Bacillus thuringiensis is a bacterium capable of differentiating into a spore, a dormant and highly resistant cellular form. During the sporulation process, this bacterium produces insecticidal toxins in the form of a crystal inclusion, usually in the sporulating cell. We previously reported that the B. thuringiensis LM1212 strain can differentiate into two distinct subpopulations of sporeformers and crystal producers and that this division-of-labor phenotype provides the bacterium with a fitness advantage in competition with a typical B. thuringiensis strain. The transcription factor CpcR was characterized as the regulator responsible for this phenotype. Here, we examined how CpcR interacts with the sporulation network to control the cell differentiation. We found that the sporulation process was inhibited prior to polar septum formation and that Spo0A activity was impaired in the presence of cpcR in strain LM1212. Using bioinformatics and genetic tools, we identified a gene positively controlled by CpcR encoding a putative phosphatase of the Spo0E family known to specifically dephosphorylate phosphorylated Spo0A (Spo0A-P). We showed that this protein (called Spo0E1) is a negative regulator of sporulation and that variations in spo0E1 expression can modulate the production of spores. Using fluorescent reporters to follow gene expression at the single-cell level, we correlated expression of cpcR and sporulation genes to the formation of the two differentiated subpopulations. IMPORTANCE Formation of spores is a paradigm for study of cell differentiation in prokaryotes. Sporulation initiation is governed by a gradual increase in the level and activity of the master regulator Spo0A. Spo0A is usually indirectly phosphorylated by a multicomponent phosphorelay, and modulation of this phosphorelay system is a critical aspect of Bacillus physiology. Though we know that this phosphorelay system is usually affected by two negative regulatory mechanisms, i.e., rap genes and spo0E family genes, the regulatory mechanisms controlling the transcription of these genes are poorly understood. Here, we report that the transcription factor CpcR positively regulates a spo0E family gene and that variations in spo0E expression can modulate the production of spores in B. thuringiensis. This work emphasizes the diversity in modes of sporulation and illustrates the diversity in the strategies employed by bacteria to control this differentiation pathway and ensure their survival.
Keywords: Bacillus thuringiensis; cell differentiation; spo0E family gene; sporulation.