The Molecular Mechanisms of Gene Regulatory Networks Controlling Cell Fate Decisions in Bacillus Subtilis
Srivastava, Priyanka 1981-
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Under nutrient starvation conditions, the soil bacterium Bacillus subtilis differentiates to form biofilms or spores. Biofilm formation and sporulation are under the control of master transcription regulator Spo0A. Spo0A is activated when phosphorylated (Spo0A~P) through a phosphorelay system which includes histidine kinases KinABC, two phosphotransferases (Spo0Fand Spo0B), and Spo0A. It has been proposed that KinA is primarily responsible for activation of sporulation while KinC is involved in controlling biofilm formation. In this study, we aimed to uncover the mechanisms of how the two distinct cell types can be independently generated by two similar kinases. We constructed two strains harboring inducible KinA and KinC with reporter genes to monitor biofilm formation and sporulation, respectively. Using these systems, we discovered that both tapA (marker gene for biofilm formation), and spoIIG (marker gene for sporulation) are induced more efficiently by KinA than KinC. Next, by analyzing the biofilm colony architecture using fluorescence microscopy, we demonstrated that distinct cell types, including motile, biofilm-forming, and sporulating cells are localized within distinct regions of a colony. We also provide evidence that biofilm formation and sporulation are mutually exclusive events, contrary to the previously published findings of sporulating cells deriving from biofilm forming cells. Further, we constructed a series of genetically engineered strains expressing reduced levels of Spo0A~P. Using these systems, we find that genes involved in biofilm formation are sufficiently turned on, leading to biofilm formation, similar to the wild type strain. However, genes involved in sporulation are strongly impaired. We also determine the novel role of Spo0E, a Spo0A~P specific phosphatase, in modulating biofilm formation by controlling the Sp0A~P levels. Based on these findings, we propose a new model in which genes involved in biofilm formation are controlled by relatively low cellular levels of Spo0A~P, while sporulation genes require relatively high levels of Spo0A~P through two kinases KinC and KinA, respectively. Thus, KinC is sufficient to induce biofilm formation by triggering the low-threshold of Spo0A~P at early times of starvation while KinA is required for triggering the high-threshold Spo0A~P to induce sporulation at relatively later times of starvation.