Thirteen posttranslational modifications convert a 14-residue peptide into the antibiotic thiocillin

Abstract

The thiazolylpeptides are a family of >50 bactericidal antibiotics that block the initial steps of bacterial protein synthesis. Here, we report a biosynthetic gene cluster for thiocillin and establish that it, and by extension the whole class, is ribosomally synthesized. Remarkably, the C-terminal 14 residues of a 52-residue peptide precursor undergo 13 posttranslational modifications to give rise to thiocillin, making this antibiotic the most heavily posttranslationally-modified peptide known to date.

Fig. 1.

Schematic of the thiocillin gene cluster. A 22-kb gene cluster from B. cereus ATCC 14579 encodes 24 genes responsible for the production of the thiocillins. Four identical structural genes encode a 52-residue peptide, of which the last 14 residues undergo 13 posttranslational modifications of 6 varieties to become the mature thiocillins. Gray squares indicate the R groups in the thiocillin family members. The peptide residues are numbered starting with the first residue after the leader peptide; thus, Ser-39 = Ser-1.

Fig. 2.

Characterization of thiocillins by HPLC and MS. (A) Schematic of the thiocillin gene cluster, showing the plasmid integration sites of insertional mutants IM1, IM2, and IM4, and control strain IM3. (B) HPLC stack plot showing methanolic extracts of cell material from cultures of B. cereus ATCC 14579, insertional mutants IM1, IM2, IM4, and control strain IM3. Peaks corresponding to compounds 1–8 are labeled. Thiocillin production is abolished in the insertional mutants, whereas in IM3, flux is shifted toward the nonhydroxylated thiocillins 1, 2, 5, and 8. (C) High-resolution MS data for compounds 1–8.

Fig. 3.

Cryptic dehydration in the proposed mechanism for pyridine and dehydropiperidine ring formation. Dehydration of Ser-1 and Ser-10 yields Dha residues at both positions. Subsequent cyclization leads to cleavage of the leader peptide and formation of the fully aromatic pyridine in the thiocillins, whereas a portion of the leader peptide is retained and the nonaromatic piperidine or dehydropiperidine is formed in the thiopeptins. The pyridine and (dehydro)piperidine rings are shown in pink, and the leader peptide fragment and leader peptide/quinaldic acid loop are shown in blue.

Fig. 4.

Thiazolylpeptide and related gene clusters. The gene clusters for thiocillin and goadsporin and the predicted gene cluster for berninamycin are shown at the top; the structural peptide sequence is shown below the strain name, with the small molecule-encoding C-terminal sequence highlighted in red. Ten other gene clusters harboring homologs of both the heterocycle-forming (tclJ/tclN) and the Dha/Dhb-forming (tclK/tclL) genes are shown.