The Burkholderia contaminans MS14 ocfC gene encodes a xylosyltransferase for production of the antifungal occidiofungin.

Burkholderia contaminans strain MS14 produces the antifungal compound occidiofungin, which is responsible for significant antifungal activities against a broad range of plant and animal fungal pathogens. Occidiofungin is a cyclic glycolipopeptide made up of eight amino acids and one xylose. A 56-kb ocf gene cluster was determined to be essential for occidiofungin production. In this study, the ocfC gene, which is located downstream of ocfD and upstream of the ocfB gene in the ocf gene cluster, was examined. Antifungal activity of the ocfC gene mutant MS14KC1 was reduced against the indicator fungus Geotrichum candidum compared with that of the wild-type strain. Furthermore, the analysis of the protein sequence suggests that the ocfC gene encodes a glycosyltransferase. Biochemical analyses using nuclear magnetic resonance (NMR) and mass spectroscopy revealed that the ocfC mutant produced the occidiofungin without the xylose. The purified ocfC mutant MS14KC1 product had a level of bioactivity similar to that of the wild-type product. The revertant MS14KC1-R of the ocfC mutant produced the same antifungal activity level on plate assays and the same antifungal compound based on high-performance liquid chromatography (HPLC) and mass spectroscopy analysis as wild-type strain MS14. Collectively, the study demonstrates that the ocfC gene encodes a glycosyltransferase responsible to add a xylose to the occidiofungin molecule and that the presence of the xylose is not important for antifungal activity against Candida species. The finding provides a novel variant for future studies aimed at evaluating its use for inhibiting clinical and agricultural fungi, and the finding could also simplify the chemical synthesis of occidiofungin variants.

Site-directed mutations in the lanthipeptide mutacin 1140.

The oral bacterium Streptococcus mutans, strain JH1140, produces the antibiotic mutacin 1140. Mutacin 1140 belongs to a group of antibiotics called lanthipeptides. More specifically, mutacin 1140 is related to the epidermin type A(I) lanthipeptides. Mutagenesis experiments of this group of lanthipeptides have been primarily restricted to the posttranslationally modified meso-lanthionine and 3-methyllanthionine residues. Site-directed mutagenesis of the core peptide of mutacin 1140 was performed using the suicide vector pVA891. Substitutions of the N-terminal residue, the charged residue in the hinge region, and residues in ring A and intertwined rings C and D were investigated. A truncation and insertion of residues in ring A and intertwined rings C and D were also performed to determine whether or not they would alter the antimicrobial activity of the producing strain. Bioassays revealed that five of 14 mutants studied had improved antimicrobial activity against the indicator strain Micrococcus luteus ATCC 10240. MICs against Streptococcus mutans UA159, Streptococcus pneumoniae ATCC 27336, Staphylococcus aureus ATCC 25923, Clostridium difficile UK1, and Micrococcus luteus ATCC 10240 were determined for three mutacin 1140 variants that had the most significant increases in bioactivity in the M. luteus bioassay. This mutagenesis study of the epidermin group of lanthipeptides shows that antimicrobial activity can be significantly improved.