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 λ 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. Received: April 17, 1998 / Accepted: June 23, 1998     

The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp.

The pH homeostasis and the sodium/proton antiport system have been studied in the newly isolated alkalophilic Bacillus sp. strain N-6, which could grow on media in a pH range from 7 to 10, and in its nonalkalophilic mutant. After a quick shift in external pH from 8 to 10 by the addition of Na2CO3, the delta pH (inside acid) in the cells of strain N-6 was immediately established, and the pH homeostatic state was maintained for more than 20 min in an alkaline environment. However, under the same conditions, the pH homeostasis was not observed in the cells of nonalkalophilic mutant, and the cytoplasmic pH immediately rose to pH 10. On the other hand, the results of the rapid acidification from pH 9 to 7 showed that the internal pH was maintained as more basic than the external pH in a neutral medium in both strains. The Na+/H+ antiport system has been characterized by either the effect of Na+ on delta pH formation or 22Na+ efflux in Na+-loaded right-side-out membrane vesicles of strain N-6. Na+- or Li+-loaded vesicles exhibited a reversed delta pH (inside acid) after the addition of electron donors (ascorbate plus tetramethyl-p-phenylenediamine) at both pH 7 and 9, whereas choline-loaded vesicles generated delta pHs of the conventional orientation (inside alkaline). 22Na+ was actively extruded from 22Na+-loaded vesicles whose potential was negative at pH 7 and 9. The inclusion of carbonyl cyanide m-chlorophenylhydrazone inhibited 22Na+ efflux in the presence of electron donors. These results indicate that the Na+/H+ antiport system in this strain operates electrogenically over a range of external pHs from 7 to 10 and plays a role in pH homeostasis at the alkaline pH range. The pH homeostasis at neutral ph was studied in more detail. K+ -depleted cells showed no delta pH (acid out) in the neutral conditions in the absence of K+, whereas these cells generated a delta pH if K+ was present in the medium. This increase of internal pH was accompanied by K+ uptake from the medium. These results suggest that electrogenic K+ entry allows extrusion of H+ from cells by the primary proton pump at neutral pH.     

Tryptophan permease gene TAT2 confers high-pressure growth in Saccharomyces cerevisiae

Hydrostatic pressure in the range of 15 to 25 MPa was found to cause arrest of the cell cycle in G(1) phase in an exponentially growing culture of Saccharomyces cerevisiae, whereas a pressure of 50 MPa did not. We found that a plasmid carrying the TAT2 gene, which encodes a high-affinity tryptophan permease, enabled the cells to grow under conditions of pressure in the range of 15 to 25 MPa. Additionally, cells expressing the Tat2 protein at high levels became endowed with the ability to grow under low-temperature conditions at 10 or 15 degrees C as well as at high pressure. Hydrostatic pressure significantly inhibited tryptophan uptake into the cells, and the Tat2 protein level was down-regulated by high pressure. The activation volume associated with tryptophan uptake was found to be a large positive value, 46.2 +/- 3.85 ml/mol, indicating that there was a net volume increase in a rate-limiting step in tryptophan import. The results showing cell cycle arrest in G(1) phase and down-regulation of the Tat2 protein seem to be similar to those observed upon treatment of cells with the immunosuppressive drug rapamycin. Although rapamycin treatment elicited the rapid dephosphorylation of Npr1 and induction of Gap1 expression, hydrostatic pressure did not affect the phosphorylation state of Npr1 and it decreased the level of Gap1 protein, suggesting that the pressure-sensing pathway may be independent of Npr1 function. Here we describe high-pressure sensing in yeast in comparison with the TOR-signaling pathway and discuss an important factor involved in adaptation of organisms to high-pressure environments.     

Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.     

Indoor environmental study and post occupancy evaluation in an office environment

1. 1. The objective of this paper is to investigate the indoor environment from the viewpoint of interaction between physical environment and the human responses. The field survey has been conducted over 1 year.

2. 2. A continuous measurement has been carried out for 1 week and distribution of variables have been measured for 1 day.

3. 3. The attitude of workers was investigated by a questionnaire.

4. 4. As the result, average luminance represented more than 1000 lx in the new building, in contrast with less tha 300 lx in the existing building.

5. 5. There was a significant difference of the occupants' response to the light environment between the two buildings.

6. 6. Measured thermal conditions are on the edge of the ASHRAE comfort envelope in summer, and in the neighborhood of the lower dry limit of the envelope in spring.

7. 7. The occupants' evaluations were remarkably changed before and after the moving. The office environment is better than that of the factory.

Mitochondrials complex I activity is reduced in latent adriamycin-induced cardiomyopathy of rat

We previously reported on the use of enzymatic analysis to impair fatty acid metabolism followed by reduced myocardial energy content, leading to severe heart failure in adriamycin (ADR)-treated rats. The aim of this study is to investigate whether impaired myocardial energy metabolism can also be detected by other methods; i.e. measuring mitochondrial complex I activity and myocardial ¹²⁵I-15-(p-iodophenyl)-3-(R,S)- methylpentadecanoic acid (BMIPP) accumulation in ADR-treated rats. Eight-week-old male Sprague-Dawley rats received 6 intraperitoneal injections of ADR (total 15 mg/kg: group ADR) or saline (control group) over 2 weeks. Left ventricular (LV) ejection fraction was assessed using echocardiography at 3- and 6-weeks after ADR injection (3 weeks and 6 weeks, respectively). Myocardial fatty acid utilization was assessed at 3 weeks and 6 weeks. The myocardial counts of BMIPP were measured after intravenous BMIPP (370 kBq) injection, and ¹²⁵I counts were measured to calculate the uptake ratio. The enzymatic activity of complex I was assessed by monitoring the oxidation of nicotinamide-adenine-dinucleotide-disodium-salt (NADH). In rats treated with ADR, significant decrease in LV ejection fraction was observed only at 6 weeks compared to control (72.5 vs. 84.5%, p < 0.01rpar;. LV ejection fraction at 3 weeks was identical between group ADR and control (81.8 vs. 84.4%). However, at 3 weeks, complex I activity was already reduced significantly in group ADR as compared to control group (p = 0.03), but the reduction in BMIPP accumulation was not (p = 0.15). Our data indicated that reduced complex I activity in a phenomenon occurred in early phase of ADR-induced cardiomyopathy, and it might play an important role in the progression of ADR-induced heart failure.     

Structural relationships of the three stimulatory factors of RNA polymerase II from Ehrlich ascites tumor cells

The structural relationships of S-II, S-II', and S-I(b) stimulatory proteins of RNA polymerase II purified from Ehrlich ascites tumor cells were investigated. From analysis of the amino acid compositions and tryptic peptide maps of these proteins labeled with radioiodinated Bolton-Hunter reagent, it was concluded that S-I(b) is a part of S-II located at either the amino- or carboxyl-terminal and that only this region mainly contains radioiodinatable amino acid residues when labeled using 125I. On chymotryptic digestion, S-II was cleaved to 21- and 18-kDa fragments in the presence of DNA. The 21-kDa fragment was found to be sufficient for stimulation of RNA polymerase II. It was suggested that S-II' is formed by phosphorylation of S-II in the domain containing the 18-kDa fragment.     

Isolation and characterization of toluene-sensitive mutants from Pseudomonas putida IH-2000

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-2000, the first known toluene-tolerant isolate, by Tn5 transposon mutagenesis. These mutants were unable to grow in the presence of toluene (log P_ow 2.8) but they could grow in medium overlaid with organic solvents having a log P_ow value higher than that of toluene such as p-xylene (log P_ow 3.1), cyclohexane (log P_ow 3.4) and n-hexane (log P_ow 3.9). The Tn5 transposable element knocked out a cyoB-like gene in one mutant and a cyoC-like gene in the other mutant. Seven open reading frames were found in a 5.5-kb region containing the cyoB- and cyoC-like genes of strain IH-2000. ORFs 3–7 showed significant identity to the cyoABCDE gene products of Escherichia coli, but ORFs 1 and 2 showed no significant homology to any protein reported so far. The growth patterns of the Tn5 mutants with the inactivated cyo-like gene were similar to that of the wild-type strain in the absence of organic solvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for toluene tolerance, although its role is still unclear.     

Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94

A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T). The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 degrees C. The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).     

Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.