H-055. Identification of the Core Components Required for σG Activation during Bacillus subtilis Sporulation

A. H. Camp, R. Losick;
Harvard Univ., Cambridge, MA.

During Bacillus subtilis sporulation, the sigma factor σG is responsible for directing gene expression at late timepoints in the forespore compartment of the developing sporangium. Ten genes are required for σG activation: the forespore-expressed spoIIQ gene, the mother-cell expressed spoIIIA operon composed of eight genes (A-H), and the vegetative spoIIIJ gene. σG activation also requires forespore engulfment, a phagocytic-like event that occurs midway through sporulation. These data suggest that SpoIIIAA-H, SpoIIQ, and SpoIIIJ comprise a novel intercellular signal transduction pathway that activates σG upon the completion of engulfment. However, in comparison to the regulatory networks that control the other sporulation-specific σ factors, this putative σG activation pathway remains poorly understood at the molecular/mechanistic level. In an effort to uncover functional interactions among the components of the σG activation network, we identified spontaneous mutations that partially restored the ability of ΔspoIIIJ cells to activate σG and form viable spores. Several isolated mutations mapped to spoIIIAE, supporting a model in which SpoIIIAE (a polytopic membrane protein) is a substrate for SpoIIIJ (a putative polytopic membrane protein chaperone). Another mutation mapped to the sporulation-specific Class A penicillin-binding protein-encoding gene pbpG. Further genetic analysis suggests that the isolated pbpG allele (pbpG*) disrupts/delays peptidoglycan synthesis in such a way that potentiates the activity of the remaining σG activation machinery. We therefore devised a strategy using pbpG* to identify the core components of the σG activation network. Strikingly, our results indicate that out of ten proteins, only SpoIIQ and SpoIIIAH represent the core of the machinery responsible for activating σG upon the completion of engulfment. Conversely, the remaining proteins appear to play accessory/regulatory roles and are dispensable (under certain genetic conditions) for σG activation. In summary, we have employed a genetic strategy to identify the core components of the σG activation pathway and we are currently testing competing models for their mechanism of action.