Sadahiro Ohmomo

Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

ABSTRACT Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

A comparative study and phage typing of silage-making Lactobacillus bacteriophages.

To investigate basic characteristics of 10 virulent phages active on silage-making lactobacilli, morphological properties, host ranges, protein composition and genome characterization were separated into five groups based on host ranges and basic properties. The seven phages of groups I, II and V were active on Lactobacillus plantarum and Lactobacillus pentosus. Phage phiPY4 (group III) infected both L. casei and Lactobacillus rhamnosus. Phage phiPY5 (group IV) specifically infected Lactobacillus casei. Morphologically, three phages of groups I belonged to the Myoviridae family, while seven other phages of groups II, III and V belonged to the Siphoviridae family. SDS-PAGE profiles, restriction analysis, G + C contents of DNA and Dot blot hybridization revealed a high degree of homology in each group. Clustering derived from host range analysis was closely related to results of DNA and protein analyses. These phages may be applicable to phage typing for silage-making lactobacilli.

Pediococcus lolii sp. nov., isolated from ryegrass silage

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, non-motile, 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) (=JCM 15055(T)=DSM 19927(T)) represents the type strain of a novel species, for which the name Pediococcus lolii sp. nov. is proposed.

Isolation of Enterocin SE-K4-Encoding Plasmid and a High Enterocin SE-K4 Producing Strain of Enterococcus faecalis K-4.

Enterococcus faecalis K-4, which produces a class IIa bacteriocin, enterocin SE-K4, carries two plasmids, pEK4S (approximately 60 kb) and pEK4L (approximately 75 kb). Plasmid-curing experiments showed that pEK4S was involved in the production of and immunity to enterocin SE-K4 in strain K-4. A derivative strain, M6, with pEK4S produced a higher amount of enterocin SE-K4 than the parental strain K-4, although its growth rate was lower than that of parental strain K-4. Phenotypic changes in strain M6 are attributed to an increase in plasmid copy number.

Identification of thermo tolerant lactic acid bacteria isolated from silage prepared in the hot and humid climate of Southwestern Japan

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.

Efficient protoplast regeneration for some homofermentative lactobacilli and pediococci

Abstract. Conditions for protoplast regeneration were examined for several strains of homofermentative lactobacilli and pediococci isolated from silage. Attempts to regenerate protoplasts using previously published agar regeneration media for lactobacilli were unsuccessful for most of the strains. Replacing or increasing colloidal substances in a medium containing raffinose and MgCl2 as osmotic stabilizers enabled efficient regeneration of the protoplasts at a frequency of 10–99%. A medium containing gelatin, polyvinylpyrrolidone (PVP) and no agar was effective for Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus rhamnosus protoplasts. An agar medium containing PVP (PVP medium) was effective for Pediococcus sp. protoplasts, and addition of agarose to the PVP medium enabled regeneration of Lactobacillus casei protoplasts. A medium containing calcium alginate gel and no agar was effective for Lactobacillus curvatus protoplasts. The type of colloidal substance required for protoplast regeneration varied from species to species. This result suggested that several kinds of media may be necessary to regenerate protoplasts for all the genera of lactobacilli and pediococci.