Search PubMed⌕ Search

Biomedical subjects

K Horikoshi

Publications and source records attributed to K Horikoshi.

At least 181 records · Page 10Linked to original sources
PubMed1989

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.

Bacillus↗
PubMed1988

Molecular cloning and nucleotide sequence of the cyclomaltodextrin glucanotransferase gene from the alkalophilic Bacillus sp. strain no. 38-2.

The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from the alkalophilic Bacillus sp. strain no. 38-2 was cloned in Escherichia coli using pBR322. A plasmid, pCS8, was isolated from a transformant producing CGTase and the cloned CGTase gene was found to be in a 5.3 kb DNA fragment. The nucleotide sequence of a 2.5 kb segment encoding the CGTase was determined. This segment showed an open reading frame which would encode a polypeptide of 712 amino acids. The pCS8 CGTase had the same enzymic properties as those of the extracellular CGTase produced by the alkalophilic Bacillus sp. strain no. 38-2. The nucleotide and amino acid sequences of this CGTase gene and gene product, respectively, have strong homology with those of the Bacillus macerans CGTase.

Amino Acid Sequence↗
PubMed1988

Isolation of Bacteria Which Can Grow at Both High pH and Low Temperature.

Fifty bacterial strains able to grow at pH 10 and 0 degrees C were isolated from soils, and growth characteristics of three selected strains were investigated. Strain 207, which showed the best growth rate of all the isolates at the conditions described above, could grow at a temperature of -5 to 39 degrees C at pH 8.5. The optimum pH for this strain changed from 9.5 at 10 degrees C to 9.0 at 20 degrees C.

Journal Article↗
PubMed1987

Bioenergetic properties of alkalophilic Bacillus sp. strain C-59 on an alkaline medium containing K2CO3.

Alkalophilic Bacillus sp. strain C-59 could grow well on an alkaline medium containing K2CO3, as well as Na2CO3, but did not grow on K+-depleted medium. Right-side-out membrane vesicles, energized in the absence of Na+, however, could not take up [14C]methylamine actively, while vesicles equilibrated with 10 mM NaCl actively took up [14C]methylamine. The uptake of [14C]serine was also stimulated by the addition of Na+, and the imposition of a sodium gradient caused transient uptake. These results indicated that an Na+/H+ antiporter was involved in pH homeostasis and generation of an electrochemical sodium gradient in strain C-59 even though a growth requirement for Na+ was not evident. The efflux of 22Na+ from 22Na+-loaded vesicles was more rapid at pH 9.5 than at pH 7 in the presence of an electron donor. On the other hand, vesicles at pH 7 showed more rapid efflux than at pH 9.5 when the antiporter was energized by a valinomycin-mediated K+ diffusion potential (inside negative).

Bacillus↗
PubMed1986

Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. strain 1139.

The cellulase gene from the alkalophilic Bacillus sp. strain 1139 was cloned in Escherichia coli using pBR322. Plasmid pFK1 was isolated from transformants producing cellulase, and the cloned cellulase gene was found to be in a 4 X 6 kb HindIII fragment. The cellulase gene was subcloned in a functional state on a 2 X 9 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame encoding 800 amino acids. The pFK1-encoded cellulase had the same enzymic properties as the extracellular cellulase produced by the alkalophilic Bacillus sp. strain 1139, but its Mr was slightly higher.

Amino Acid Sequence↗
PubMed1986

Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9.

Most of the cloned penicillinase from alkalophilic Bacillus sp. strain 170 and alkaline phosphatase were released into the culture medium by Escherichia coli strains bearing plasmid pEAP1 or pEAP2 (T. Kudo, C. Kato, and K. Horikoshi, J. Bacteriol. 156:949-951, 1983). We analyzed the basis for excretion of periplasmic enzymes in the cells bearing these plasmids. Several experiments such as subcloning, insertion of a chloramphenicol acetyltransferase cartridge, and DNA sequencing were done. A dormant kil gene in plasmid pMB9 was expressed by a promoter of the inserted DNA fragment of alkalophilic Bacillus sp. strain 170, and as a result, the outer membrane of E. coli became permeable, allowing the proteins to be excreted without cell lysis.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequences of two cellulase genes from alkalophilic Bacillus sp. strain N-4 and their strong homology.

Two genes for cellulases of alkalophilic Bacillus sp. strain N-4 (ATCC 21833) have been sequenced. From the DNA sequences the cellulases encoded in the plasmids pNK1 and pNK2 consist of 488 and 409 amino acids, respectively. The DNA and protein sequences of the pNK1-encoded cellulase are related to those of the pNK2-encoded cellulase. The pNK2-encoded cellulase lacks the direct repeat sequence of a stretch of 60 amino acids near the C-terminal end of the pNK1-encoded cellulase. The duplication of the cellulase genes and the formation of the direct repeat in the pNK1-encoded cellulase occurred at almost the same time.

Amino Acid Sequence↗
PubMed1985

Molecular cloning and expression of a xylanase gene of alkalophilic Aeromonas sp. no. 212 in Escherichia coli.

A gene coding for a xylanase activity of alkalophilic Aeromonas sp. no. 212 (ATCC 31085) was cloned in Escherichia coli HB101 with pBR322. Plasmid pAX1 was isolated from transformants producing xylanase, and the xylanase gene was located in a 6.0 kb Hind III fragment. The pAX1-encoded xylanase activity in E. coli HB101 was about 80 times higher than that of xylanase L in alkalophilic Aeromonas sp. no. 212. About 40% of the enzyme activity was observed in the periplasmic space of E. coli HB101. The pAX1-encoded xylanase had the same enzymic properties as those of xylanase L produced by alkalophilic Aeromonas sp. no. 212, but its molecular weight was lower (135 000 vs 145 000, as estimated by SDS polyacrylamide gel electrophoresis).

Aeromonas↗
PubMed1985

Nucleotide sequence of the beta-lactamase gene of alkalophilic Bacillus sp. strain 170.

A gene for the beta-lactamase from alkalophilic Bacillus sp. strain 170 was cloned in a functional state on a 1.0 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame of 257 amino acids, which represents the beta-lactamase precursor protein. It is considered that the signal peptide consisted of 30 amino acids including 12 hydrophobic amino acids.

Amino Acid Sequence↗
PubMed1985

Cloning of a developmentally regulated element from alkalophilic Bacillus subtilis DNA.

An alkalophilic Bacillus DNA bank cloned in an expression probe plasmid, pGR71, was screened for the presence of developmentally regulated genetic elements. A 508-base pair HindIII fragment isolated from this bank in plasmid pGR71-5 expressed plasmid-encoded chloramphenicol resistance only during the sporulation phase of a Bacillus subtilis host grown on Schaeffer medium. This developmentally regulated expression was altered in spo0E and spo0H mutants which had very low levels of chloramphenicol acetyltransferase activity relative to the wild type or other spo0 mutants. We determined the nucleotide sequence of the entire 508-base pair fragment and located the site of regulated transcription initiation by high-resolution S1 nuclease mapping of the in vivo transcript. The deduced promoter sequences upstream from this start site were 5'C-G-A-A-T-C-A-T-G-A3' at -10 and 5' A-G-G-A-A-T-C3' at -35. This transcript was not detected in spo0E or spo0H mutants, indicating that the products of these genes control developmentally regulated chloramphenicol acetyltransferase expression at the level of transcription.

Acetyltransferases↗
PubMed1984

Production of mitogen-free immune interferon and T-cell growth factor by human peripheral blood lymphocytes induced with biotin-labeled staphylococcal enterotoxin A.

A method for the facile removal of mitogens or inducers of lymphokine production from cell culture medium of stimulated cells is described. The technique is based on the covalent attachment of biotin to mitogen or inducer and the removal of the biotinylated products from stimulated cell culture medium using immobilized avidin. Using this procedure, biotin-labeled staphylococcal enterotoxin A (SEA-B) was shown not to differ significantly from unmodified SEA in its capacity to stimulate mitogenesis and induce production of immune interferon (IFN) and T-cell growth factor (TCGF) in cultures of human mononuclear cells from peripheral blood. SEA-B was also shown not to differ from SEA in its binding to SEA antibodies. Results of mitogenicity studies and competitive radioimmune assay (RIA) measurements indicate that SEA-B is essentially completely removed from stimulated cell culture medium by absorption with avidin coupled to Sepharose 4B.

Biotin↗
PubMed1984

Cloning of leucine genes of alkalophilic Bacillus No. 221 in E. coli and B. subtilis.

The leucine genes of alkalophilic Bacillus No. 221 were cloned into the HindIII sites of pBR322 and pGR71, and recombinant plasmids pHK101 and pHK111 were constructed. The cloned 7.2 kb DNA consisting of HindIII fragments of 4.0 kb and 3.2 kb could substitute for leucine genes (alpha-isopropylmalate (alpha-IPM) synthetase gene, beta-IPM dehydrogenase gene, and alpha-IPM isomerase gene) of E. coli and B. subtilis, but not ilvB gene of B. subtilis. The 4.0 kb HindIII fragment could substitute for alpha-IPM synthetase gene of E. coli, and the 3.2 kb HindIII fragment could substitute for alpha-IPM isomerase gene of E. coli. Both 4.0 kb and 3.2 kb fragments are necessary to substitute for beta-IPM dehydrogenase gene. The expression of beta-IPM dehydrogenase gene of alkalophilic Bacillus No. 221 was repressed by the addition of leucine in the culture medium of B. subtilis carrying the plasmid pHK111. These results indicated that the 7.2 kb fragment contains the leucine gene cluster and its regulatory region.

3-Isopropylmalate Dehydrogenase↗
PubMed1984

Composition of the peptidoglycan of alkalophilic Bacillus spp.

Peptidoglycans of 10 alkalophilic Bacillus strains were isolated as trichloroacetic acid-insoluble materials from cell walls prepared by treatment with sodium dodecyl sulfate, disruption with a sonic oscillator, and trypsin digestion. Major constituents detected commonly in hydrolysates of the peptidoglycans were glucosamine, muramic acid, D- and L-alanine, D-glutamic acid, meso-diaminopimelic acid, and acetic acid. Ammonia derived from amide was found in a portion of the hydrolysates. The composition of peptidoglycan was not changed whether the strain was cultured at pH 7 or 10. All the peptidoglycan examined was of the A1 gamma type of peptidoglycan found in most strains of the genus Bacillus.

Amino Acids↗
PubMed1984

Molecular cloning and expression of cellulase genes of alkalophilic Bacillus sp. strain N-4 in Escherichia coli.

The genes for cellulases of alkalophilic Bacillus sp. strain N-4 were cloned in Escherichia coli with pBR322. Plasmids pNK1 and pNK2 were isolated from the transformants producing carboxymethyl cellulase, and the carboxymethyl cellulase genes cloned were in 2.0- and 2.8-kilobase-pair HindIII fragments, respectively. On the DNA level, the pNK1 fragment had a different restriction map from that of the pNK2 fragment, but the genomic hybridization experiments showed partial homology among these fragments. A total of 74 and 34% of the enzyme activities were observed in the periplasmic space of E. coli carrying the plasmids pNK1 and pNK2 , respectively. The carboxymethyl cellulase thus produced had broad pH activity curves (pH of 5 to 10.9) and was stable up to 75 degrees C.

Bacillus↗
PubMed1983

Oxidation of acetylpolyamines by extracellular polyamine oxidase produced by Penicillium sp. No. PO-1.

The oxidation of acetylpolyamines by an extracellular polyamine oxidase of Penicillium sp. No. PO-1 was investigated. The optimal pH value for oxidation of acetylpolyamines was 6.0. The purified enzyme oxidized spermidine, spermine, N1-acetylspermidine, N8-acetylspermidine, N1,8-diacetylspermidine, N1-acetylspermine and N1,12-diacetylspermine. The relative velocities for oxidation of acetylpolyamines were lower than those of spermidine and spermine. The Km values for oxidation of acetylpolyamines were higher than those of spermidine and spermine. The enzyme split N1-acetylspermidine and N8-acetylspermidine at the same position of the linkage as in spermidine oxidation. N1-Acetylspermine was changed to N1-acetylspermidine. This oxidation mechanism was different from that of rat liver polyamine oxidase. N1-Acetylspermine inhibited the oxidation of spermine. Putrescine, N8-acetylspermidine and N1,12-diacetylspermine also inhibited the N1-acetylspermidine oxidation by the enzyme.

Animals↗