Characterization of a Novel Plasmid-Borne Thiopeptide Gene Cluster in Staphylococcus epidermidis Strain 115

Abstract

bThiopeptides are small (12-to 17-amino-acid), heavily modified peptides of bacterial origin. This antibiotic family, with more than 100 known members, is characterized by the presence of sulfur-containing heterocyclic rings and dehydrated residues within a macrocyclic peptide structure. Thiopeptides, including micrococcin P1, have garnered significant attention in recent years for their potent antimicrobial activity against bacteria, fungi, and even protozoa. Micrococcin P1 is known to target the ribosome; however, like those of other thiopeptides, its biosynthesis and mechanisms of self-immunity are poorly characterized. We have discovered an isolate of Staphylococcus epidermidis harboring the genes for thiopeptide production and self-protection on a 24-kb plasmid. Here we report the characterization of this plasmid, identify the antimicrobial peptide that it encodes, and provide evidence of a target replacement-mediated mechanism of self-immunity.

Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

“…A number of incisive studies have delineated key players from the 24-gene tcl cluster and have provided insights into how the Tclprocessing machinery tolerates alterations in the core peptide sequence (25)(26)(27)(28). More recently, we identified a second micrococcin gene cluster in a strain of Staphylococcus epidermidis (now classified as Macrococcus caseolyticus) strain 115 (29). This gene cluster is considerably smaller (12 genes) ( Fig.…”

“…subtilis transformation. Transformation of B. subtilis was accomplished by using the previously described starvation-induced competence protocol (29). Following selection, colonies were verified for correct integration of transgenes using a modified colony PCR protocol as follows: 1 ml of a saturated overnight culture was pelleted and resuspended in 200 l of B. subtilis lysis buffer (50 mM Tris with 0.5 mg/ml lysozyme).…”

Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

“…Successful integration into B. subtilis was confirmed by PCR with primers spanning chromosomal integration junctions. Bioassays of methanolic extracts from B. subtilis micrococcin expression strains were conducted as described previously (13,14).…”

“…It is additionally oxidatively decarboxylated at the C-terminal Thr residue, initially to a ketone to form micrococcin P2 (MP2), which may be rereduced to an alcohol, yielding micrococcin P1 (MP1) (13). Micrococcin/thiocillin (tcl) biosynthetic gene clusters have been identified in Macrococcus caseolyticus strain 115 (Mc) and Bacillus cereus ATCC 14579 (Bc) (14,15). The Bc cluster is comparatively large (24 chromosomal genes) and controls the synthesis of a mixture of eight micrococcin/thiocillin variants, whereas the simpler Mc gene cluster (12 plasmid-encoded genes) dictates the synthesis of a single micrococcin (MP1) product (14,15).…”

“…Micrococcin/thiocillin (tcl) biosynthetic gene clusters have been identified in Macrococcus caseolyticus strain 115 (Mc) and Bacillus cereus ATCC 14579 (Bc) (14,15). The Bc cluster is comparatively large (24 chromosomal genes) and controls the synthesis of a mixture of eight micrococcin/thiocillin variants, whereas the simpler Mc gene cluster (12 plasmid-encoded genes) dictates the synthesis of a single micrococcin (MP1) product (14,15). Mc requires only 9 of its 12 tcl genes to synthesize MP1 (SI Appendix, Fig.…”