Date

2022-9-15

Author

Akaysoy, Sergen

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Bacillus subtilis is the Gram-positive model bacterium that enzymatically produces the dipeptide antibiotic bacilysin. Bacilysin is the simplest bioactive peptide known composed of L-alanine at its N-terminal and L-anticapsin at its C-terminal. In a former work in our laboratory, a mutant strain of B. subtilis, namely OGU1 was constructed by bacA-targeted pMutin T3 insertion into the parental strain PY79 genome resulting in a genomic organization bacA′::lacZ::erm::bacABCDEF and unable to synthesize bacilysin. RT-qPCR, transcriptome, secretome, and proteome studies conducted so far revealed that there are significant differences in the expression of a vast number of genes between OGU1 and PY79, including gerE and glmS which are significantly downregulated in OGU1. In B. subtilis, GerE, a member of LuxR-FixJ family of transcription regulators, is expressed late during sporulation in the mother cell compartment and acts as the master protein of its own regulon. It is involved in spore germination and spore coat assembly. glmS, on the other hand, encodes glutamine-fructose-6-phosphate transaminase for cell wall synthesis. These two genes were cloned in E. coli DH5α, next subcloned and expressed in E. coli BL21 and B. subtilis OGU1. The effects of these genes were determined by comparing the parental strain PY79, the mutant strain OGU1, and the OGU1 complemented with cloned and expressed gerE and glmS with each other, respectively. Phenotypic analyses were performed, including the resistance of endospores against different chemicals, the germination profile of endospores as well as colony morphology and pigmentation of each strain. Moreover, the possible interrelations between bac operon and gerE and glmS functions were investigated by performing electrophoretic mobility shift assays (EMSA). These assays were conducted to elucidate the possible DNA-protein interactions between the bac promoter and the purified GerE and GlmS proteins as well as the promoter regions of gerE and glmS genes and the purified bacilysin. The results indicated that GerE, but not GlmS binds to bac promoter while bacilysin did not display any interaction with gerE and glmS promoters.

Subject Keywords

Bacillus subtilis, Bacilysin, Gene cloning, bac operon, Phenotypic analyses, Electrophoretic Mobility Shift Assay (EMSA)

URI

https://hdl.handle.net/11511/99456

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Graduate School of Natural and Applied Sciences, Thesis