when the usage was analyzed relative to the transcription direction of individual genes.

Archaea↗
PubMed1999

Computer analyses of complete genomes suggest that some archaebacteria employ both eukaryotic and eubacterial mechanisms in translation initiation.

The translation initiation mechanism of archaebacteria is still not clearly understood. Our previous work showed that ATG triplets before start codons have been strongly depleted in eukaryotic genomes, presumably because ribosome of eukaryotes scans mRNA from the 5' to 3' direction to find proper start codons. Extra ATG triplets before start codons would confuse the process and thus they have been negatively selected in eukaryotic genomes. In eubacterial genomes, on the other hand, ribosome binds to the Shine-Dalgarno (SD) sequence at once without mRNA scanning, and the characteristic patterns of ATG triplet depletion were not observed (Saito, R., Tomita, M., 1999. On negative selection against ATG triplets near start codons in eukaryotic and procaryotic genomes. J. Mol. Evol. 48, 213-217). The ATG triplet analysis on archaebacterial genomes revealed that Methanococcus jannaschii and Pyrococcus horikoshii show patterns similar to eukaryotes, implying that these species employ scanning of mRNA from the 5' to 3' direction in the process of translation initiation. On the other hand, our earlier study found that these archaea have SD-like sequences, which are complementary to the 3' end sequence of 16S rRNA, as in eubacterial translation initiation (Osada, Y., Saito, R., Tomita, M. Analysis of base-pairing potentials between 16S rRNA and 5' UTR for translation initiation in various procaryotes. Bioinformatics, in press). These two results combined lead us to conclude that these archaea probably use a hybrid mechanism; their ribosome scans mRNAs from the 5' to 3' direction and then 16S rRNA binds to the SD-like sequence of the 5' UTR.

5' Untranslated Regions↗
PubMed2000

Polypurine.polypyrimidine sequences in complete bacterial genomes: preference for polypurines in protein-coding regions.

The genomes of Methanococcus jannaschii, Mycoplasma genitalium, Haemophilus influenzae, Archaeoglobus fulgidus, Helicobacter pylori, Treponema pallidum, Borrelia burgdorferri, Rickettsia prowazekeii, Mycobacterium tuberculosis, Methanobacterium thermoautotrophicum, Synechocystis sp. PCC6803, Bacillus subtilis, Chlamydia trachomatis, Pyrococcus horikoshii, Aquifex aeolicus, Mycoplasma pneumoniae and Escherichia coli have been analysed for the presence of polypurine.polypyrimidine tracts, in order to understand their distribution in these genomes. We observed a variation in abundance of such sequences in these bacteria, with the archaeal genomes forming a high-abundance group and the canonical eubacteria forming a low-abundance group. The genomes of M. tuberculosis and A. aeolicus are unique among the organisms analysed here in the abnormal underrepresentation and overrepresentation of polypurine.polypyrimidine, respectively. We also observe a strand bias, i.e., a preferential occurrence of polypurines in coding strands. It varies widely among the bacteria, from the very high bias in M. jannaschii to the slightly inverse bias in the parasitic genomes of T. pallidum and C. trachomatis. The extent of strand bias, however, cannot be explained on the basis of the GC-content of the genome, use of all-purine codons or an excess in the amino acids that are encoded by such codons. The probable causes and effects of this phenomenon are discussed.

Bacteria↗