Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

Abstract

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

Production, stability, gene sequencing and in situ anti-Listeria activity of mundticin KS expressed by three Enterococcus mundtii strains

“…Genomic DNA from E. mundtii WFE3, WFE20 and WFE31 were used as templates for amplification with the primer pair Mnt-1F (5 0 -TGAGA-GAAGGTTTAAGTTTTGAAGAA-3 0 )/Mnt-1R (5 0 -TCCACTGAAATCCAT-GAATGA-3 0 ) mapping upstream of the coding sequence of known bacteriocins KS (Kawamoto et al, 2002), CRL35 (Saavedra, Minahk, de Ruiz Holgado, & Sesma, 2004), QU2 (Zendo et al, 2005) and MunL (Feng, Guron, Churey, & Worobo, 2009), applying the conditions described by Zendo et al (2005). DNA from E. mundtii PON10063 was used as negative control in PCRs.…”

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis

“…lactis IL1403 (pJP214) and L. lactis subsp. cremoris NZ9000 (pJR199) is, in most of the cases, comparatively higher than production of a number of other bacteriocins in different LAB hosts using dedicated ABC transport systems Van Belkum et al 1997;Axelsson et al 1998;Biet et al 1998;Horn et al 1998Horn et al , 1999Martínez et al 2000;Kawamoto et al 2002;Morisset and Frere 2002) or the sec pathway (Worobo et al 1995;Biet et al 1998;McCormick et al 1998McCormick et al , 1999, indicating that production of EntP by the sec-dependent pathway is an efficient process in L. lactis. Protein secretion is a preferred means of protein expression in the development of LAB as vehicles for the delivery of biologically active molecules (Dieye et al 2003).…”

“…Among the LAB, the enterococci produce a diverse and heterogeneous group of bacteriocins, coined enterocins, which are mutually different with respect to their antimicrobial activity, structure, processing, and secretion (Franz et al 1999a;Cintas et al 2001;Kawamoto et al 2002;Nilsen et al 2003). However, since enterocins may be produced by enterococcal species carrying antibiotic resistance genes and/or genes coding for potential virulence factors (Eaton and Gasson 2001;Franz et al 2001;Shankar et al 2002), interest in the heterologous production and functional expression of enterocins in other bacterial hosts is growing rapidly.…”

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

“…Peptides in subgroup 3 as well as enterocin SE-K4 and carnobacteriocin B2 in subgroup 4 exceptionally have neither the conserved tryptophan at the C-tails, nor the cysteine residues to form the C-terminal disulfide bridge (Table 2). Marrec et al 2000Metivier et al 1998Aymerich et al 1996Henderson et al 1992Marugg et al 1992Tichaczek et al 1994Kalmokoff et al 2001Birri et al 2010Feng 2009Kawamoto et al 2002Bennik et al 1998Yamazaki et al 2005Bhugaloo-Vial et al 1996Jack et al 1996Vaughan et al 2001Fimland et al 2002bHeng et al 2007 Subgroup 2 Kwaadsteniet et al 2006Quadri et al 1995Eguchi et al 2001Diep et al 2006 The YGNGV/L "pediocin box" is marked (bold), the leucine residue (L), odd, and uncertain amino acids (X) are marked as bold and red. Names of the six newly identified class IIa bacteriocins are in red (Rea et al 2011).…”