Discovery of a widely distributed toxin biosynthetic gene cluster

Abstract

Bacteriocins represent a large family of ribosomally produced peptide antibiotics. Here we describe the discovery of a widely conserved biosynthetic gene cluster for the synthesis of thiazole and oxazole heterocycles on ribosomally produced peptides. These clusters encode a toxin precursor and all necessary proteins for toxin maturation and export. Using the toxin precursor peptide and heterocycle-forming synthetase proteins from the human pathogen Streptococcus pyogenes, we demonstrate the in vitro reconstitution of streptolysin S activity. We provide evidence that the synthetase enzymes, as predicted from our bioinformatics analysis, introduce heterocycles onto precursor peptides, thereby providing molecular insight into the chemical structure of streptolysin S. Furthermore, our studies reveal that the synthetase exhibits relaxed substrate specificity and modifies toxin precursors from both related and distant species. Given our findings, it is likely that the discovery of similar peptidic toxins will rapidly expand to existing and emerging genomes.

Fig. 1.

Conservation of toxin biosynthesis operons in S. pyogenes and E. coli. (A) Genetic organization of the streptolysin S-associated gene cluster (sagA–I) from S. pyogenes and the E. coli microcin B17 gene cluster (mcbA–G). (B) Through the action of a trimeric synthetase complex, oxazole and thiazole heterocycles are incorporated into a peptidic protoxin scaffold (black) and are active in vitro. Chemical transformations carried out by SagC/McbB (green, cyclodehydratase) and SagB/McbC (yellow, dehydrogenase) orthologs are shown. Molecular mass change for each reaction is shown in daltons. SagD/McbD (blue) serves as an enzymatic scaffold and facilitates substrate binding. (C) Heterocycle formation on E. coli McbA by S. pyogenes SagBCD. Shown are calculated (calcd) and observed (obsd) molecular masses for MBP-McbA after reaction with SagBCD and thrombinolysis. Formation of a single thiazole/oxazole leads to the loss of 20 Da from the mass of the peptide. (D) Linear mode MALDI-TOF mass spectrum of McbA treated with SagBCD. The number of heterocycles on the protoxin peptide is indicated above the respective mass (singly charged species).

Fig. 2.

Cytolytic activity of in vitro synthetase reactions. (A) Hemolytic assays of SagA plus SagBCD synthetase reactions in microtiter wells containing defibrinated sheep blood. Bars indicate lysis normalized to a positive control (Triton X-100). Levels indicated are 1:1, 3:4, 1:2, and 1:4 ratios of synthetase reaction to blood (left to right) of 16-h reactions (n = 3). Lane 1, SagA plus SagBCD; lane 2, SagA alone; lane 3, SagBCD alone; lane 4, SagA plus SagBC; lane 5, SagA plus SagCD; lane 6, SagA plus SagBD; lane 7, vehicle. Inset demonstrates typical appearance of lytic (L) and nonlytic (N) reactions. (B) Fluorescence microscopy and DIC images of HEK293a cells treated as indicated. Actin filaments (red) and cytoplasm (green) are merged in Upper, and DIC images are in Lower. (C) LDH release assay of HEK293a cells treated with SagA plus SagBCD. Lysis is measured as A₄₉₀ normalized to positive control (Triton X-100). Lane 1, SagA plus SagBCD; lane 2, SagA alone; lane 3, SagA plus SagBC; lane 4, SagA plus SagCD; lane 5, SagA plus SagBD; lane 6, SagBCD alone; lane 7, SagA-panC/S plus SagBCD.

Fig. 3.

The biosynthetic operon for producing thiazole/oxazole-containing toxins is widely distributed. (A) Gene clusters from organisms containing SLS- and MccB17-like bacteriocins. Members are sorted by prokaryotic phylum. Relative gene length and directionality are shown (scale for actinobacteria and cyanobacteria is reduced by 50%). Each gene cluster contains a protoxin (A, black), dehydrogenase (B, yellow), cyclodehydratase (C, green), and a docking scaffold (D, blue). These genes produce both single-domain and fusion proteins. Numerous ancillary enzymes are included and increase the chemical diversity of the toxin family. (B) Select protoxin amino acid sequences. Predicted leader peptide cleavage sites are denoted with an asterisk. Hyphens indicate a known cleavage site. Potential sites of heterocyclization are indicated in blue, and known sites of heterocycle formation are indicated in red. Green text signifies conversion to dehydroalanine. Toxins similar to SagA (top) are predicted cytolysins.

Fig. 4.

Disparate heterocycle synthetases accept substrates from related and distant prokaryotes. (A) The S. pyogenes synthetase SagBCD accepts the C. botulinum protoxin and produces a cytolytic toxin. (Left) Hemolytic assay. (Center and Right) Fluorescence microscopy and DIC images of HEK293a cells treated as indicated. (B) Hemolytic assay of SagA and ClosA treated with the synthetase complex from E. coli (McbBCD) and the hyperthermophile P. furiosus (PagBCD). Recombinant MBP-McbD must be first treated with thrombin (thr) before synthetase activity is observed.