Monitoring of the multiple bacteriocin production by Enterococcus faecium NKR-5-3 through a developed liquid chromatography and mass spectrometry-based quantification system

Pérez, Rodney; Himeno, Kunio; Ishibashi, Naoki; Masuda, Yoshito; Zendo, Takeshi; Fujita, Koji; Wilaipun, Pongtep; Leelawatcharamas, Vichien; Nakayama, Jiro; Sonomoto, Kenji

doi:10.1016/j.jbiosc.2012.06.003

Cited by 33 publications

(27 citation statements)

References 29 publications

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“…25) E. faecium NKR-5-3 was found to control the production of multiple bacteriocins, except for enterocin NKR-5-3B, via a two-component regulatory system using with the peptides, enterocin NKR-5-3D, as induction factor. 24,45) In addition, this strain controls the production of bacteriocins depending on the culture temperature. 45) Bacteriocins from E. faecium NKR-5-3 show various spectra of activity.…”

Section: Multiple Bacteriocin-producing Labmentioning

confidence: 99%

“…24,45) In addition, this strain controls the production of bacteriocins depending on the culture temperature. 45) Bacteriocins from E. faecium NKR-5-3 show various spectra of activity. Although enterocin NKR-5-3B is active against a wide range of Gram-positive bacteria, the others have rather narrow activity spectra, but with different patterns.…”

Section: Multiple Bacteriocin-producing Labmentioning

confidence: 99%

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Screening and Characterization of Novel Bacteriocins from Lactic Acid Bacteria

Zendo

2013

Bioscience, Biotechnology, and Biochemistry

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Bacteriocins produced by lactic acid bacteria (LAB) are expected to be safe antimicrobial agents. While the best studied LAB bacteriocin, nisin A, is widely utilized as a food preservative, various novel ones are required to control undesirable bacteria more effectively. To discover novel bacteriocins at the early step of the screening process, we developed a rapid screening system that evaluates bacteriocins produced by newly isolated LAB based on their antibacterial spectra and molecular masses. By means of this system, various novel bacteriocins were identified, including a nisin variant, nisin Q, a two-peptide bacteriocin, lactococcin Q, a leaderless bacteriocin, lacticin Q, and a circular bacteriocin, lactocyclicin Q. Moreover, some LAB isolates were found to produce multiple bacteriocins. They were characterized as to their structures, mechanisms of action, and biosynthetic mechanisms. Novel LAB bacteriocins and their biosynthetic mechanisms are expected for applications such as food preservation and peptide engineering.

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Section: Multiple Bacteriocin-producing Labmentioning

confidence: 99%

Section: Multiple Bacteriocin-producing Labmentioning

confidence: 99%

Screening and Characterization of Novel Bacteriocins from Lactic Acid Bacteria

Zendo

2013

Bioscience, Biotechnology, and Biochemistry

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“…Relative bioactivity (%) was determined by measuring the diameter of the zone of inhibition of each mutant and then dividing it by the size of the zone of inhibition of the wild-type (WT) and multiplying by 100. In the case of the Ent53B Leu1 derivatives, estimation of production was done using the electrospray ionization LC/MS (ESI-LC/MS) quantification system we described previously [24]. Briefly, the bacteriocin from a 10 ml culture supernatant was concentrated and partially purified using a Sep-Pak C18 cartridge (Waters).…”

Section: Rna Isolation and Semi-quantitative Reverse Transcription Pcmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.

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“…Since then, a number of circular bacteriocins from various bacterial species have been isolated: gassericin A (24), subtilosin A (25), circularin A (3), butyrivibriocin AR10 (26), uberolysin (27), carnocyclin A (28), lactocyclicin Q (29), garvicin ML (30), leucocyclicin Q (31), acidocin B (32), and, most recently, enterocin NKR-5-3B (33). Enterocin NKR-5-3B (Ent53B), one of several bacteriocins produced by Enterococcus faecium NKR-5-3 previously isolated from the Thai fermented fish pla-ra (34)(35)(36), is a 64-amino-acid novel circular bacteriocin with a molecular mass of 6,316.4 Da. A nuclear magnetic resonance (NMR)-derived three-dimensional solution structure of Ent53B revealed the presence of four helical segments that enclose a tightly packed hydrophobic core (33).…”

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confidence: 99%

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

et al. 2016

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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...

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Monitoring of the multiple bacteriocin production by Enterococcus faecium NKR-5-3 through a developed liquid chromatography and mass spectrometry-based quantification system

Cited by 33 publications

References 29 publications

Screening and Characterization of Novel Bacteriocins from Lactic Acid Bacteria

Screening and Characterization of Novel Bacteriocins from Lactic Acid Bacteria

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

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

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