Early sporulation transcription factor rsfa has a strong impact on the regulation of bac operon activity in Bacillus subtilis

Date

2025-8-28

Author

Belen, Doğay

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Bacillus subtilis is a Gram-positive, endospore-forming bacterium widely employed in both fundamental research and various biotechnological applications. This organism produces bacilysin, the simplest known peptide antibiotic. Previous - omics-based studies conducted in our laboratory demonstrated that a bacilysindeficient mutant derivative of the PY79 strain, namely OGU1, exhibits lower expression of numerous sporulation-related genes, suggesting a potential regulatory role of bacilysin in sporulation processes. The rsfA gene encodes a transcription factor which has its own regulon. Although the rsfA regulon remains poorly characterized in the literature, RsfA is known to participate in two major sporulation regulons governed by the sigma factors SigF and SigG. Previous findings have shown that rsfA is transcriptionally upregulated in OGU1 relative to PY79, while its protein product RsfA is downregulated, suggesting a possible post-transcriptional regulation. The present thesis investigates the molecular interactions between RsfA protein and the bacABCDEF operon responsible for bacilysin biosynthesis. In this study, rsfA was first cloned into E. coli cells using a suitable cloning plasmid vector and subsequently subcloned into OGU1 using an appropriate expression vector. The consequences of RsfA overproduction on sporulation, bac operon expression, and bacilysin production were then assessed through comparative phenotypic analyses, RT-qPCRs, β-galactosidase assay, and bacilysin bioassay. Furthermore, electrophoretic mobility shift assays (EMSA) were conducted to explore potential direct interactions between purified RsfA protein and the bac operon promoter, as well as those between bacilysin molecule and the rsfA promoter.

Subject Keywords

Bacillus subtilis, Transcription factors, Sporulation, Germination, RsfA, Bacilysin

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https://hdl.handle.net/11511/115675

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Graduate School of Natural and Applied Sciences, Thesis