Characterization of a novel plasmid-borne thiopeptide gene cluster in Staphylococcus epidermidis strain 115

Abstract

Thiopeptides 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.

FIG 1

Structures of various thiopeptide antibiotics. Panels: A, MP1; B, thiostrepton; C, GE2270A.

FIG 2

The antimicrobial activity of S. epidermidis strain 115 is plasmid encoded. (A) Examples of flanking-patch assays for production and immunity. In each triplet, the central patch was applied to the agar 18 h prior to the flanking patches. 115C is a pBac115-cured derivative. Bs corresponds to B. subtilis 168, a susceptible indicator strain. (B) Plasmid preparations from S. epidermidis 115 and HS derivatives. Lane M, molecular size markers.

FIG 3

pBac115 contains thiopeptide biosynthetic (tcl) genes. (A) Plasmid map of pBac115 with annotation based upon results from BLASTx and direct alignments with previously identified tcl genes. (B) Amino acid sequence of the TclE precursor peptide. The asterisk indicates the predicted cleavage site; the core peptide, which undergoes modification to produce the mature thiopeptide, is underlined.

FIG 4

Biochemical analysis of S. epidermidis 115 antimicrobial activity. (A) RP-HPLC analysis of purified material from strain 115 and material similarly prepared from pBac115-cured strain 115C. Abs, absorbance. (B) ESI-MS fragmentation of the major HPLC peak from strain 115. The inset shows a closeup of the region from m/z 1,142 to m/z 1,168. (C) Comparison of observed m/z values with those predicted for MP1.

FIG 5

Comparisons of the tcl gene clusters from S. epidermidis 115 and B. cereus ATCC 14579. Connectors indicate homologous genes based on BLASTp comparisons of their predicted protein products. Predicted functions are shown in the color-coded key.

FIG 6

Allelic-exchange experiments at the L11-encoding rplK locus. (A) Map of rplK alterations of the B. subtilis 168 (Bs) chromosome, with strain names indicated. ΔP22 is an abbreviation for the B. subtilis rplKΔP22 allele, which is known to give resistance to MP1. The tclQ allele was amplified from S. epidermidis 115. (B) Spot dilution assays showing growth at 24 h on LB agar with or without MP1 (18 μg/ml). The rows correspond to the genotypes indicated in panel A. Cm, chloramphenicol.