Lantibiotic nukacin

The lantibiotic nukacin ISK-1 exerts antimicrobial activity through binding to lipid II. Here, we perform NMR analyses of the structure of nukacin ISK-1 and the interaction with lipid II. Unexpectedly, nukacin ISK-1 exists in two structural states in aqueous solution, with an interconversion rate on a time scale of seconds. The two structures differ in the relative orientations of the two lanthionine rings, ring A and ring C. Chemical shift perturbation induced by the titration of lipid II reveals that only one state was capable of binding to lipid II. On the molecular surface of the active state, a multiple hydrogen-bonding site formed by amino acid residues in the ring A region is adjacent to a hydrophobic surface formed by residues in the ring C region, and we propose that these sites interact with the pyrophosphate moiety and the isoprene chain of the lipid II molecule, respectively.

Nukacin 3299 (formerly designated simulancin 3299), produced by a Staphylococcus simulans strain involved in bovine mastitis in Brazil, is the first peptide bacteriocin described in this staphylococcal species. With the intent to elucidate some aspects of its biology, nukacin 3299 was purified and characterized. The mass of the purified bacteriocin was shown to be 2957.3 Da, and the peptide N-terminal amino acids (KKKSGVI) were identified by Edman degradation. The nukacin 3299 structural gene, nukA, was detected by PCR and DNA sequencing, showing that this bacteriocin is identical to nukacin ISK-1, a 27-amino acid type-A (II) lantibiotic produced by Staphylococcus warneri ISK-1, isolated from a "nukadoko", in Japan. The genes involved in nukacin 3299 biosynthesis are located on plasmid pRJ97 (>27 kb). They have an organization similar to that of the nukacin ISK-1 gene cluster, excepted for the presence of an IS257/431 element (791 bp) present between the orf1 and nukA genes of the nukacin 3299 gene cluster. The presence of this insertion sequence is expected to affect the expression of orf1, whose function is presently unknown. Nukacin 3299 proved to be sensitive to proteolytic enzymes and relatively stable at different temperatures and between pH 3.0-9.0. Nukacin 3299 exhibited activity towards staphylococcal strains involved in bovine mastitis, showing a potential application on mastitis control, a disease with great economic impact.

We determined the mode of action of nukacin ISK-1. It did not cause membrane potential dissipation or the efflux of ATP or K(+) ions from the cells of a sensitive bacterial strain; however, it blocked the membrane depolarization activity of nisin. Nukacin ISK-1-treated cells had single arrangements of cells without the formation of a complete septum. A remarkable reduction in cell wall width was also observed, but cytoplasmic content was unaffected. We concluded that nukacin ISK-1 is bacteriostatic.