Characterization of Mundticin L, a Class IIa Anti- Listeria Bacteriocin from Enterococcus mundtii CUGF08

Abstract

Enterococcus mundtii CUGF08, a lactic acid bacterium isolated from alfalfa sprouts, was found to produce mundticin L, a new class IIa bacteriocin that has a high level of inhibitory activity against the genus Listeria. The plasmid-associated operons containing genes for the mundticin L precursor, the ATP binding cassette (ABC) transporter, and immunity were cloned and sequenced. The fifth residue of the conservative consensus sequence YGNGX in the mature bacteriocin is leucine instead of valine in the sequences of the homologous molecules mundticin KS (ATO6) and enterocin CRL35. The primary structures of the ABC transporter and the immunity protein are homologous but unique.Bacteriocins are ribosomally synthesized proteinaceous compounds that inhibit closely related bacteria (19). Due to consumer concerns with chemical and irradiation preservation methods and due to the rising demand for minimally processed food products, alternative methods for shelf life extension and enhanced safety are needed. Bacteriocins are considered "natural" antimicrobials since many bacteriocins are produced by food grade lactic acid bacteria, which are generally recognized as safe. Bacteriocins can be divided into three main classes: the class I lanthionine-containing lantibiotics, exemplified by nisin; the class II non-lanthionine-containing bacteriocins; and the class III heat-labile, large proteins (6). Class III bacteriocins have limited application due to their thermal instability and cytolytic activity against eukaryotic cells. Class II can be further divided into class IIa containing pediocin-like bacteriocins, class IIb containing two-peptide bacteriocins, and class IIc containing other bacteriocins (8). Class IIa bacteriocins have been extensively studied since pediocin PA-1 was first discovered (12) and characterized (20). Currently, only nisin in class I has been approved by the FDA as a natural food additive. Bacteriocins belonging to class IIa are promising alternative antimicrobials since they are more stable over a broader range of heating regimens and pH conditions. In addition, these bacteriocins exhibit stronger antimicrobial activity against the genus Listeria than nisin (17) but have a narrower antimicrobial spectrum.The potential applications of class IIa bacteriocins in both meat and plant-based foods as a means to provide protection against potential food-borne pathogens and extend shelf life continue to expand. In an attempt to use biological methods for controlling food-borne pathogens on fresh sprouts, a number of food grade lactic acid bacteria were isolated from the indigenous microbiota on alfalfa sprouts. Some of these isolates were found to be bacteriocinogenic. This study describes a new class IIa bacteriocin, mundticin L produced by Enterococcus mundtii CUGF08 isolated from alfalfa sprouts.Bacteriology. The mundticin L producer E. mundtii CUGF08 was obtained from alfalfa sprouts. The genus and species were determined by BLAST homology analysis (http://blast.ncbi.nlm .nih.gov/blast.cgi) of the 16S rRNA...

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

“…Further analyses are necessary to characterize the entire bacteriocin genetic loci of E. mundtii WFE3, WFE20, and WFE31. Some mundticins have been reported to be encoded by gene clusters carried by plasmids (Feng et al, 2009;Kawamoto et al, 2002), but some E. mundtii strains have been found to express bacteriocins by chromosomal genes . A character residing on chromosomal DNA is more stable than plasmid encoded information and may be significant for future applications of these strains at industrial level.…”

“…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.…”

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).…”

Natural Antimicrobials for Biopreservation of Sprouts

“…However, in spite of its capacity for growth and bacteriocin production under MA, this strain failed to inhibit the growth of L. monocytogenes on sprouts and for this reason was not likely to be feasible for application as a protective culture (Bennik et al, 1999). More recently, the bacteriocin-producing strain E. mundtii CUGF08 isolated from alfalfa sprouts was shown to produce an anti-listerial bacteriocin called mundticin L (Feng et al, 2009). This bacteriocin is highly similar to mundticin ATO6, differing in only by one amino acid residue in the YYGNGV (YYGNGL) motif and the last two amino acid residues, which are reversed as in the almost identical mundticin SK (Kawamoto et al, 2002).…”