A total of 75 strains (including 5 reference strains) of Bacillus amyloliquefaciens, B. cereus, B. circulans, B. licheniformis, B. megaterium, B. pumilus, B. sphaericus, B. subtilis , and B. thuringiensis and 36 species-unidentified... more
A total of 75 strains (including 5 reference strains) of Bacillus amyloliquefaciens, B. cereus, B. circulans, B. licheniformis, B. megaterium, B. pumilus, B. sphaericus, B. subtilis , and B. thuringiensis and 36 species-unidentified Bacillus strains were surveyed for plasmids by cesium chloride-ethidium bromide equilibrium centrifugation of cell lysates in a study of antibiotic resistance in host cells. Of the 111 strains, 13 (including 3 reference strains) were found to harbor plasmids, and 5 of the 13 showed antibiotic resistance. This antibiotic resistance appeared not to be due to the plasmids, however, because the trait was not cured by cultivation of cells in nutrient medium containing ethidium bromide (1 μg/ml), sodium dodecyl sulfate (0.2 μg/ml), or novobiocin (1 μg/ml), except in one strain, in which kanamycin and streptomycin resistances were cured by novobiocin. One strain of B. amyloliquefaciens , S294, was found to harbor a plasmid, pFTB14, which differed from the plasm...
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PCR mutagenesis of a 0.9-kbp fragment, containing a repressor gene, traR, and its target promoter, Ptra, from Streptomyces nigrifaciens plasmid pSN22, produced Streptomyces lividans clones with temperature-inducible Ptra expression. Using... more
PCR mutagenesis of a 0.9-kbp fragment, containing a repressor gene, traR, and its target promoter, Ptra, from Streptomyces nigrifaciens plasmid pSN22, produced Streptomyces lividans clones with temperature-inducible Ptra expression. Using the promoterless gene for the thermostable Thermus flavus malate dehydrogenase as an indicator, an induction of enzyme activity of as much as was observed in a temperature shift from 28 to 37 degrees C. Temperature downshift reestablished repression of Ptra, making these promoter cassettes very attractive for the temporally regulated expression of cloned genes in Streptomyces spp.
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Growth of some xylose fermenting yeasts, Candida shehatae, Pichia stipitis CBS5773, fusant F101 and fusant F198, was completely inhibited in xylose medium added with 0.5% v/v acetic acid which caused the reduction of pH to 4.1. Only one... more
Growth of some xylose fermenting yeasts, Candida shehatae, Pichia stipitis CBS5773, fusant F101 and fusant F198, was completely inhibited in xylose medium added with 0.5% v/v acetic acid which caused the reduction of pH to 4.1. Only one xylose fermenting strain, Pachysolen tannophilus NRRL-Y2460, showed relatively low growth and ethanol fermentation. However, in the medium added with 1.0% v/v acetic acid (pH 3.7) all of these strains were completely inhibited. When the medium was adjusted by hydrochloric acid to pH 4.1 and 3.7, all xylose fermenting strains showed almost the same growth as in the medium without pH adjustment (pH 6.2). In glucose medium added with 0.5% v/v acetic acid, various strains of Saccharomyces cerevisiae, M30, Sc90, N1, G/3, G/5, G/2, TJ3 and SH1089, grew with lower specific growth rate and provided lower maximal cell concentration rate than in medium without adding acetic acid (pH 6.2). All strains, except N1, produced slightly higher maximal ethanol concent...
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Research Interests: Microbiology and Biology
The use of a lysine-overproducing strain ofLactobacillus plantarumin food or feed fermentations may lead to the production of lysine-rich products. The availability of functional genes and information on the regulation of lysine... more
The use of a lysine-overproducing strain ofLactobacillus plantarumin food or feed fermentations may lead to the production of lysine-rich products. The availability of functional genes and information on the regulation of lysine biosynthesis are required to develop a lysine-overproducing strain. The genome sequence ofL. plantarumrevealed putative lysine biosynthetic genes, some of which may produce isozymes. This study examined the functionality of the genes and the regulation of the first four enzymes of lysine biosynthesis, together with homoserine dehydrogenase, inL. plantarum. The genes were expressed inEscherichia coli, and the regulation of the enzymes was studied in cell extracts of both recombinantE. coliandL. plantarum. Among seven lysine biosynthetic genes studied (aspartokinase genes,thrA1andthrA2; aspartate semialdehyde dehydrogenase genes,asd1andasd2; dihydrodipicolinate synthase genes,dapA1anddapA2; and the dihydrodipicolinate reductase gene,dapB) plus two homoserine d...
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Research Interests: Biology and Japonica Rice
... Fig. 5. Modification of the pSN22 traB gene in order to add an amino-terminal c-Myc epitope to the TraB protein. Following the procedure of Johannes et al. ... Note that wild-type TraB and TraBmB are expressed from a promoter upstream... more
... Fig. 5. Modification of the pSN22 traB gene in order to add an amino-terminal c-Myc epitope to the TraB protein. Following the procedure of Johannes et al. ... Note that wild-type TraB and TraBmB are expressed from a promoter upstream of traA (Kataoka et al., 1991b). ...
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Four halotolerant yeast strains, M21T, M34-1, HS054 and D41, were isolated from various foods in South-East Asia. These isolates were most closely related to Pichia anomala, with which each strain had from zero to two differences in the... more
Four halotolerant yeast strains, M21T, M34-1, HS054 and D41, were isolated from various foods in South-East Asia. These isolates were most closely related to Pichia anomala, with which each strain had from zero to two differences in the 26S rDNA D1/D2 domain nucleotide sequence; for this reason, they were thought to be the same as, or sister species of, P. anomala. Of the four yeast isolates, only one strain, M21T, had an 18S rDNA sequence that differed from those of P. anomala IFO 10213T and the other three isolates, having 20 substitutions and two gaps. Strain M21T showed lower cation (Li+) tolerance (⩽0·3 M LiCl) than P. anomala IFO 10213T or the other three strains (⩽0·5 M LiCl). Furthermore, the DNA–DNA hybridization data indicated that M21T was clearly distinct from P. anomala IFO 10213T and the other three isolates. The ability of strain M21T to assimilate d-arabinose distinguished it from P. anomala IFO 10213T and the other three isolates; it also differed in that it was abl...
Research Interests: Evolutionary Biology, Microbiology, Medical Microbiology, Biology, Lithium, and 14 moreMedicine, Food Microbiology, Myanmar, Phylogeny, Nucleic acid hybridization, Fungal Spores, Temperature, ribosomal RNA, Antifungal Agents, CARBOHYDRATES, Ribosomal DNA, Molecular Sequence Data, Ubiquinone, and Anomala
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Research Interests: Biochemistry, Molecular Biology, Biology, Polysaccharides, Medicine, and 15 moreProtein Engineering, Mice, Animals, Larva, Protein Conformation, SIGNAL PEPTIDE, Molecular weight, Protein isoforms, Species Specificity, Structure activity Relationship, Recombinant Proteins, Biochemistry and cell biology, Bombyx mori, sf, and Medical biochemistry and metabolomics
The rolling circle (RC) mechanism of DNA replication generating single-stranded DNA (ssDNA) intermediates is common in various high-copy circular plasmids in Streptomyces, and the ssDNA released after leading strand synthesis is converted... more
The rolling circle (RC) mechanism of DNA replication generating single-stranded DNA (ssDNA) intermediates is common in various high-copy circular plasmids in Streptomyces, and the ssDNA released after leading strand synthesis is converted to its double-stranded form (dsDNA) by the host proteins. The in vivo and in vitro lagging strand syntheses from ssDNA replicative intermediates of RC plasmid pSN22 in Streptomyces lividans was characterized. The presence or absence of the single-strand origin (sso), the replication initiation site of lagging strand synthesis, did not significantly affect the copy numbers of pSN22 derivatives. In vivo lagging strand synthesis was not affected by the rifampicin inhibition of S. lividans RNA polymerase. Likewise, in vitro lagging strand synthesis using cell-free extracts revealed sso-independent, rifampicin-resistant lagging strand synthesis in S. lividans. Although all four dNTPs are usually required for the initiation of such synthesis, the presence of only one NTP was sufficient to carry outlagging strand synthesis in vitro. Interestingly, the cell-free extract of exponential-phase cells required less ATP than that of stationary-phase cells. These results reveal a predominant RNA polymerase-independent priming system in S. lividans that may be a result of the stabilization of RC plasmids lacking sso in S. lividans.
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pSN22 is an 11-kb multicopy plasmid from Streptomyces nigrifaciens which is being studied in Streptomyces lividans. A segment of about 7 kb of pSN22 contains five genes involved in conjugation. Three of them, traA, traB, and traR, are... more
pSN22 is an 11-kb multicopy plasmid from Streptomyces nigrifaciens which is being studied in Streptomyces lividans. A segment of about 7 kb of pSN22 contains five genes involved in conjugation. Three of them, traA, traB, and traR, are essential for plasmid transfer and for the mobilization of chromosomal markers (fertility), while the remaining two genes, spdA and spdB, merely enhance the efficiency of plasmid transfer, resulting in the formation of larger pocks. In vitro promoter-probing experiments identified a 550-bp BglII-SmaI DNA fragment with promoter activity in both orientations; Northern (RNA blot) hybridization identified corresponding divergent transcripts of 1 and 5.2 kb for traR and the traA-traB-spdB operon, respectively. The traR gene product repressed its own transcription and also the transcription of the traA-traB-spdB operon. Plasmids containing a functional traB gene could not "survive" without traR being present in the same cell either in cis or in tra...
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ABSTRACT
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Expression of the tra operon, essential for conjugative transfer of the 11-kb Streptomyces nigrifaciens plasmid pSN22, is negatively regulated by traR, which is located upstream of the tra operon and transcribed in the opposite... more
Expression of the tra operon, essential for conjugative transfer of the 11-kb Streptomyces nigrifaciens plasmid pSN22, is negatively regulated by traR, which is located upstream of the tra operon and transcribed in the opposite orientation. The transcriptional start points for the tra and traR mRNAs were determined by primer extension; they are 72 bp apart and have identical -10 promoter sequences. The TraR protein was overexpressed in Escherichia coli and used for gel retardation and DNase I protection experiments. It bound specifically to the bidirectional tra-traR promoter region and protected four DNA regions, each of which contains a similar 12-bp sequence. The binding was strongest to the region downstream of the tra promoter, probably ensuring that expression of the potentially lethal traB gene is turned off before traR. The efficiency of intramycelial plasmid transfer was decreased by the mutation at the downstream region.
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The envelope protein of dengue virus is the major protein involved in host cell receptor binding for viral entry and induction of immunity. A gene fragment encoding domain III of the dengue 2 envelope protein (D2EIII, amino acids 298–400)... more
The envelope protein of dengue virus is the major protein involved in host cell receptor binding for viral entry and induction of immunity. A gene fragment encoding domain III of the dengue 2 envelope protein (D2EIII, amino acids 298–400) was successfully expressed in Nicotinana benthamiana plant using a tobacco mosaic virus (TMV)-based transient expression system. The N-terminal 5′ untranslated region-omega
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The taxonomic relationships among 56 strains of 16 species of the genus Bacillus were studied by deoxyribonucleic acid (DNA) -DNA hybridization. In general, no significant DNA homology was detected between two strains of different... more
The taxonomic relationships among 56 strains of 16 species of the genus Bacillus were studied by deoxyribonucleic acid (DNA) -DNA hybridization. In general, no significant DNA homology was detected between two strains of different species, except for a group of species consisting of B. subtilis, B. amyloliquefaciens, B. lichenifonis, and B. pumilus and for another group of species including B.
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Partial sequences of farnesyl diphosphate (FPP) synthase genes derived from the Rhodobacter-Rhodovulum group and from the Rhodopseudomonas palustris-Bradyrhizobium japonicum group of the alpha-Proteobacteria were subjected to phylogenetic... more
Partial sequences of farnesyl diphosphate (FPP) synthase genes derived from the Rhodobacter-Rhodovulum group and from the Rhodopseudomonas palustris-Bradyrhizobium japonicum group of the alpha-Proteobacteria were subjected to phylogenetic analysis to investigate the relationships of phototrophic and non-phototrophic bacteria in the alpha-Proteobacteria . The four Rhodovulum species formed a monophyletic group within the Rhodobacter cluster, and Agrobacterium ferrugineum IAM 12616(T) intermingled with the Rhodobacter species. This topology is in good agreement with the 16S rRNA phylogeny, although the FPP synthase gene was more divergent than the 16S rRNA. On the other hand, strains of the phototrophic Rps. palustris formed a cluster far from that of the non-phototrophic Bradyrhizobium japonicum strains. Moreover, Rps. palustris strains were differentiated from the nodule-forming B. japonicum, Mezorhizobium loti MAFF 303099 and Sinorhizobium sp. NGR 234 in the FPP synthase phylogeny....
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To investigate the frequency of heterogeneity among the multiple 16S rRNA genes within a single microorganism, we determined directly the 120-bp nucleotide sequences containing the hypervariable alpha region of the 16S rRNA gene from 475... more
To investigate the frequency of heterogeneity among the multiple 16S rRNA genes within a single microorganism, we determined directly the 120-bp nucleotide sequences containing the hypervariable alpha region of the 16S rRNA gene from 475 Streptomyces strains. Display of the direct sequencing patterns revealed the existence of 136 heterogeneous loci among a total of 33 strains. The heterogeneous loci were detected only in the stem region designated helix 10. All of the substitutions conserved the relevant secondary structure. The 33 strains were divided into two groups: one group, including 22 strains, had less than two heterogeneous bases; the other group, including 11 strains, had five or more heterogeneous bases. The two groups were different in their combinations of heterogeneous bases. The former mainly contained transitional substitutions, and the latter was mainly composed of transversional substitutions, suggesting that at least two mechanisms, possibly misincorporation durin...
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ABSTRACT Four bacterial strains were isolated from palm brown sugar and ragi collected in Bali and Yogyakarta, Indonesia, by an enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences... more
ABSTRACT Four bacterial strains were isolated from palm brown sugar and ragi collected in Bali and Yogyakarta, Indonesia, by an enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the four isolates constituted a cluster separate from the genera Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter and Asaia with a high bootstrap value in a phylogenetic tree. The isolates had high values of DNA-DNA similarity (78-100%) between one another and low values of the similarity (7-25%) to the type strains of Acetobacter aceti, Gluconobacter oxydans, Gluconacetobacter liquefaciens and Asaia bogorensis. The DNA base composition of the isolates ranged from 56.8 to 57.2 mol% G+C with a range of 0-4 mol%. The major quinone was Q-10. The isolates oxidized acetate and lactate to carbon dioxide and water, but the activity was weak, as with strains of Asaia bogorensis. The isolates differed from Asaia bogorensis strains in phenotypic characteristics. The name Kozakia baliensis gen. nov., sp. nov., is proposed for the four isolates. Strain Yo-3T (= NRIC 0488T = JCM 11301T = IFO 16664T = DSM 14400T) was isolated from palm brown sugar collected in Bali, Indonesia, and was designated as the type strain.
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The lysine biosynthetic genes asd, dapA, and dapB, encoding aspartate semialdehyde dehydrogenase (ASADH), dihydrodipicolinate synthase (DHPS), and dihydrodipicolinate reductase (DHPR), respectively, have been cloned from Lactobacillus... more
The lysine biosynthetic genes asd, dapA, and dapB, encoding aspartate semialdehyde dehydrogenase (ASADH), dihydrodipicolinate synthase (DHPS), and dihydrodipicolinate reductase (DHPR), respectively, have been cloned from Lactobacillus plantarum IAM 12477 by heterologous ...
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Forty-six strains of acetic acid bacteria newly isolated from flowers, fruits, and fermented foods collected in Indonesia were taxonomically studied. They were Gram-negative rods, produced acetic acid from ethanol, oxidized acetate and... more
Forty-six strains of acetic acid bacteria newly isolated from flowers, fruits, and fermented foods collected in Indonesia were taxonomically studied. They were Gram-negative rods, produced acetic acid from ethanol, oxidized acetate and lactate to CO(2) and H(2)O, and had Q-9 as the major ubiquinone system. On the basis of DNA-DNA similarity, all strains studied, including type strains and reference strains of the genus Acetobacter, were separated into eleven groups (Groups I to XI). Of the 46 isolates, two isolates were included in Group II and identified as Acetobacter pasteurianus, five in Group IV as A. orleanensis, 16 in Group V as A. lovaniensis, five in Group VII as A. indonesiensis, and three in Group VIII as A. tropicalis. The remaining 15 isolates constituted three new groups based on DNA-DNA similarity; four isolates were included in Group IX, two in Group X, and nine in Group XI. No isolates were identified as A. aceti (Group I), A. peroxydans (Group III), and A. estunensis (Group VI). Phylogenetic analysis based on 16S rDNA sequences of representative strains of the Groups indicated belonging to the strains of the genus Acetobacter. On the basis of DNA base composition, DNA-DNA similarity, and 16S rDNA sequences, three new species of the genus Acetobacter are proposed: Acetobacter syzygii sp. nov. for Group IX, Acetobacter cibinongensis sp. nov. for Group X, and Acetobacter orientalis sp. nov. for Group XI. The distribution of Acetobacter strains in Indonesia is discussed in light of isolation sources.
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Thirty-one Acetobacter strains obtained from culture collections and 45 Acetobacter strains isolated from Indonesian sources were investigated for their phenotypic characteristics, ubiquinone systems, DNA base compositions, and levels of... more
Thirty-one Acetobacter strains obtained from culture collections and 45 Acetobacter strains isolated from Indonesian sources were investigated for their phenotypic characteristics, ubiquinone systems, DNA base compositions, and levels of DNA-DNA relatedness. Of 31 reference strains, six showed the presence of ubiquinone 10 (Q-10). These strains were eliminated from the genus Acetobacter. The other 25 reference strains and 45 Indonesian isolates were subjected to a systematic study and separated into 8 distinct groups on the basis of DNA-DNA relatedness. The known species, Acetobacter aceti, A. pasteurianus, and A. peroxydans are retained for three of these groups. New combinations, A. orleanensis (Henneberg 1906) comb. nov., A. lovaniensis (Frateur 1950) comb. nov., and A. estunensis (Carr 1958) comb. nov. are proposed for three other groups. Two new species, A. indonesiensis sp. nov. and A. tropicalis sp. nov. are proposed for the remaining two. No Indonesian isolates were identified as A. aceti, A. estunensis, and A. peroxydans. Phylogenetic analysis on the basis of 16S rDNA sequences was carried out for representative strains from each of the groups. This supported that the eight species belonged to the genus Acetobacter. Several strains previously assigned to the species of A. aceti and A. pasteurianus were scattered over the different species. It is evident that the value of DNA-DNA relatedness between strains comprising a new species should be determined for the establishment of the species. Thus current bacterial species without data of DNA-DNA relatedness should be reexamined for the stability of bacterial nomenclature.
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Two cation-tolerant yeasts with powdered colonies, K28-3-2T and K26-1-4, were isolated from dry salted shrimp and sewage, respectively, in Siem Reap province, Cambodia. The D1/D2 sequences of the 26S rDNA data showed that the two isolates... more
Two cation-tolerant yeasts with powdered colonies, K28-3-2T and K26-1-4, were isolated from dry salted shrimp and sewage, respectively, in Siem Reap province, Cambodia. The D1/D2 sequences of the 26S rDNA data showed that the two isolates were conspecific and related to the Pichia burtonii and Candida fennica. Two isolates were examined by a polyphasic taxonomic approach, including molecular phylogenetic analysis, morphological, physiological and biochemical tests, DNA hybridization and MSP-PCR fingerprinting, in comparison with P. burtonii and C. fennica. The two isolates were found to grow by multilateral budding with true and pseudo-mycelium, to not produce ascospores, and to contain ubiquinone Q-8 similar to that of P. burtonii and C. fennica. The two isolates were not differentiated from the two closest species, P. burtonii and C. fennica, by the phenotypic character examined, except for the cation (Li+)-tolerance. From DNA-DNA reassociation studies, however, the two isolates showed low similarities to the closest two species. Based on D1/D2 sequences of 26S rDNA and DNA-DNA reassociation data, they were shown to be a new distinct species from P. burtonii and C. fennica. Therefore, a novel species is proposed, Candida khmerensis sp. nov., represented by strain K28-3-2T (=JCM 13262(T)=CBS 9784T). The novel species, Candida khmerensis sp. nov. can be clearly distinguished from P. burtonii and C. fennica by either the 26S rDNA D1/D2 or ITS region with 5.8S rDNA sequencing, or by the MSP-PCR fingerprinting pattern.
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beta 1,4-Galactosyltransferase (UDP galactose: beta -N-acetylglucosaminide: beta 1,4-galactosyltransferase; EC 2.4.1.22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal... more
beta 1,4-Galactosyltransferase (UDP galactose: beta -N-acetylglucosaminide: beta 1,4-galactosyltransferase; EC 2.4.1.22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mammalian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells,
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Research Interests: Microbiology, Molecular Evolution, Horizontal Gene Transfer, Multidisciplinary, Phylogeny, and 10 moreNitrogen Fixation, Sequence alignment, Rhodobacter sphaeroides, Oxidoreductases, Molecular Diversity, Nitrogen, Evolutionary History, Alphaproteobacteria, Amino Acid Sequence, and Lateral Gene Transfer
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Almond α-mannosidase was purified by separation on columns of DEAE-Sephadex A50 and hydroxyapatite, and characterized. Its optimum pH was approximately 3.8. It was also shown to be stable from pH 6 to 8. Its activity was stable up to... more
Almond α-mannosidase was purified by separation on columns of DEAE-Sephadex A50 and hydroxyapatite, and characterized. Its optimum pH was approximately 3.8. It was also shown to be stable from pH 6 to 8. Its activity was stable up to 60°C. The thermostability of almond α-mannosidase at 73°C appeared to be superior to that of jack bean a-mannosidase. We examined the substrate specificity of the former toward high-mannose-type N-glycan Man9GlcNAc2, and showed that the deduced trimming pathway was more diverse than that of the latter. We could use almond α-mannosidase as well as jack bean α-mannosidase for analysis of sugar chain structures.