RESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Bacteriocinas/metabolismo , Enterococcus faecium/metabolismo , Transporte Biológico , Fermentación , Señales de Clasificación de ProteínaRESUMEN
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.
RESUMEN
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 Qß. 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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Bacteriocinas/metabolismo , Señales de Clasificación de Proteína/fisiología , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Transporte Biológico/genética , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Mutagénesis Sitio-Dirigida , Mutación Puntual/fisiología , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/genética , Vías Secretoras/genéticaRESUMEN
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.
Asunto(s)
Bacteriocinas/genética , Enterococcus faecium/metabolismo , Secuencia de Aminoácidos/genética , Bacteriocinas/metabolismo , Bacteriocinas/farmacología , Hidrocarburos Aromáticos con Puentes/metabolismo , Hidrocarburos Aromáticos con Puentes/farmacología , Clonación Molecular , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Enterococcus faecium/genética , Pruebas de Sensibilidad Microbiana , Mutación , Señales de Clasificación de Proteína/genéticaRESUMEN
UNLABELLED: 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. IMPORTANCE: 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.
Asunto(s)
Bacteriocinas/biosíntesis , Enterococcus faecium/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Secuencia de Aminoácidos , Bacteriocinas/genética , Bacteriocinas/metabolismo , Clonación Molecular , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Datos de Secuencia MolecularRESUMEN
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 Leu(24) and Trp(87) to yield the mature (circular) enterocin NKR-5-3B.
Asunto(s)
Bacteriocinas/química , Enterococcus faecium/química , Bacteriocinas/biosíntesis , Bacteriocinas/genética , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Familia de Multigenes , Vías Biosintéticas , Hidrocarburos Aromáticos con Puentes/farmacología , Enterococcus faecium/aislamiento & purificación , Microbiología de Alimentos , Regulación Bacteriana de la Expresión Génica , TailandiaRESUMEN
Enterococcus faecium NKR-5-3 produces four antimicrobial peptides referred here as enterocins NKR-5-3A, B, C and D. A two-step electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS)-based quantification system was developed to monitor its multiple bacteriocin production profiles, which is essential in understanding the complex production regulation mechanism of strain NKR-5-3. The developed ESI-LC/MS-based quantification system can easily monitor the multiple bacteriocin production of this strain. Using the developed system, the production of enterocin NKR-5-3B was found to be not as variable as those of the other enterocins in different cultivation media. Production of enterocin NKR-5-3B was also found to have a wider optimum incubation temperature (20-30°C) than enterocins NKR-5-3A, C and D (25°C). Furthermore, at least 2 nM of the bacteriocin-like inducing peptide, enterocin NKR-5-3D, regulated the production of NKR-5-3 enterocins except enterocin NKR-5-3B. These findings taken together suggest that enterocin NKR-5-3B has an independent production regulation mechanism from the other NKR-5-3 enterocins. The developed system could effectively pin-point the production profiles of the multiple bacteriocins of E. faecium NKR-5-3 under different fermentation conditions.
Asunto(s)
Antibacterianos/biosíntesis , Bacteriocinas/biosíntesis , Cromatografía Liquida/métodos , Enterococcus faecium/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Antibacterianos/análisis , Bacteriocinas/análisis , Fermentación , Péptidos/aislamiento & purificación , TemperaturaRESUMEN
The structure of enterocin NKR-5-3C, an anti-listerial bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3, was determined. Enterocin NKR-5-3C is a novel class IIa bacteriocin that possesses an YGNGL motif sequence and two disulfide bridges in its structure. It is encoded on gene ent53C together with an 18-amino-acid-residue double glycine leader peptide.
Asunto(s)
Bacteriocinas/química , Enterococcus faecium/genética , Genes Bacterianos , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Bacteriocinas/genética , Bacteriocinas/farmacología , Secuencia de Bases , Disulfuros/química , Enterococcus faecium/metabolismo , Listeria/efectos de los fármacos , Listeria/crecimiento & desarrollo , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Análisis de Secuencia de ProteínaRESUMEN
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.
Asunto(s)
Bacteriocinas/aislamiento & purificación , Enterococcus faecium/metabolismo , Peces/microbiología , Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Bacteriocinas/biosíntesis , Bacteriocinas/genética , Secuencia de Bases , Cromatografía de Fase Inversa , Enterococcus faecium/genética , Fermentación , Lactobacillus/efectos de los fármacos , Lactobacillus/metabolismo , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
Enterococcus faecalis NKR-4-1 isolated from pla-ra produces a novel two-peptide lantibiotic, termed enterocin W, comprising Wα and Wß. The structure of enterocin W exhibited similarity with that of plantaricin W. The two peptides acted synergistically, and their order of binding to the cell membrane was important for their inhibitory activity.
Asunto(s)
Bacteriocinas/aislamiento & purificación , Bacteriocinas/metabolismo , Enterococcus faecalis/metabolismo , Secuencia de Aminoácidos , Antibacterianos/farmacología , Bacteriocinas/química , Bacteriocinas/farmacología , Secuencia de Bases , Hidrocarburos Aromáticos con Puentes/química , Hidrocarburos Aromáticos con Puentes/aislamiento & purificación , Hidrocarburos Aromáticos con Puentes/metabolismo , Hidrocarburos Aromáticos con Puentes/farmacología , Membrana Celular/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Dipéptidos/química , Dipéptidos/aislamiento & purificación , Dipéptidos/metabolismo , Dipéptidos/farmacología , Sinergismo Farmacológico , Genes Bacterianos/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
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 KQU-131, one residue in the leader peptide and the other two in the mature peptide.