Background: Streptolysin S (SLS) is a cytolytic virulence factor produced by the human pathogen Streptococcus pyogenes and other Streptococcus species. Related “SLS-like” toxins have been characterized in select strains of Clostridium and Listeria, with homologous clusters bioinformatically identified in a variety of other species. SLS is a member of the thiazole/oxazole-modified microcin (TOMM) family of natural products. The structure of SLS has yet to be deciphered and many questions remain regarding its structure-activity relationships. Results: In this work, we assessed the hemolytic activity of a series of C-terminally truncated SLS peptides expressed in SLS-deficient S. pyogenes. Our data indicate that while the N-terminal poly-heterocyclizable (NPH) region of SLS substantially contributes to its bioactivity, the variable C-terminal region of the toxin is largely dispensable. Through genome mining we identified additional SLS-like clusters in diverse Firmicutes, Spirochaetes and Actinobacteria. Among the Spirochaete clusters, naturally truncated SLS-like precursors were found in the genomes of three Lyme disease-causing Borrelia burgdorferi sensu lato (Bbsl) strains. Although unable to restore hemolysis in SLS-deficient S. pyogenes, a Bbsl SLS-like precursor peptide was converted to a cytolysin using purified SLS biosynthetic enzymes. A PCR-based screen demonstrated that SLS-like clusters are substantially more prevalent in Bbsl than inferred from publicly available genome sequences. Conclusions: The mutagenesis data described herein allowed us to define the minimal cytolytic unit of SLS as the NPH region. Interestingly, this region is found in all characterized TOMM cytolysins, as well as the novel putative TOMM cytolysins we discovered. We propose that this conserved region represents the defining feature of the SLS-like TOMM family. We demonstrate the cytolytic potential of a Bbsl SLS-like precursor peptide, which is of similar length to the SLS minimal cytolytic unit, when modified with purified SLS biosynthetic enzymes. As such, we speculate that some Borrelia have the potential to produce a TOMM cytolysin, although the biological significance of this finding remains to be determined. In addition to providing new insight into the structure-activity relationships of SLS, this study greatly expands the cytolysin group of TOMMs.