Phenotypic rescue of a Bacilysin-silenced strain of Bacillus Subtilis with Bacilysin supplementation

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2020-9

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Ertekin, Ozan

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Bacillus subtilis, the model organism for the gram positive bacteria, is distinguished for its secondary metabolite synthesis. Remarkably, most of these metabolites are small peptides which are generally synthesized non-ribosomally with multifunctional enzyme complexes. Bacilysin, a dipeptide composed of L-alanine and L-anticapsin, is the smallest peptide antibiotic which is produced by several enzymatical steps, however, its biosynthesis does not fully fit the general mechanisms of NRPSs. This small molecule acts specifically as an glucosamine-6-phosphate synthase inhibitor, thus interfering with cell wall formation in target microbes. Previously, it was found that the synthesis of bacilysin is governed by bacA operon and regulation of its synthesis is related with sporulation and quorum-sensing pathways. However, the effect of this dipeptide on its producer is obscure. To elucidate this effect, our research group constructed a bacilysin non-producer strain of B. subtilis named as OGU1. By the use of proteomic methods of 2DE MALDI-TOF MS/MS and nanoLC-MS/MS the differentially expressed proteins of OGU1 in comparison to parental strain PY79 was identified. The number of differentialy expressed proteins was rather high, being generaly underrepresented, and these proteins were mostly related with sporulation and germination pathways. In the present study, the results of comparative proteomics were validated with RT-qPCR analysis of selected 20 genes. Certain phenotypic deficiencies/inadequacies regarding spore pigmentation, lysozyme and chloroform resistance of spores, spore coat protein profile as well as spore germination kinetics were next observed in OGU1. Protein-free and bacilysin containing culture broth concentrate supplementation to growing OGU1 cultures partially or entirely rescued all of these deficient phenotypes, indicating a pleiotropic role of bacilysin on sporulation and germination of its producer.

Subject Keywords

B. Subtilis, Bacilysin, Sporulation, Germination, Chemical Complementation, RT-qPCR

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https://hdl.handle.net/11511/69287

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Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats

O. Ertekin, “Phenotypic rescue of a Bacilysin-silenced strain of Bacillus Subtilis with Bacilysin supplementation,” M.S. - Master of Science, Middle East Technical University, 2020.