Regulation of bacilysin production and rescue of sporulation defects in a bacilysin negative strain by the sporulation transcription factors GerR and SpoVT in Bacillus subtilis

Date

2024-9

Author

Doğan, Berk Alan

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Bacillus subtilis is a Gram-positive bacteria notable for its transformability, extremely resistant endospores and antimicrobial secondary metabolites. Bacilysin is a nonribosomally synthesized dipeptide produced by B. subtilis and consists of the amino acids L-alanine and L-anticapsin. This small antibiotic acts as a glucosamine-6-phosphate synthetase inhibitor, blocking cell wall synthesis in bacteria and fungi. Our previous findings showed that the regulation of the bacilysin biosynthetic operon, the bac operon, is upregulated by sporulation initiation regulators such as Spo0A and sigma H and is inhibited by sporulation inhibiting regulators such as AbrB and CodY. We have also shown that the bacilysin silenced strain OGU1 is defective in multiple sporulation phenotypes including spore resistance, germination and sporulating colony morphology. Proteomic and transcriptomic studies have shown that OGU1 differentially expresses many genes, and underexpresses many sporulation genes. These suggest a two way link between bacilysin production and sporulation. In the present study, two underrepresented sporulation transcription factors, GerR and SpoVT, were cloned and overexpressed in OGU1 to see if their overexpression could rescue the bacilysin-negative defects. GerR and SpoVT were found to partially rescue these defects, though neither could fully rescue the defective phenotypes. To observe the effects of GerR and SpoVT on bac, beta-galactosidase assays, bacilysin bioassays and EMSAs were employed. GerR appears to be a positive regulator of bac, While SpoVT’s role on bac regulation remains uncertain, as the beta-galactosidase assay and bioassay produce conflicting results.

Subject Keywords

B. subtilis, Bacilysin, Sporulation, Germination, Transcription factors

URI

https://hdl.handle.net/11511/111404

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Graduate School of Natural and Applied Sciences, Thesis