Regulatory interactions between the transcription factors GerE and SpoIIID and Bac operon expression, sporulation and germination in Bacillus subtilis

Date

2024-9

Author

Koçak Karadöller, Selin

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Bacillus subtilis is a Gram-positive model organism capable of forming endospores and producing various antimicrobial secondary metabolites. One of these secondary metabolites is bacilysin, a nonribosomally synthesized dipeptide consisting of the amino acids L-alanine and L-anticapsin. Previous works by our group have shown the connection between bacilysin production and sporulation. Such that, the major sporulation regulators were shown to regulate the expression of the bac operon. In addition, a strain lacking bac operon expression, OGU1, was shown to form endospores with lowered resistances to heat, chloroform, and lysozyme as well as lowered germination rates. OGU1 was also shown to be underexpressing many major sporulation regulators suggesting a two-way connection between bacilysin production and sporulation. Two transcription factors that were downregulated in OGU1, GerE and SpoIIID were selected for this study. These genes were cloned and overexpressed in OGU1 to see if they could restore its sporulation defects where they were found to partially rescue these defects. In addition, the potential roles of these genes in bac regulation were studied. -galactosidase assays were employed in OGU1 to see how GerE and SpoIIID overexpression affected bac expression. Disc diffusion bioassays using gerE and spoIIID knockout strains were utilized to observe the effects of the knockouts on bacilysin production. These experiments showed conflicting results, with the -galactosidase assay suggesting both are negative regulators and the bioassay revealing that spoIIID and gerE knockout strains might positively regulate bacilysin production. Finally, EMSAs were performed to observe whether SpoIIID and GerE can directly regulate the bac operon expression. Both were shown to bind bac operon on their own and in co-existence. Moreover, adding external bacilysin enhanced the DNA binding of both proteins.

Subject Keywords

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

URI

https://hdl.handle.net/11511/111398

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Graduate School of Natural and Applied Sciences, Thesis