Mutations in the gerP Locus of Bacillus subtilis and Bacillus cereus Affect Access of Germinants to Their Targets in Spores

ThegerP1 transposon insertion mutation ofBacillus cereus is responsible for a defect in the germination response of spores to bothl -alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization ofgerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC ) can be found in theBacillus subtilis genome sequence; null mutations inyisD andyisF , constructed by integrational inactivation, result in a mutant phenotype similar to that seen inB. cereus , though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to agerP mutant by the introduction of acotE mutation, which renders the spore coats permeable to lysozyme. TheB. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of theBacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.