Each peptide of the two-component lantibiotic lacticin 3147 requires a separate modification enzyme for activity

The genetic determinants for production and immunity to the two-component l antibiotic lacticin 3147 are encoded by a 12.6 kb region of the plasmid pMR C01. this region contains ten genes arranged in two divergent clusters; the se include the structural genes and a number of genes whose products show s ignificant similarity to proteins involved in the biosynthesis of other lan tibiotics, Using a strategy of deletion and mutational analysis, the effect of disruption of a number of these genes was investigated. Inactivation of either of the structural genes, ItnA1 or ItnA2, resulted in mutants that w ere incapable of producing active lacticin 3147; however, the combination o f the cell-free supernatant from both mutants resulted in a restoration of bacteriocin activity, confirming that processing and export of the structur al peptides can occur independently. An unusual feature of the lacticin 314 7 gene cluster is the presence of two lanM homologues, whose gene products are proposed to be involved in the dehydration and thioether-forming reacti ons which result in lanthionine bridge formation. Mutants created in the It nM1 and ItnM2 genes were also incapable of lantibiotic production, confirmi ng an essential role for these enzymes in the lacticin 3147 biosynthetic pa thway and supporting the assertion that these proteins are modification enz ymes. Interestingly, addition of purified LtnA1, but not purified LtnA2, to the cell-free supernatant of the ItnM1 mutant restored bacteriocin activit y; in contrast, only purified LtnA2 could complement the cell-free supernat ant of the ItnM2 mutant. Creation of a number of double mutants supported t hese findings, and confirmed that LtnM1 is required to produce mature LtnA1 , while LtnM2 is required to produce mature LtnA2.