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

Abstract

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-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 of gerP 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 the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. 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 a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. 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 the Bacillus 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.Spore germination is initiated by the interaction of the germinant molecule with a receptor in the spore. The nature of this receptor is not yet proven, but the available evidence suggests that the genes of the gerA family whose products are required for the response to specific germinants are likely to encode this receptor (16,20). The trigger reaction commits spores to undergo a series of successive events which result in the loss of spore dormancy and resistance properties. Spores of Bacillus cereus initiate germination in response to L-alanine or ribosides, of which inosine is the most effective (8). Inhibition of the alanine racemase activity associated with spores by Ocarbamyl D-serine is necessary to observe maximum rates of L-alanine-triggered germination, as D-alanine is a competitive inhibitor (8). The first measurable event after commitment is the loss of heat resistance (a rise in spore internal pH, a release of monovalent ions, and a release of dipicolinic acid (DPA) and calcium ions from spores are also early events), and later events include the activation of spore lytic enzymes (7, 17)...

Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis

“…While this role has been appreciated for some time (4), it is only recently that its molecular basis has begun to become understood. For example, in addition to the discovery that the cortex-lytic enzyme CwlJ is in the coat (7), the Moir laboratory has shown that the gerP operon, likely encoding coat protein(s), participates in germination (9). Specifically, that study indicates that the coat does more than merely act as a passive sieve through which germinants flow.…”

The ExsA Protein of Bacillus cereus Is Required for Assembly of Coat and Exosporium onto the Spore Surface

“…On addition of L-alanine or inosine to initiate germination of spores of mutant AM1605, OD was lost more slowly than in wild type, and spores became phase grey, but dipicolinic acid release and loss of heat resistance were equally fast in the wild type and the mutant (data not shown). This is characteristic of spores that have incomplete spore coats, as demonstrated for B. cereus (3). The precise reason for the block is not demonstrated, but such spores could lack the full complement of cortex lytic enzymes, such as the CwlJ protein in B. subtilis, which is extracted during coat removal (1,4,20).…”

Characterization of a Major Bacillus anthracis Spore Coat Protein and Its Role in Spore Inactivation

“…A sample was removed for viable counts, and then DPC was added to a 0.025 M concentration and the tubes were incubated at 27°C for 1 h. Sensitivities to 10% n-octanol, 5 and 10% phenol, 10% hydrogen peroxide, and 4% sodium hypochlorite (0.5% available chlorine) were measured as described elsewhere (16,23). Spore germination in L-alanine plus inosine was also done as described elsewhere (5).…”

“…In addition to a protective function, the coat has some role in germination (5,6), perhaps by providing access for germinants to the germination proteins located in the inner forespore membrane. A lytic enzyme required for digestion of the spore cortex during germination may also be located in the coat (8,19).…”