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.

FIG. 1.

Separation of purified mundticin KS by Tricine-SDS-PAGE. Lane 1, Rainbow protein size markers; lane 2, mundticin KS stained with Coomassie brilliant blue R250; lane 3, mundticin KS overlaid with cells of E. faecium IFO13712 embedded in MRS agar (0.8% [wt/vol] agar). The arrow indicates the active peptide band.

FIG. 2.

(A) Gene organization of the mun locus. Horizontal arrows that designate the direction of transcription represent the ORFs. Filled and open bars indicate putative promoters. Two arrowheads in opposite directions indicate terminators. Relevant nucleotide numbers and restriction sites are also shown. (B) Nucleotide sequence of the region encoding mundticin KS and the deduced amino acid sequence. Putative promoter regions are indicated in lowercase letters, and ribosome binding sites (RBS) are underlined. The N-terminal amino acid sequence of purified mundticin KS determined in this study is underlined. The arrow indicates the processing site of the peptide. The nucleotide sequence with arrows in opposite directions represents a transcriptional terminator with a stem and loop structure. The nucleotide sequence of the mun locus has been deposited in DDJB under accession number AB066267.

FIG. 3.

Deletion mutant analysis of the mun locus. Deletion mutants of the mun locus constructed in the vector plasmid, pRH100, by PCR are depicted on the left. Filled and open bars represent putative promoters. Two arrowheads in opposite directions indicate terminators. E. faecium IFO13712 transformants harboring the indicated deletion mutant plasmids were tested for production of and immunity against mundticin KS, as described in Materials and Methods. Symbols: +, production of or resistance to mundticin KS; −, no production of or sensitivity to mundticin KS. ND, not determined.

FIG. 4.

Bacteriocin activity of the recombinant peptides of mundticin KS and mundticin ATO6 expressed in E. coli. (A) Specific activities of the peptides against E. faecium IFO13712. Amino acid sequences of the recombinant peptides with and without rTEV digestion, along with that of native mundticin KS, are shown on the left. The His tag sequence is written in small capital letters, and the rTEV protease cleavage site is underlined. (B) Western analysis of the peptides. Each lane contained 0.1 μg of the peptides. After Tricine-SDS-PAGE, Western blotting and detection were done using the anti-mundticin KS serum as described in Materials and Methods. Lane 1, His-rMunKS peptide; lane 2, His-rMunATO6 peptide; lane 3, rMunKS peptide; lane 4, rMunATO6 peptide; lane 5, native mundticin KS. (C) Bacteriocin activity of rMunKS and rMunATO6 peptides to LAB strains. In 20 μl of solution, 200, 67, and 40 ng of the peptides were added to wells 1, 2, and 3, respectively, of each MRS agar plate seeded with the indicated LAB strain cells. The plates were then incubated overnight at 30°C.

FIG. 5.

Mundticin KS production in heterogeneous hosts by transformation with pRK1 containing mun locus. (A) Bacteriocin activity of culture supernatants from transformants. pRH100 (vector) or pRK1 transformants of the indicated LAB strains were grown in MRS broth containing erythromycin (1 μg/ml) at 30°C overnight. Each well of the MRS agar plate seeded with E. faecium IFO13712 cells contained 20 μl of the culture supernatants. (B) Western analysis of culture supernatants from transformants. Western blotting and detection were done using the anti-mundticin KS serum as in Fig. 4. Each lane contained 20 μl of the culture supernatants except that lane 1 contained 0.1 μg of purified mundticin KS. Lane 2, pRH100 transformant of E. faecium; lane 3, pRK1 transformant of E. faecium; lane 4, pRH100 transformant of L. lactis; lane 5, pRK1 transformant of L. lactis; lane 6, pRH100 transformant of L. curvatus; lane 7, pRK1 transformant of L. curvatus.

FIG. 6.

Localization of the mun locus in a large plasmid, pML1, of E. mundtii NFRI 7393. (A) Plasmid profiles of E. mundtii NFRI 7393. The plasmids were separated by 0.6% agarose gel electrophoresis. Lane 1, a marker plasmid, pV1, of 28 kb (20); lane 2, plasmids of E. mundtii NFRI 7393. (B) Localization of the mun locus by PCR analysis. pML1 and pMS1 plasmid DNAs were carefully gel purified. Using the purified pML1 plasmid DNA (lanes 2, 4, and 6) or pMS1 plasmid DNA (lanes 3, 5, and 7) as the template, PCR amplification for the mun locus was done with three sets of primer combinations. Combination 1, primers ORF3-F and ORF2-R (lanes 2 and 3); combination 2, primers BacN-F and ORF3-NR (lanes 4 and 5); combination 3, primers ORF1-F and ORF2-R (lanes 6 and 7). The mun locus was expected to generate PCR products of 0.8, 2.7, or 3.4 kb in length when primer combination 1, 2, or 3 was used, respectively. Lanes 1 and 8 contain 100-bp and 1-kb DNA ladder markers, respectively.