UC San Diego

Thiazole/Oxazole Modified Microcins (TOMMs) represent a large, widely distributed family of ribosomally encoded natural products. This family of enzymes selectively modify cysteine, serine and threonine amino acid residues to five-membered heterocyclic rings. A diverse set of bacterial organisms have utilized the TOMM biosynthetic pathway to create a multitude of natural products with a wide range of activities. Chapter II describes how the TOMM family was created using bioinformatic searches to expand the known set of natural product biosynthetic gene clusters from four to over 250, each encoding a different natural product. Included in this new family is streptolysin S (SLS) a virulent toxin produced by human pathogenic bacteria Streptococcus pyogenes. We showed through in vitro analysis that SLS is a heterocycle containing TOMM natural product. In Chapter III we used biochemical studies to identified the precursor peptide residues that were essential for SLS hemolytic activity. From this information we were able to generate artificial precursor peptides that had hemolytic activity upon TOMM posttranslational modification. It was shown that SLS biosynthesis relies on the same substrate binding motif as microcin B17. In Chapter IV a SLS-like TOMM cluster in Clostridium botulinum was characterized. Mass spectrometry was used to identify sites of heterocyclization in SLS as well as in its close relative clostridiolysin S. Chapter V described the generation of an E. coli based bioengineering strategy for reconstituting SLS activity ex vivo, the first example of an active SLS analog being produced in another organism outside of the firmicutes phylum. This represents a method for generating high yields of TOMM natural products in a high throughput manner. In Chapter VI we validated the expression of the unannotated TOMM precursor peptides identified in Chapter II. RT-PCR, gene knockouts and in vitro activity assays were used to demonstrate expression