Lactobacillus sakei and other lactic acid bacteria were studied on the change of the type of stereoisomers (the ratio of L-form to D-form) of lactic acid produced in the presence of sodium acetate and under other cultural conditions. Of 49 strains tested, only L. sakei NRIC 1071T and L. coryniformis subsp. coryniformis NRIC 1638T changed the type in the presence of 50 mM sodium acetate compared with the absence of sodium acetate. The type produced by L. sakei NRIC 1071T was shifted 30% or more from the DL-type to the L-type in the presence of 50 mM sodium acetate. L. sakei NRIC 1071T produced not only twice or more the amount of L-lactic acid but decreased the amount of D-lactic acid compared with the absence of sodium acetate. The shift of the DL-type to the L-type by L. sakei is due to the high production of L-lactic acid and the low production of D-lactic acid. The type of stereoisomers produced by 11 L. sakei strains was also shifted from the DL-type to the L-type in the presence of 50 mM sodium acetate. The shift of stereoisomers by the majority of L. sakei strains seems interesting from the viewpoint of the delineation of this species.
A pair of primers, NV35 and NV36, and another pair of primers, NV81 and NV82/SM82, are commonly used for polymerase chain reaction (PCR) detection of Norwalk-like virus (NLV) genome RNA sequences in authorized test laboratories in Japan. However, the efficiency of NLV genome RNA detection with these primer pairs has been less than satisfactory. In the present study, we attempted to establish more appropriately matched primer pairs for improved detection of NLV genome RNA sequences using a combination of primers including NV35, NV36, NV81, NV82/SM82, SR33, and SRs (a mixture of 4 primers SR46, SR48, SR50, and SR52). We also evaluated appropriate primers for improved reverse transcription of NLV genome RNA. Stool samples used for detection of NLV included 18 samples collected from NLV-infected patients who ingested oysters (group 1) and 13 samples collected from those who did not ingest oysters (group 2). Reverse transcription of RNA genome with primer NV35 was less efficient compared with that with primer SR33 or NV81. When PCR products obtained with NV35 and NV36 as a pair of primers were subjected to gel electrophoresis, a strong extra band was detected compared with those obtained with other primer pairs. Since this extra band may represent heterodimeric or homodimeric hybrids, or intramolecular hybrids derived from these primers, this template-independent hybridization could lower the efficiency of primer-dependent polymerase reaction. Of 18 primer pairs, a pair of NV81 and SRs provided the best detection of PCR products following reverse transcription of NLV RNA with SR33 or NV81. The detection rate was 61% for both reverse transcription with SR33 and that with NV81. After reverse transcription using SR33 as a primer, nested PCR using a pair of NV81 and SRs following primary PCR using a pair of NV81 and NV82/SM82 increased the detection rate to 89% in group 1 and 100% in group 2.
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 0164T. 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 0180T) 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.
A tannase-producing soil bacteria has been isolated and identified as Bacillus cereus. It can degrade tannic acid and produce maximum tannase (0.22 U/ml) at stationary phases of growth (24 h). Maximum growth and enzyme production occurred with initial medium pH of 4.5–5.0. Partial purified tannase showed optimum activity at pH 4.5 and 40°C. It remains stable up to 30°C and pH 4.5 to 5.0. The enzyme is salt tolerant, stable up to 2 M of NaCl and retains 82% original activity in 3 M.
Pseudomonas putida KT2442TOL (formerly designated TOL), a toluene-resistant variant of strain KT2442 constitutively overexpressed several proteins. The most abundantly produced 24-kDa soluble protein was found to be similar to AhpC, the small subunit of alkyl hydroperoxide reductase. Molecular cloning of the P. putida ahpC based on the N-terminal sequence allowed cloning of closely located ahpF, the large subunit of alkyl hydroperoxide reductase. The deduced amino acid sequences of these genes showed high similarity with corresponding bacterial homologues. Results of RNA transcriptional analyses suggested that P. putida ahpC and ahpF were co-transcribed. A lower level expression of the ahpF may result from an attenuation of transcription by stem-and-loop structures located between two genes. oxyR, the known expression regulatory gene of ahpC-ahpF, was separately cloned and a point mutation that rendered an amino acid change (Phe106 to Ile) in OxyR was observed. Reverse mutation of the oxyR gene by allelic exchange in P. putida KT2442TOL revealed that this mutation was the cause of the overexpression. About 50% of the reverse mutated cells lost colony-forming ability under toluene, indicating the mutation of oxyR that contributes to overexpression of the oxyR-regulated genes has some relationship with the solvent resistance, but their contribution was not significant.