Functional Analysis of Genes Involved in the Biosynthesis of Enterocin NKR-5-3B, a Novel Circular Bacteriocin.
A putative biosynthetic gene cluster of the enterocin NKR-5-3B (Ent53B), a novel circular bacteriocin, was analyzed by sequencing the flanking regions around enkB, the Ent53B structural gene, using a fosmid library. A region approximately 9 kb in length was obtained, and the enkB1, enkB2, enkB3, and enkB4 genes, encoding putative biosynthetic proteins involved in the production, maturation, and secretion of Ent53B, were identified. We also determined the identity of proteins mediating self-immunity against the effects of Ent53B. Heterologous expression systems in various heterologous hosts, such as Enterococcus faecalis and Lactococcus lactis strains, were successfully established. The production and secretion of the mature Ent53B required the cooperative functions of five genes. Ent53B was produced only by those heterologous hosts that expressed protein products of the enkB, enkB1, enkB2, enkB3, and enkB4 genes. Moreover, self-immunity against the antimicrobial action of Ent53B was conferred by at least two independent mechanisms. Heterologous hosts harboring the intact enkB4 gene and/or a combination of intact enkB1 and enkB3 genes were immune to the inhibitory action of Ent53B.


OBJECTIVE
In addition to their potential application as food preservatives, circular bacteriocins are now considered possible alternatives to therapeutic antibiotics due to the exceptional stability conferred by their circular structure. The successful practical application of circular bacteriocins will become possible only if the molecular details of their biosynthesis are fully understood. The results of the present study offer a new perspective on the possible mechanism of circular bacteriocin biosynthesis. In addition, since some enterococcal strains are associated with pathogenicity, virulence, and drug resistance, the establishment of the first multigenus host heterologous production of Ent53B has very high practical significance, as it widens the scope of possible Ent53B applications.
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Rodney H
RH
Perez
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Naoki
N
Ishibashi
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Tomoko
T
Inoue
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Kohei
K
Himeno
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Yoshimitsu
Y
Masuda
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Narukiko
N
Sawa
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan zendo@agr.kyushu-u.ac.jp.
Takeshi
T
Zendo
Department of Fishery Products, Faculty of Fisheries, Kasetsart University, Chatuchak, Bangkok, Thailand.
Pongtep
P
Wilaipun
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok, Thailand.
Vichien
V
Leelawatcharamas
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Jiro
J
Nakayama
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, Fukuoka, Japan.
Kenji
K
Sonomoto
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D013293:Enterococcus faecalis / Q000235:genetics / Q000378:metabolism; D016984:Enterococcus faecium / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data
D001430:Bacteriocins