Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp. A-01.

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.

Taxonomic homogeneity of a salt-tolerant lactic acid bacteria isolated from shoyu mash.

Forty-seven salt-tolerant lactic acid bacteria, which had been isolated from different places and grown in 15% NaCl, were examined to assess their taxonomic heterogeneity. Among the isolates, 42 were isolated from shoyu mash during the acid fermentation phase, 2 were from miso and 3 were from anchovy pickles. All isolates were identified as Tetragenococcus halophilus on the basis of DNA relatedness values. We further examined 102 phenotypic characteristics of them. The isolates exhibited differences in only 16, supporting the conclusion obtained from the DNA relatedness analysis.

Recharacterization of Pseudomonas fulva Iizuka and Komagata 1963, and proposals of Pseudomonas parafulva sp. nov. and Pseudomonas cremoricolorata sp. nov.

Seven Pseudomonas fulva strains obtained from culture collections were taxonomically studied. The seven strains were separated into three clusters (Clusters I to III) on the basis of 16S rRNA gene sequences, and located phylogenetically in the genus Pseudomonas sensu stricto. Further, the strains were classified into 4 groups (Groups I to IV) on the basis of DNA-DNA similarity. As a result, Cluster I was split into Groups I and II. Group I included the type strain of P. fulva and two strains, and levels of DNA-DNA similarity ranged from 88 to 100% among the strains. Group II contained two strains, and the level between the two strains ranged from 91 to 100%. Group III consisted of one strain. Group IV included one strain, and this strain showed a high level of DNA-DNA similarity with the type strain of Pseudomonas straminea NRIC 0164(T). Clusters II and III corresponded to Groups III and IV, respectively. The four groups were separated from one another and from related Pseudomonas species at the level from 3 to 45% of DNA-DNA similarity. The strains of Groups I, II, and III had ubiquinone 9 as the major quinone. According to numerical analysis by the use of 133 phenotypic characteristics, the seven P. fulva strains were split into four phenons (Phenons I to IV). The groups by DNA-DNA similarity corresponded well with the phenons produced by numerical taxonomy, and differential characteristics were recognized. Consequently, Group I was regarded as P. fulva because the type strain (NRIC 0180(T)) of this species was included in this group. Strains in Group II were identified as a new species, Pseudomonas parafulva sp. nov., and the type strain is AJ 2129 (=IFO 16636=JCM 11244=NRIC 0501). NRIC 0181 in Group III was identified as a new species, Pseudomonas cremoricolorata sp. nov., and the type strain is NRIC 0181 (=IFO 16634=JCM 11246). NRIC 0182 in Group IV was identified as P. straminea on the basis of the high level of DNA-DNA similarity with the type strain of this species.

Rhodococcus tukisamuensis sp. nov., isolated from soil.

A novel strictly aerobic, heterotrophic, mesophilic bacterium, strain Mb8(T), was isolated from soil in Sapporo City, Hokkaido, Japan. The G+C content of strain Mb8(T) was 66.0 mol%. It had mycolic acids with 44-52 carbon atoms and C16 : 0 and C18 : 1 (9) as the major fatty acids. The major isoprenoid quinone was MK-8(H(2)). The cell wall contained meso-diaminopimelic acid, arabinose and galactose. 16S rDNA, chemotaxonomic and morphological data indicated that this strain clearly belonged to the genus RHODOCOCCUS: Based on phenotypic properties and DNA-DNA hybridization data, strain Mb8(T) (=JCM 11308(T)=NCIMB 13903(T)) has been assigned to the genus Rhodococcus as the type strain of Rhodococcus tukisamuensis sp. nov.

Bacillus vietnamensis sp. nov., a moderately halotolerant, aerobic, endospore-forming bacterium isolated from Vietnamese fish sauce.

Five strains of Gram-positive, endospore-forming, moderately halotolerant bacteria were studied taxonomically. Four were isolated from Vietnamese fish sauce and one from the Gulf of Mexico. Phylogenetic analysis based on 16S rRNA gene sequences showed that these strains clustered within the radiation of the genus Bacillus but separately from recognized Bacillus species. DNA G+C composition of the isolates ranged from 43 to 44 mol%. Strains 15-1(T) and NRRL B-14850 showed high levels of DNA-DNA relatedness (82-100 %) to each other and to the other strains isolated here; they displayed low levels of DNA-DNA relatedness (<29 %) to the type strains of selected recognized Bacillus species. They grew in 15 % NaCl and optimally in 1 % NaCl, which is characteristic of moderately halotolerant bacteria. The isolates grew at pH 6.5 to 10.0 but not at pH 6.0. Their cell walls contained meso-diaminopimelic acid. The major isoprenoid quinone was MK-7 and the principal cellular fatty acids were anteiso-C(15 : 0), iso-C(15 : 0) and anteiso-C(17 : 0). Based on these results, the strains tested were regarded as members of a novel Bacillus species for which the name Bacillus vietnamensis sp. nov. is proposed. The type strain is 15-1(T) (=JCM 11124(T)=NRIC 0531(T)=NRRL 23890(T)).

Bacillus okuhidensis sp. nov., isolated from the Okuhida spa area of Japan.

Two Gram-positive, endospore-forming, alkaliphilic bacteria were isolated from water samples obtained from the Okuhida hot spa area of Japan. The unknown bacteria were characterized using phenotypic and molecular taxonomic methods. On the basis of phylogenetic evidence and phenotypic distinctiveness, a new species, Bacillus okuhidensis sp. nov., is proposed. The type strain of Bacillus okuhidensis is GTC 854T (= JCM 10945T = DSM 13666T).

Alicyclobacillus sendaiensis sp. nov., a novel acidophilic, slightly thermophilic species isolated from soil in Sendai, Japan.

An acidophilic, slightly thermophilic bacterium, designated strain NTAP-1T, that produces a thermostable extracellular acid collagenase activity with potential industrial applications was isolated from soil of Aoba-yama Park, Sendai, Japan. The temperature range for growth was 40-65 degrees C, with an optimum at 55 degrees C, and the pH range for growth was 2.5-6.5, with an optimum at pH 5.5. Analysis of the 16S rDNA sequence of strain NTAP-1T showed that it is most closely related to strains of the genus Alicyclobacillus. Consistently, the major constituents of the cell-membrane lipid of strain NTAP-1T were omega-alicyclic fatty acids. However, DNA-DNA reassociation studies showed only low similarities (less than 33%) to any type strain of Alicyclobacillus. On the basis of the phenotypic and genotypic properties, a novel species is proposed, Alicyclobacillus sendaiensis sp. nov., represented by strain NTAP-1T (=JCM 11817T=ATCC BAA-609T).