Expression of the Immunity Protein of Plantaricin 423, Produced by Lactobacillus plantarum 423, and Analysis of the Plasmid Encoding the Bacteriocin

Abstract

Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.Bacteriocins produced by lactic acid bacteria (LAB) have received considerable attention as food preservatives and as potential replacements of antibiotics (28). These peptides are classified based on their primary structure, and one of the largest groups, class II, containing small unmodified heat-stable proteins, is further divided into four subgroups (20). The pediocin-like bacteriocins of class IIa have a strong inhibitory effect on Listeria spp. and share a conserved sequence motif in their N termini (9). Bacteriocin genes may be present on the chromosome or on plasmids and usually occur in one or two operons encoding a structural protein, an immunity protein that protects the producer from its own bacteriocin, and several translocation genes.Plantaricin 423, a class IIa pediocin-like bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer, has been characterized at a protein and genetic level (31, 32). The genetic determinants for this bacteriocin are located on an 8,135-bp plasmid. The genes encoding plantaricin 423 are arranged in an operon with four open reading frames (ORFs) encoding the structural protein PlaA, putative immunity protein PlaB, and two putative membrane translocation proteins, PlaC and PlaD (31, 32). The first 31 amino acid residues in the N terminus of the prepeptide PlaA, which includes an 18-amino-acid leader peptide and 13 amino acid residues of the mature peptide (32), are identical to that of coagulin, pediocin PA-1, and pediocin AcH (...

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

“…The operons coding for the bacteriocins produced by L. plantarum 423 and E. mundtii ST4SA strains are situated on plasmids (46,51). The replicons of the bacteriocin-containing plasmids and the plasmid vectors used to introduce the mCherry marker are compatible, as was confirmed by plasmid isolations.…”

“…Enterococcus mundtii ST4SA was transformed using a modified version of the method described for Pediococcus (46). Ten milliliters of an overnight culture of E. mundtii ST4SA was inoculated into 100 ml MRS broth supplemented with 4.5% glycine, 0.5 M sucrose, and 40 mM threonine.…”

Cloning and Expression of Plantaricin W Produced by Lactobacillus plantarum U10 Isolate from “Tempoyak” Indonesian Fermented Food as Immunity Protein in Lactococcus lactis

“…Heterologous expression of several plantaricin peptides have been studied in Escherichia coli [16,17], L. sakei [18] and Saccharomyces cerevisiae [19]. In this paper, we report for the first time the cloning of plnW into lactococcal Nisin Controlled Expression (NICE) system.…”

Screening of lactic acid bacteria for bacteriocins by microbiological and PCR methods

“…Class II contains small (<10 kDa), heat-stable bacteriocins that are formed directly, without posttranslational modification. Examples are pediocin from Pediococcus pentosaceus and P. acidilactici [17], and enterocin and plantaricin 423 from Enterococcus faecium [18] and Lactobacillus plantarum 423 [19], respectively. Class III contains large bacteriocins (>30 kDa) that are heat-labile proteins.…”