Functional Analysis of Genes Involved in the Biosynthesis of Enterocin NKR-5-3B, a Novel Circular Bacteriocin

Abstract

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. IMPORTANCEIn 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. Bacteriocins are ribosomally synthesized antimicrobial peptides that generally exert their antagonistic activity toward strains that are closely related to the producer strain (1, 2), although an increasing number of bacteriocins have been reported to have a broad activity range (3, 4). In the last decade, the interest in bacteriocins, especially those from lactic acid bacteria (LAB), has increased considerably, as they potentially can be used as natural food preservatives and therapeutic antibiotics (5-8).Over the years, various classification schemes of bacteriocins from Gram-positive bacteria have been suggested, which commonly divide bacteriocins into two groups: class I (lantibiotics) and class II (nonlantibiotics) (9-12). Lantibiotics are small heatstable peptides which contain unusual amino acids, such as lanthionine and/or methyllanthionine, as a result of posttranslational modifications of some common amino acid residues (13,14). In contrast, class II bacteriocins, which are also heat-stable peptides, do not undergo extensive postt...

Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis

“…The head-to-tail circularization of Ent53B occurs between the Leu1 and Trp64 [22]. We recently reported that the maturation of Ent53B from its precursor peptide requires cooperative functioning of four gene products, EnkB1234, that form an enzyme complex [21]. However, very little is known about the molecular mechanism of its biosynthesis.…”

“…Synthesized cDNAs were then used as template for the RT-PCR to confirm the presence of messenger transcripts. Since the Ent53B precursor and its biosynthetic machinery are transcribed in a single polycistronic mRNA [21], introduction of mutations in the enkB gene should have no effect on the upstream genes within the enkB biosynthetic operon as long as these mutations will not affect the proper framing of these genes. This was easily confirmed through DNA sequencing.…”

“…Detection, quantification assay and MIC determination of mature Ent53B and derivatives The production of mature Ent53B was evaluated using a direct-colony-overlay assay as described previously [21] with slight modification. Briefly, recombinant Enterococcus faecalis JH2-2 harbouring the respective plasmids were precultivated in 5 ml GM17 medium containing 10 µg ml À1 cm incubated at 30 C overnight and sub-cultured into fresh medium containing the same antibiotic and kept under the same conditions for 24 h. The amount of cells of each recombinant strain was normalized to the same optical density (OD 600 of 3.0) using sterile distilled water.…”

“…To gain insights into the essential Ent53B leader peptide regions, Ent53B precursor peptides with truncated leader peptides were constructed by inverse PCR using the plasmid pNK-B1234 as template DNA [21]. Molecular cloning techniques were performed using standard methods [25].…”

“…Enterocin NKR-5-3B (Ent53B) is a 64-amino acid novel circular bacteriocin produced by Enterococcus faecium NKR-5-3 [21][22][23][24]. The Ent53B structural gene, enkB, encodes an 87-amino acid prepeptide, which consists of a 23-amino acid leader peptide and a 64-amino acid propeptide domain.…”