Renkichi Takata

Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1

Bacillus ehimensis EAG1 (IFO15659) produced and secreted chitosanase in the presence of exogenous chitosan. The chitosanase was purified from the culture filtrate of the bacterium to apparent homogeneity in SDS-polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 31,000. A 1.9-kbp DNA fragment containing the chitosanase gene was cloned and the complete nucleotide sequence was determined. The sequence was found to contain a single open reading frame encoding a protein of 302 amino acids. The deduced amino acid sequence showed significant homology with the chitosanase from Bacillus circulans MH-K1.

Bacillus ehimensis sp. nov. and Bacillus chitinolyticus sp. nov., new chitinolytic members of the genus Bacillus.

Five chitin-degrading bacteria were isolated from soil. These organisms were strictly aerobic and rod shaped, formed spores, contained menaquinone 7 as the major isoprenoid quinone and 12-methyltetradecanoic acid as the major cellular fatty acid, and had guanine-plus-cytosine contents of 51.3 to 54.9 mol%, characteristics which indicate that they belong to the genus Bacillus. These five strains were divided into two groups on the basis of physiological characteristics and the results of a DNA-DNA hybridization study. As low levels of DNA relatedness were found between our isolates and previously described Bacillus strains, we propose that our isolates should be classified in two new Bacillus species, Bacillus ehimensis and Bacillus chitinolyticus. The type strains of B. ehimensis and B. chitinolyticus are strains IFO 15659 and IFO 15660, respectively.

Microbial interactions responsible for dissolved DNA production in a hypereutrophic pond

Concentration of dissolved DNA, microbial biomass, and consumption of bacteria by heterotrophic nanoflagellates (HNF) and ciliates were examined in a hypereutrophic pond for over 7 months to elucidate the main factors which influenced the release of dissolved DNA. Changes in concentration of dissolved DNA correlated well with both abundance of ciliates ( r = 0.788, p < 0.01) and rotifers ( r = 0.738, p < 0.01). A significant correlation was also found between dissolved DNA concentration and ciliate community ingestion rates ( r = 0.668, p <0.01). These results suggest that consumption of bacteria by ciliates is an important reason for the release of dissolved DNA.

Isolation and characterization of a collagenolytic enzyme from Bacillus licheniformis N22

A bacterium having collagenolytic activity was isolated from soil and identified as Bacillus licheniformis. The collagenolytic enzyme was obtained in a medium containing gelatin and purified to apparent homogeneity on the basis of SDS-polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 29,000, and hydrolyzed native, insoluble collagen. In addition, the enzyme hydrolyzed soluble collagen, gelatin, hemoglobin and casein, but not synthetic oligopeptides, usually used as substrates for collagenase assay.

Isolation and characterization of Pz-peptidase from Bacillus licheniformis N22

Abstract Pz-peptidase is an endopeptidase that cleaves the synthetic substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (Pz-peptide), which was originally developed for the assay of Clostridium histolyticum collagenase (Wunsch and Heidrich, Hoppe-Seylers Z. Physiol. Chem., 333, 149–151, 1963; Morales and Woessner, J. Biol. Chem., 252, 4855–4860, 1977). Pz-peptidase was purified from the culture filtrate of Bacillus licheniformis N22. The purified Pz-peptidase showed a molecular weight of 70,000 in SDS-polyacryl-amide gel electrophoresis and 150,000 in gel filtration. Optimal pH for cleavage of Pz-peptide was 7.8. The Pz-peptidase activity was strongly inhibited by metal chelators such as EDTA and O -phenanthroline. Substrate specificity studies indicated that Pz-peptidase cleaved oligopeptides at the Xaa-Gly site in Xaa-Gly-Pro. However, Pz-peptidase failed to hydrolyze native collage, denatured collagen, hemoglobin and casein.

Cloning and sequencing of the Pz-peptidase gene from Bacillus licheniformis N22

Pz-peptidase is an endopeptidase that cleaves the synthetic substrate Pz-peptide (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg), which was originally developed for the assay of collagenase. The Pz-peptidase gene of Bacillus licheniformis N22 was cloned and sequenced. The gene consists of 628 amino acids with a motif for zinc-dependent metalloprotease, and shares 42% amino acid identity with the oligoendopeptidase of Lactococcus lactis. This is the first report on the gene structure of a Pz-peptidase.

Targeted disruption of sti35, a stress-responsive gene in phytopathogenic fungus Fusarium oxysporum.

Abstractsti35 is one of the heat-shock genes in Fusarium oxysporum, which is a fungal pathogen for wilt disease in plants. We have isolated a genomic clone of sti35 and used it to create disruption mutations. Disruption of the sti35 coding region resulted in the loss of a 32-kDa protein present in heat-shocked cells. The disruption had no detectable effect on growth and development at various temperatures, nor on the ability to acquire thermotolerance in nutrient medium. But the sti35 disruptants showed increased thermotolerance, relative to the wild-type strain, when incubated in minimal medium after heat treatment.

Fate of a PCBS degrading recombinant pseudomonas putida AC30(PMFB2) and its effect on the densities of microbes in marine microcosms contaminated with PCBS

The fate of a genetically engineered microorganism, Pseudomonas putida AC30 (pMFB2) [AC30(pMFB2)], which is capable of degrading PCBs, and its effect on the densities of microbes were studied in seawater and seawater‐sediment microcosms contaminated with PCBs. In both microcosms AC30(pMFB2) had no apparent effect on the population densities of indigenous microbes such as virus, bacteria and phytoplankton. It was considered that this was caused by a rapid decrease of AC30(pMFB2). Obtained results confirmed that non‐indigenous genetically engineered microbes could not survive and had no significant effect on the population changes of indigenous microbes, as seen in other experiments.

Detection and cloning of the gene encoding a protein produced by nonpathogenic mutants of Fusarium oxysporum.

Treatment of Fusarium oxysporum with 5-azacytidine, a potent inhibitor of DNA methylation, induced nonpathogenic mutants. Analysis of the protein expression pattern by two-dimensional gel electrophoresis revealed a protein that is present in yeast-form cells of the mutants but absent in those of the wild-type strain. N-terminal amino acid analysis indicated that this protein is identical to a region of a polypeptide encoded by a cDNA clone, sti35, previously identified as a heat shock gene in F. oxysporum. A genomic clone for sti35 was isolated and sequence analysis revealed an intron and two heat shock elements upstream of sti35. The analysis also revealed the presence of a leader sequence composed of 27 amino acid residues, which shares a common amino acid composition with leader sequences of the proteins located in the mitochondrial matrix. Different expression patterns of sti35 in the mutants and wild-type strain were demonstrated.

Factors affecting the survival of pseudomonas putida bearing pcbs‐degrading recombinant plasmid in marine microcosms contaminated with pcbs

It was found in our previous study that Pseudomonas putida AC30 (pMFB2) [AC30(pMFB2)] which was introduced into seawater and seawater‐sediment microcosms at initial densities of 1.5 x 103 cells/ml, decreased and died out within 4 and 7 days, respectively. Accordingly, we studied the reason for the failure of AC30 (pMFB2) to survive. We concluded that the predation of organisms such as protozoa were not the main reason for the decline of AC30 (pMFB2), but the filtrates of seawater which passed through the 0.2um pore sized filters were factors inhibiting the growth and survival of AC30(pMFB2).