Isolation of a novel bacteriocin-producing lactic acid bacteria and its use in food preservation

从日本米糠膏中分离出粪肠球菌N15，作为发酵蔬菜的发酵剂，发现其产生的细菌素具有广谱活性，包括抗单核增生李斯特菌和环状芽孢杆菌JCM2504。细菌素的特征表明细菌素N15属于IIa类细菌素。然后，根据IIa类细菌素的保守氨基酸序列设计退化PCR引物，用PCR扩增出2 kbp的DNA片段。发现粪肠杆菌N15可产生肠球菌A，并获得肠球菌A免疫蛋白编码基因entI。粪肠球菌N15产生肠球菌蛋白A，发现至少有三种质粒，pEFNP1、pEFNP2和pEFNP3。测定最小质粒pEFNP1的核苷酸序列。利用pEFNP1构建穿梭载体，可在大肠杆菌和粪肠杆菌中复制。构建了由pUC19和pEFNP1组成的嵌合质粒pKU201。将红霉素耐药基因插入pKU201，获得穿梭载体质粒pKU203。pKU203在粪肠杆菌N15中转化，并证实了其复制能力。而pKU203在菌株N15中不稳定。接下来，将粪肠杆菌N15的内操纵子连接到pKU203中，构建质粒pKU204。将pKU204引入粪肠杆菌N15、粪肠杆菌JCM8726和粪肠杆菌EFN204中，获得粪肠杆菌ECL204转化体。pKU204在未添加Em的EFN204菌株中保持稳定，表明entI基因提高了质粒的稳定性。以粪肠球菌ECL204菌株为例，pKU204在肠球菌A压力下比在没有选择压力下稳定得多。这些结果表明，entI基因可以作为一种选择标记基因。

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.