Genome mining for radical SAM protein determinants reveals multiple sactibiotic-like gene clusters

Abstract

Thuricin CD is a two-component bacteriocin produced by Bacillus thuringiensis that kills a wide range of clinically significant Clostridium difficile. This bacteriocin has recently been characterized and consists of two distinct peptides, Trnβ and Trnα, which both possess 3 intrapeptide sulphur to α-carbon bridges and act synergistically. Indeed, thuricin CD and subtilosin A are the only antimicrobials known to possess these unusual structures and are known as the sactibiotics (sulplur to alpha carbon-containing antibiotics). Analysis of the thuricin CD-associated gene cluster revealed the presence of genes encoding two highly unusual SAM proteins (TrnC and TrnD) which are proposed to be responsible for these unusual post-translational modifications. On the basis of the frequently high conservation among enzymes responsible for the post-translational modification of specific antimicrobials, we performed an in silico screen for novel thuricin CD-like gene clusters using the TrnC and TrnD radical SAM proteins as driver sequences to perform an initial homology search against the complete non-redundant database. Fifteen novel thuricin CD-like gene clusters were identified, based on the presence of TrnC and TrnD homologues in the context of neighbouring genes encoding potential bacteriocin structural peptides. Moreover, metagenomic analysis revealed that TrnC or TrnD homologs are present in a variety of metagenomic environments, suggesting a widespread distribution of thuricin-like operons in a variety of environments. In-silico analysis of radical SAM proteins is sufficient to identify novel putative sactibiotic clusters.

Figure 1. Diagrammatic representation of the thuricin CD-like gene clusters found in the TrnC and TrnD screens.

Genes involved in transport are coloured red, structural genes in blue, radical SAM encoding genes in yellow and genes involved in spore germination in green.

Figure 2. Protein sequence alignment of TrnD and all the TrnD homologs identified in this study.

Figure 3. Protein sequence alignment of TrnC and all the TrnC homologs identified in this study.

Figure 4. Rectangular cladogram of TrnD and all the TrnD homologs encountered during the screen.

Cladogram was generated using the R package and visualised in DENDROSCOPE.

Figure 5. Rectangular cladogram of TrnC and all the TrnC homologs encountered during the screen.

Cladogram was generated using the R package and visualised in DENDROSCOPE.