Taxonomic position of new bacteriocin (nukacin ISK-1) producer isolated from long-aged Nukadoko.

Abstract

The new lantibiotic nukacin producers, strain ISK-1 previously labeled as Pediococcus sp. ISK-1 and strain Nagano, were characterized biochemically and genetically. Cells were spherical, 0.6 µm in size, and occurred in pairs. The strains were gram-positive, catalase positive and produced more than 75% of DLlactic acid from glucose and their growth was stimulated by mevalonic acid. The strains tolerated 18% NaCl. GϩC content was 36%. 16S rRNA homology revealed that the strains resembled Staphylococcus warneri. Both strains were confirmed by quantitative DNA-DNA hybridization to be variant strains of S. warneri. These strains have been deposited in the Japan Collection of Microorganism as S. warneri JCM 10004 for strain ISK-1 and S. warneri JCM 10005 for strain Nagano.In the northern Kyushu area, many old aged Nukadoko (bed of fermented rice bran) have been maintained until now and some are believed to have lasted for a few hundred years (Kimura et al., 1997). Nukadoko is usually used for pickle fermentation using washed vegetables without any aseptic treatment. We therefore suspected that Nukadoko must possess a native microflora control system that attacks undesirable microorganisms that comes into contact with the bed from the environment. We isolated two organisms, strain ISK-1 and strain Nagano, from an old aged bed owned by Mrs. Sato in Kitakyushu-shi, Japan, a cooking instructor for Iwashino-Nukamisodaki which is a local traditional Japanese cuisine, consisting of cooked sardine with old aged Nukadoko. These strains were isolated by the following procedure. One gram of Nukadoko was suspended in 10 ml of pure water and stood for 24 h at 30°C. One milliliter of the suspension was suspended in 100 ml of thioglycolate (TGC, Difco Laboratories, Detroit, MI, USA) with 1.5% agar and 1.5% CaCO 3 to which NaN 3 10 ppm and cycloheximide 10 ppm were supplemented. The agar plates were incubated in an anaerobic jar for 2 days. The organism was purified by repeating single colony isolation on TGC agar plate.Strain ISK-1 and strain Nagano grew in both anaerobic and microaerophilic conditions. These strains were gram-positive cocci occurring in pairs, catalase positive, and produced more than 75% of DL-lactic acid from glucose with only a little amount of acetic acid. Lactic acid was determined in the form of L-and D-in-

Characterization of a Gene Cluster ofStaphylococcus warneriISK-1 Encoding the Biosynthesis of and Immunity to the Lantibiotic, Nukacin ISK-1

“…S. warneri ISK-1 (JCM 11004) isolated from long-aged Nukadoko 3) and Pediococcus pentosaceus JCM 5885 (formerly reported as Pediococcus acidilactici JCM 5885 T ) were grown in MRS medium (Oxoid, Hampshire, U.K.), as described previously. 6) Escherichia coli JM 109 (Toyobo, Osaka, Japan) 17) was grown in LB medium 18) at 37 C. Plasmid pUC18 (Toyobo) was used for cloning of the fragments of the nukacin ISK-1 biosynthetic gene cluster.…”

“…[3][4][5][6] The N-terminal amino acid sequence analysis, amino acid composition analysis, mass spectrum analysis, and structural gene sequence analysis suggested that nukacin ISK-1 consists of 27 amino acids including 2 molecules of lanthionine, 1 molecule of 3-metyllanthionine, and 1 residue of dehydrobutyrine ( Fig. 1).…”

Lantibiotic Engineering: Molecular Characterization and Exploitation of Lantibiotic-Synthesizing Enzymes for Peptide Engineering

“…Staphylococcus warneri ISK-1 was isolated from a wellaged Nukadoko , a bed of fermented rice bran [8] . This bacterium produces a novel type-A(II) lantibiotic, nukacin ISK-1 [9] ( fig.…”

A Novel Type of Immunity Protein, NukH, for the Lantibiotic Nukacin ISK-1 Produced byStaphylococcus warneriISK-1

“…The bacterial strains and plasmids used in this study are listed in Table 1. S. warneri ISK-1 (JCM 11004) 15) was cultured in half concentrated MRS medium (Oxoid, Hampshire, United Kingdom) 18) with shaking at 37 C. L. lactis subsp. lactis CNRZ481 25) was cultured in MRS medium without shaking at 30 C. Escherichia coli JM109 26) used as a cloning host and S. carnosus TM300 27) were grown in LB medium 28) at 37 C. L. lactis NZ9000 29) was grown in M17 medium (Merck, Darmstadt, Germany) supplemented with 0.5% glucose (GM17) at 30 C. Plasmid pNZ8048 30) was used for the expression of nukFEGH.…”

“…14) Nukacin ISK-1 is a type-A(II) lantibiotic produced by Staphylococcus warneri ISK-1 (JCM 11004). [15][16][17][18] The putative structure of nukacin ISK-1 (MW 2960 Da) y To whom correspondence should be addressed. Fax: +81-92-642-3019; E-mail: sonomoto@agr.kyushu-u.ac.jp Abbreviation: NICE, nisin-controlled expression agreed with that of lacticin 481 (MW 2901 Da) in terms of number of amino acid residues and position of unusual amino acids.…”