A Gram-positive, coccus-shaped, lactic acid bacterium, strain NGRI 0510Q T , was isolated from ryegrass silage produced in Okinawa Prefecture, Japan. The cell is non-spore-forming, nonmotile, and occurs in pairs or tetrads. The strain is homofermentative and produces D-and L-lactic acid from glucose. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NGRI 0510Q T belongs to the genus Pediococcus and clusters within the Pediococcus acidilactici and Pediococcus pentosaceus group, with 98.2 and 96.9 % sequence identity, respectively. DNA-DNA relatedness between strain NGRI 0510Q T and P. acidilactici JCM 8797 T and P. pentosaceus JCM 5890 T was 19.3 and 17.3 %, respectively. Based on its phenotypic characteristics, phylogenetic relationship and DNA-DNA relatedness, NGRI 0510Q T (5JCM 15055 T 5DSM 19927 T ) represents the type strain of a novel species, for which the name Pediococcus lolii sp. nov. is proposed.
To develop high-quality silage starters adapted to hot and humid weather, 12 LAB isolates from silage produced in Kyushu and Okinawa, Japan were characterized based on their morphological features, growth curves and sugar utilization. In addition, the nucleotide sequences of the V2-V3 region of their 16S rRNA genes and the 16S-23S rRNA intergenic spacer (ITS) regions were determined. The isolates were also subjected to RAPD-PCR analysis, DNA-DNA hybridization, G+C content analysis and immuno-identification using species-specific monoclonal antibodies and SDS-PAGE profiling. Nearly all of the isolates exhibited high thermotolerance and rapid growth. Combining ITS sequence analysis, RAPD-PCR and immuno-identification enabled rapid and accurate identification of closely related LAB strains that other methods failed to appropriately differentiate; for example, L. plantarum was distinguished from L. pentosus, and L. casei was distinguished from L. rhamonsus. Using the aforementioned techniques, the isolated strains were identified as L. plantarum, L. rhamonsus, L. rapi, Pediococcus pentosaceus and P. lolii. Our findings also showed that there is greater diversity among thermophilic LABs in silage prepared in a hot and humid environment.
The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His(6)-tagged acetate kinase purified from E. coli JM109 was optimal at 35 degrees C.
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