Recently, foodborne Staphylococcus equorum WS2733 was isolated from a French red smear cheese on account of its strong inhibitory activity against Gram-positive pathogens such as Listeria. The antagonistic substance was identified as macrocyclic
peptide antibiotic micrococcin P1, which had previously not been reported for the genus Staphylococcus.
Micrococcin P1, also a potent inhibitor of the
malaria parasite Plasmodium falciparum, is structurally related to
thiostrepton, thiocillins and
nosiheptide. Although all of these
peptide antibiotics have been known for quite a long time, their mode of biosynthesis had not been determined in detail yet. By using degenerated PCR, a gene fragment encoding a
nonribosomal peptide synthetase (NRPS) could be amplified from S. equorum. The corresponding chromosomal locus was disrupted by insertional mutagenesis, and it could be shown that all mutants obtained displayed a
micrococcin P1-deficient phenotype. Sequence analysis of a coherent 2.8-kb fragment revealed extensive homology to known NRPSs, and allowed the assignment of the domain organization 'condensation-adenylation-thiolation-condensation'; an arrangement predicted only for two loci within the presumably 14-modular, 1.6-MDa biosynthetic NRPS template. Biochemical characterization of the adenylation domain exhibited selectivity for the substrate
amino-acid threonine. All of these data substantiate that the macrocyclic
peptide antibiotic is biosynthesized nonribosomally, and provide the basis for the characterization of the entire biosynthetic gene cluster. The biosynthetic machinery of
micrococcin will serve as a model system for structurally related, pharmacologically important pyridinyl polythiazole class
peptide antibiotics. Furthermore, this knowledge will enable the manipulation of its NRPS template, which in turn may grant the targeted engineering of even more potent anti-listerial and anti-
malaria drugs.