EnkT is a novel ATP-binding cassette (ABC) transporter responsible for secretion of four bacteriocins, enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z), produced by Enterococcus faecium ...NKR-5-3. It is generally recognized that the secretion of a bacteriocin requires a dedicated ABC transporter, although molecular mechanisms of this secretion are yet to be revealed. In order to characterize the unique ability of EnkT to secrete multiple bacteriocins, the role of N-terminal leader peptides of bacteriocin precursors was evaluated using Ent53C precursor as a model. The 18-amino acid leader peptide of Ent53C (Lc) was modified by site-directed mutagenesis to generate various point mutations, truncations, or extensions, and substitutions with other leader peptides. The impact of these Lc mutations on Ent53C secretion was evaluated using a quantitative antimicrobial activity assay. We observed that Ent53C production increased with Ala substitution of the highly conserved C-terminal double glycine residues that are recognized as the cleavage site. In contrast, Ent53C antimicrobial activity decreased, with decrease in the length of the putative α-helix-forming region of Lc. Furthermore, EnkT recognized and transported Ent53C of the transformants possessing heterologous leader peptides of enterocin A, pediocin PA-1, brochocins A and B, and lactococcins Qα and Qβ. These results indicated that EnkT shows significant tolerance towards the sequence and length of leader peptides, to secrete multiple bacteriocins. This further demonstrates the functional diversity of bacteriocin ABC transporters and the importance of leader peptides as their recognition motif.
EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, ...C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems.
Enterocin NKR-5-3B, one of the multiple bacteriocins produced by Enterococcus faecium NKR-5-3, is a 64-amino acid novel circular bacteriocin that displays broad-spectrum antimicrobial activity. Here ...we report the identification, characterization, and three-dimensional nuclear magnetic resonance solution structure determination of enterocin NKR-5-3B. Enterocin NKR-5-3B is characterized by four helical segments that enclose a compact hydrophobic core, which together with its circular backbone impart high stability and structural integrity. We also report the corresponding structural gene, enkB, that encodes an 87-amino acid precursor peptide that undergoes a yet to be described enzymatic processing that involves adjacent cleavage and ligation of Leu24 and Trp87 to yield the mature (circular) enterocin NKR-5-3B.
Enterococcus faecium NKR-5-3 produces multiple-bacteriocins, enterocins NKR-5-3A, B, C, D, and Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). However, the biosynthetic mechanisms on how their ...productions are regulated are yet to be fully understood. In silico analysis revealed putative promoters and terminators in the enterocin NKR-5-3ACDZ gene cluster, and the putative direct repeats (5′-ATTTTAGGATA-3′) were conserved upstream of each promoter. Transcriptional analysis by quantitative real-time polymerase chain reaction (PCR) of the biosynthetic genes for the enterocins NKR-5-3 suggested that an inducing peptide (Ent53D) regulates the transcription of the structure genes and corresponding biosynthetic genes of enterocins NKR-5-3, except for Ent53B (a circular bacteriocin), thus consequently regulating their production. Moreover, transcriptional analysis of some knock-out mutants showed that the production of Ent53A, C, D and Z is controlled by a three-component regulatory system (TCS) consisting of Ent53D, EnkR (response regulator), and EnkK (histidine kinase). The production of the circular bacteriocin Ent53B appeared to be independent from this TCS. Nevertheless, disrupting the TCS by deletion of a single component (enkD, enkR and enkK) resulted in a slight increase of enkB transcription and consequently the production of Ent53B, presumably, as an indirect consequence of the increase of available energy to the strain NKR-5-3. Here, we demonstrate the regulatory control of the multiple bacteriocin production of strain NKR-5-3 likely through the TCS consisting of Ent53D, EnkR, and EnkK. The information of the sharing of the regulatory machinery between bacteriocins in strain NKR-5-3 can be useful in its future application such as designing strategies to effectively dispense its multiple bacteriocin arsenal.
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.
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.
Enterocins NKR-5-3A, B, C, and D were purified from the culture supernatant of Enterococcus faecium NKR-5-3 and characterized. Among the four purified peptides, enterocin NKR-5-3A (5242.3 Da) was ...identical to brochocin A, produced by Brochothrix campestris ATCC 43754, in mature peptides, and its putative synergistic peptide, enterocin NKR-5-3Z, was found to be encoded in ent53Z downstream of ent53A, encoding enterocin NKR-5-3A. Enterocin NKR-5-3B (6316.4 Da) showed a broad antimicrobial spectrum, and enterocin NKR-5-3C (4512.8 Da) showed high activity against Listeria. Enterocin NKR-5-3D (2843.5 Da), showing high homology to an inducing peptide produced by Lactobacillus sakei 5, induced the production of the enterocins. The enterocins showed different antimicrobial spectra and intensities. E. faecium NKR-5-3 concomitantly produced enterocins NKR-5-3A, B, C, and D which probably belong to different classes of bacteriocins. Furthermore, NKR-5-3 production was induced by enterocin NKR-5-3D.
Enterococcus faecium NKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, ...Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA ent53A, enkC ent53C, enkD ent53D, and enkZ ent53Z) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIaz and enkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies of enkT and ΔenkT mutant strains showed that enkT is responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRK mutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter.
Malachite green (MG) has been focused on as a biotreatment target and its biological properties have also been an issue in food fish aquaculture. An MG-removing bacterium was isolated from ...aquaculture fish pond sediment samples in Thailand. The isolate, strain T-5-2, is a Gram-negative, aerobic rod-shaped bacterium, and has been identified as a member of the
Pseudomonas putida
group. Proton nuclear magnetic resonance spectroscopy (
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H-NMR) analysis of a broth culture medium containing MG showed that the concentration of MG decreased markedly and that other molecules, including leucomalachite green (LMG), were generated. Moreover, liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis showed that the MG concentration in the broth culture medium continuously decreased. This analysis also demonstrated that the concentration of LMG initially increased and then gradually decreased. Furthermore, gas chromatography–mass spectrometry (GC–MS) analysis showed 4-(dimethylamino)benzophenone (4DABP) as a degradation component of MG, which was confirmed by
1
H-NMR and LC–MS/MS analysis. These findings suggest that this bacterial strain can remove MG in broth culture and degrade it to certain metabolites, including LMG and 4DABP. This study is the first detailed evaluation by the combination of LC–MS/MS, GC–MS, and
1
H-NMR analyses of an MG-removing bacterium isolated from Thai aquaculture fish ponds.
Staphylococcus hominis KQU-131, isolated from Thai fermented marine fish, produces a heat stable bacteriocin. Structural and genetic analysis indicated that the bacteriocin is a variant of nukacin ...ISK-1, a type-A(II) lantibiotic, and we termed the bacteriocin nukacin KQU-131. There were three different amino acid residues between nukacin ISK-1 and nukacin KQU131, one residue in the leader peptide and the other two in the mature peptide.
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