|Budget Amount *help
¥11,000,000 (Direct Cost : ¥11,000,000)
Fiscal Year 1999 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1998 : ¥8,200,000 (Direct Cost : ¥8,200,000)
Enterococcus faecium N15 was isolated from nuka (Japanese rice bran paste), utilized as starter for fermented vegetables, and was found to produce a bacteriocin which exhibited a broad spectrum of activity including against Listeria monocytogenes and Bacillus circulans JCM2504. The characteristics of the bacteriocin suggest that bacteriocin N15 belongs to class IIa bacteriocins. Then, a 2-kbp DNA fragment was amplified by PCR with degenerated PCR primers, which were designed based on the conserved amino acid sequence of class IIa bacteriocins. It was found that E. faecium N15 produced enterocin A and entI gene encoding immunity protein for enterocin A was obtained.
Enterococcus faecium N15 produced enterocin A and was found to have at least three plasmids, pEFNP1, pEFNP2, and pEFNP3. The nucleotide sequence of the smallest plasmid, pEFNP1, was determined. pEFNP1 was used to construct shuttle vectors that could replicate in Escherichia coli and E. faecium. The chimeric plasmid pKU201, composed of pUC19 and pEFNP1, was constructed. The shuttle vector plasmid pKU203 was then obtained by inserting the erythromycin (Em) resistance gene into pKU201. pKU203 was transformed in E. faecium N15 and its replication was confirmed. However, pKU203 was not stable in strain N15. Next, the entAI operon of E. faecium N15 was ligated into pKU203 and the plasmid pKU204 was constructed. pKU204 was also introduced into E. faecium N15 and E. faecalis JCM8726 and E. faecium EFN204 as a result of which the transformants E. faecalis ECL204 were obtained. pKU204 was stably maintained in E. faecium strain EFN204 without Em, suggesting that the entI gene improved the plasmid stability. In the case of E. faecalis strain ECL204, pKU204 was considerably more stable under the enterocin A pressure than without selection pressure. These findings indicated that the entI gene could be utilized as a selection marker gene.