Mundticin

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.

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.

The antilisterial class IIa bacteriocins, plantaricin 423 and mundticin ST4SA, have previously been purified from the cell-free supernatants of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, respectively. Here, we present the fusions of mature plantaricin 423 and mundticin ST4SA to His-tagged green fluorescent protein (GFP) for respective heterologous expression in Escherichia coli. Fusion of plantaricin 423 and mundticin ST4SA to His-tagged GFP produced the fusion proteins GFP-PlaX and GFP-MunX, respectively. Both fusion proteins were autofluorescent, circumvented inclusion body formation and lowered the toxicity of class IIa bacteriocins during heterologous expression. Not only did GFP-class IIa fusion stabilize heterologous expression and boost yields, the fluorescent intensity of GFP-PlaX and GFP-MunX could be monitored quantitatively and qualitatively throughout expression and purification. This robust fluorometric property allowed rapid optimization of conditions for expression and bacteriocin liberation from GFP via the incorporated WELQut protease cleavage sequence. Incubation temperature and IPTG concentration had a significant effect on bacteriocin yield, and was optimal at 18°C and 0.1-0.2 mM, respectively. GFP-MunX was approximately produced at a yield of 153.30 mg/L culture which resulted in 12.4 mg/L active mundticin ST4SA after liberation and HPLC purification. While GFP-PlaX was produced at a yield of 121.29 mg/L culture, evidence suggests heterologous expression resulted in conformation isomers of WELQut liberated plantaricin 423.