Biomedical subjects

K Nakasone

Publications and source records attributed to K Nakasone.

At least 19 recordsLinked to original sources

PubMed2001

Isolation of the rpoD gene encoding the principal sigma factor of the deep-sea piezophilic bacterium Shewanella violacea strain DSS12 and its overexpression in Escherichia coli.

The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively. Comparison with E. coli sigma70 and P. putida sigma70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S. violacea sigma70 factor was overexpressed in E. coli and successfully purified to near homogeneity.

Amino Acid Sequence↗

PubMed2001

Piezoresponse of the cyo-operon coding for quinol oxidase subunits in a deep-sea piezophilic bacterium, Shewanella violacea.

We have isolated the genes for quinol oxidase from a deep-sea piezophilic bacterium, Shewanella violacea. Analysis of the deduced amino acid sequences of the cyo subunits showed that this oxidase has high similarity to Escherichia coli bo-type quinol oxidase. Northern blot analysis showed that these genes are expressed at a high level when the bacterium is grown at elevated pressure. Upstream in the cyo-operon, a sigma54-binding motif and an octamer sequence unit were found, suggesting that these elements may play a role in regulation of expression of the cyo-operon in response to changes in pressure.

Base Sequence↗

PubMed2000

Isolation and piezoresponse of the rpoA gene encoding the RNA polymerase alpha subunit from the deep-sea piezophilic bacterium Shewanella violacea.

The rpoA gene encoding the alpha subunit of RNA polymerase from the deep-sea piezophilic bacterium Shewanella violacea DSS12 was cloned and sequenced. The rpoA gene was found to encode a polypeptide consisting of 329 amino acids with a molecular mass of 36238 Da. S. violacea alpha protein was expressed in a ts Escherichia coli mutant, to confirm whether the rpoA gene is functional. It complemented this mutation, indicating a chimeric RNA polymerase is assembled at the non-permissive temperature. Recombinant alpha protein was overexpressed using an expression plasmid harboring the rpoA gene and purified to near homogeneity. Primer extension analysis revealed that two transcriptional initiation sites are recognized by sigma(70) RNA polymerase. It also indicated that pressure response (piezoresponse) in the alpha operon occurred at the transcriptional level, suggesting some positive regulators may interact with the transcriptional apparatus and regulate the expression of the operon at different pressure conditions.

Amino Acid Sequence↗

PubMed2000

Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis.

The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18. 3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B. halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 sigma factors which belong to the extracytoplasmic function family, 10 are unique to B. halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.

ATP-Binding Cassette Transporters↗

PubMed2000

Glutamine synthetase gene expression at elevated hydrostatic pressure in a deep-sea piezophilic Shewanella violacea.

A glutamine synthetase gene (glnA) was isolated from a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12. A 7.5-kb SacI fragment containing the complete glnA gene was cloned and sequenced. The glnA gene was found to encode a protein consisting of 469 amino acid residues, showing 75.0% identity to the glutamine synthetase of Escherichia coli. Primer extension analyses revealed two transcription initiation sites in glnA and expression from each site was positively regulated by pressure. Putative promoters recognized by sigma(70) and sigma(54) were identified in the region upstream of glnA. An electrophoretic mobility shift assay demonstrated that S. violacea sigma(54) specifically binds to the promoter region of glnA, suggesting that sigma(54) may play an important role in pressure-regulated transcription in this piezophilic bacterium.

Base Sequence↗

PubMed2000

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea.

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.

Amino Acid Sequence↗

PubMed2000

Characterization and comparative study of the rrn operons of alkaliphilic Bacillus halodurans C-125.

The ribosomal RNA operons (rrn) of alkaliphilic Bacillus halodurans C-125 were characterized and compared with those of B. subtilis. We isolated clones containing rrn operons from a lambda phage library of the C-125 chromosome, and the complete nucleotide sequence of each was determined. Eight rrn operons were identified by PFGE analysis of the C-125 chromosome digested with I-CeuI. The transcriptional orientation of the rrn operons mapped on the chromosome by Southern hybridization analysis was the same as the direction of replication of the chromosome. These operons were designated as rrnA-H, starting from the oriC locus in clockwise rotation. Sequence and structural analyses of these operons suggested that six of the rrn operons in the C-125 chromosome, rrnA, rrnB, rrnC-rrnD, rrnE, and rrnH, correspond to rrnO, rrnA, rrnJ-rrnW, rrnI, and rrnD in B. subtilis, whereas the other rrn operons (rrnF and rrnG) were specifically observed in C-125. The rrn loci were positioned from 0 degrees to 90 degrees on the physical map, with the oriC locus assigned the position zero degrees. Two ORFs annotated as tnpA and ykfC, whose gene products are likely to act as transposases, were found downstream of these six operons. Comparative analysis of the 16S-23S and 23S-5S ITS (internally transcribed sequence) regions of B. halodurans C-125 and those of B. subtilis revealed that the ITS regions in C-125 were much longer than those in B. subtilis. There was no substantial difference in the length of potential promoter sequences in B. halodurans and B. subtilis.

Alkalies↗

PubMed2000

Phage conversion of exfoliative toxin A production in Staphylococcus aureus.

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (phiETA) that encodes ETA and determined the complete nucleotide sequence of the phiETA genome. phiETA has a head with a hexagonal outline and a non-contractile and flexible tail. The genome of phiETA is a circularly permuted linear double-stranded DNA, and the genome size is 43 081 bp. Sixty-six open reading frames (ORFs) were identified on the phiETA genome, including eta, which was found to be located very close to a putative attachment site (attP). phiETA converted ETA non-producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that phiETA or related phages are responsible for the acquisition of eta genes in S. aureus.

Animals↗

PubMed2000

Pressure regulation of soluble cytochromes c in a deep-Sea piezophilic bacterium, Shewanella violacea.

Two c-type cytochromes from the soluble fraction of a deep-sea moderately piezophilic bacterium, Shewanella violacea, were purified and characterized, and the genes coding for these cytochromes were cloned and sequenced. One of the cytochromes, designated cytochrome c(A), was found to have a molecular mass of approximately 8.3 kDa, and it contained one heme c per molecule. The other, designated cytochrome c(B), was found to have a molecular mass of approximately 23 kDa, and it contained two heme c molecules per protein molecule. The amount of cytochrome c(B) expressed in cells grown at high hydrostatic pressure (50 MPa) was less than that in cells grown at atmospheric pressure, whereas cytochrome c(A) was constitutively expressed under all pressure conditions examined. The results of Northern blotting analysis were consistent with the above-mentioned observations and suggested that the pressure regulation of cytochrome c(B) gene expression occurred at the transcriptional level. These results suggest that the components of the respiratory chain of moderately piezophilic S. violacea could be exchanged according to the growth pressure conditions.

Amino Acid Sequence↗

PubMed2000

Structural analysis of the ntrBC genes of deep-sea piezophilic Shewanella violacea.

The ntrBC genes coding for the bacterial signal-transducing protein NtrB and the bacterial enhancer-binding protein NtrC of deep-sea piezophilic Shewanella violacea were cloned and their nucleotide sequences were analyzed. The conserved regions of NtrB and those of NtrC are well conserved in the case of the ntrBC products of S. violacea.

Amino Acid Sequence↗

PubMed1999

Cloning of two cold shock genes, cspA and cspG, from the deep-sea psychrophilic bacterium Shewanella violacea strain DSS12.

We cloned and characterized two cold shock inducible genes from the deep-sea psychrophilic bacterium Shewanella violacea strain DSS12. The cloned genes, designated cspA and cspG, encode proteins each consisting of 70 amino acid residues which show 62 and 67% sequence identity with Escherichia coli CspA and CspG, respectively. AT-rich UP elements were found immediately upstream of the promoter region and the cspA and cspG mRNA contained unusually long 5' untranslated regions like that in the E. coli cspA, cspB, cspG and cspI genes. Following a temperature downshift to 4 degrees C or -1 degree C, the levels of cspA and cspG mRNA increased and the level of expression of cspG was greater than that of cspA both before and after cold shock. These results suggest that CspA and CspG may function as RNA chaperones, the mRNAs encoded by these two genes may be regulated post-transcriptionally and they may function as regulators of other cold shock inducible genes like in E. coli.

Amino Acid Sequence↗

PubMed1999

An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125.

Among alkaliphilic bacteria reported so far, Bacillus sp. C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically. A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25Mb. Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb. Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map. All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125. Several ORFs showing significant similarities to those of B. subtilis in the AscI linking clones were positioned on the physical map. The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region.

Bacillus↗

PubMed1999

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125.

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15-20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a lambda phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis.

Bacillus↗

PubMed1999

Genetic analysis of the chromosome of alkaliphilic Bacillus halodurans C-125.

Seventeen Sse8387I linking clones isolated from the chromosome of Bacillus halodurans C-125 for the purpose of constructing a physical map were sequenced and analyzed by comparison with the BSORF database and the nonredundant protein databank. The orientations of Sse8387I or AscI linking clones serving to join adjacent fragments were determined by southern blot analysis using specific DNA probes. One-third of the open reading frames (ORFs) identified in the Sse8387I linking clones showed no significant similarity to any protein so far reported. The ORFs showing significant similarities to those of Bacillus subtilis were mapped in the chromosome of strain C-125, and the locations of the putative genes on the map were not well conserved between B. halodurans C-125 and B. subtilis.

Bacillus↗

PubMed1999

Replication origin region of the chromosome of alkaliphilic Bacillus halodurans C-125.

An 18.5-kb DNA fragment containing the oriC region of the chromosome of the alkaliphilic Bacillus halodurans C-125 was obtained by PCR and sequenced. Sixteen open reading frames (ORFs) were identified in this region. A sequencing similarity search using the BSORF database found that ORF1 to 13 all had significant similarities to gene products of Bacillus subtilis. Three other ORFs (ORF14-16) of unknown function were positioned down-stream of gyrB instead of rrnO, which is found in the same region in the case of B. subtilis. The ORF organization from gidA to gyrA was the same as that of B. subtilis. The gene organization and the location of the DnaA-box region were also similar to those of the chromosomes of other bacteria, such as Escherichia coli and Pseudomonas putida. There were two DnaA-box clusters (Box-region C and R) with a consensus sequence TTATCCACA on both sides of the dnaA gene but another DnaA box cluster (Box-region L) which is found in the region between thdF and jag in B. subtilis was not found in the corresponding region in the case of alkaliphilic Bacillus halodurans C-125.

Alkalies↗

PubMed1999

Sequence analysis of a 32-kb region including the major ribosomal protein gene clusters from alkaliphilic Bacillus sp. strain C-125.

Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the alpha cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.

Bacillus↗

PubMed1998

Cloning and expression of the gene encoding RNA polymerase alpha subunit from alkaliphilic Bacillus sp. strain C-125.

The rpoA gene, encoding the alpha subunit of RNA polymerase, was isolated from alkaliphilic Bacillus sp. strain C-125 by the PCR method. A 3-kb HindIII fragment containing the complete rpoA gene was cloned and sequenced. The alpha subunit gene was found to encode a protein consisting of 314 amino acid residues with a molecular mass of 34,805 Da. Compared with the amino acid sequences of other known eubacterial RNA polymerase alpha subunits, the gene has 84% identity to that of B. subtilis, while showing 48% and 47% identity to that of Streptomyces coelicolor and Escherichia coli, respectively. Six conserved regions, which are observed in the case of other eubacteria, were found in the RNA polymerase alpha subunit of this strain. Five of them are located in the N-terminal domain involved in assembly of the core enzyme, while one is located in the C-terminal domain, which interacts with several transcriptional factors and a specific DNA element. By means of recombinant plasmids, a hexahistidine-tagged derivative of the RNA polymerase alpha subunit of strain C-125 and two deletion derivatives (C- and N-terminal domains) of this protein were overexpressed in E. coli cells and purified to near homogeneity.

Amino Acid Sequence↗

PubMed1998

Mechanisms of gene expression controlled by pressure in deep-sea microorganisms.

A pressure-regulated operon has been cloned and sequenced from deep-sea barophilic Shewanella strains. To understand pressure-regulated mechanisms of gene expression, a regulatory element upstream of the pressure-regulated operon from Shewanella sp. strain DSS12 was studied. Regions A and B were classified by sequence analysis. A unique octamer motif, AAGGTAAG, was found to be repeated in tandem 13 times in region B. An electrophoretic mobility shift assay demonstrated that a O54-like factor recognizes region A and other unknown factors recognize region B. Different shift patterns of the protein-DNA complexes were observed when extracts of cells cultured at 0.1 MPa or 50 MPa were incubated with a DNA probe specific for region B. These results indicate that the deep-sea strain DSS12 expresses different DNA-binding factors under different pressure conditions.

Bacterial Proteins↗