The Bacillus cereus GerN and GerT Protein Homologs Have Distinct Roles in Spore Germination and Outgrowth, Respectively

Abstract

The GerT protein of Bacillus cereus shares 74% amino acid identity with its homolog GerN. The latter is a Na ؉ /H ؉ -K ؉ antiporter that is required for normal spore germination in inosine. The germination properties of single and double mutants of B. cereus ATCC 10876 reveal that unlike GerN, which is required for all germination responses that involve the GerI germinant receptor, the GerT protein does not have a significant role in germination, although it is required for the residual GerI-mediated inosine germination response of a gerN mutant. In contrast, GerT has a significant role in outgrowth; gerT mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in B. cereus therefore contributes to the success of spore outgrowth from the germinated state during alkaline or Na ؉ stress.Bacillus cereus ATCC 10876 spores germinate in inosine or in L-alanine as sole germinants (5). They require both GerI and GerQ germinant receptors for germination in inosine as the sole germinant, whereas the GerL receptor is responsible for most of the response to L-alanine as the sole germinant, with a smaller contribution from the GerI receptor (3). The GerN protein is needed for successful inosine germination, as spores of a gerN mutant of B. cereus ATCC 10876 demonstrate a slow and abnormally ordered germination response in inosine as the sole germinant (24). The residual germination was very asynchronous, and some spores appeared to become phase dark before losing heat resistance (24). This GerN protein is a homolog of a widely distributed family of cation transporters (9) and has been demonstrated to mediate Na ϩ /H ϩ and Na ϩ / H ϩ -K ϩ antiport activity when expressed at a low level in Escherichia coli (21). Such GerN-mediated antiport activity is therefore apparently required for the germination response mediated by the GerI germinant receptor in B. cereus. The role of such an ion transport protein in germination remains unclear, especially as other germinant receptors in the same strain do not require a functional GerN protein; for example, the gerL-dependent alanine response is almost identical to that of the wild type in a gerN mutant (24). Both GerN and a closely related homolog are encoded in B. cereus ATCC 14579 (13) and in other members of the family, including Bacillus anthracis (17) and Bacillus thuringiensis (16). Both genes appear to be monocistronic, and their coding sequences are widely separated on the genome. We have called this second gerN-like gene gerT (19). An entirely different (spore coat) protein in B. subtilis has recently been called gerT (7), but that gene does not have a homolog in B. cereus, and as B subtilis does not carry an equivalent homolog of the B. cereus gerN or gerT gene, the nomenclature will not overlap. We describe here the spore germination and outgrowth phenotypes of mutants of B. cereus ATCC 10876 carrying an insertionally inactivated gerT gene. Resulting phenotypes s...

GerO, a Putative Na + /H + -K + Antiporter, Is Essential for Normal Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

“…Thus, the sporulation-specific expression of gerO and gerQ suggests that GerO and GerQ might be spore-specific proteins. There is also evidence suggesting that the spore germination-associated likely monovalent cation antiporters GerN and GerT of B. cereus are encoded by sporulation-specific genes (12,29). While the latter results suggest that all of these cation transport proteins are present in spores, gerO and gerQ appear to be expressed only in the mother cell compartment of sporulating C. perfringens cells, although this analysis has not been carried out with gerN and gerT.…”

“…(iii) Finally, the loss of GerO and to a lesser extent of GerQ results in defects in C. perfringens spore germination. These results suggesting roles for one or more cation transport proteins in C. perfringens spore germination are similar to those suggested for spores of some Bacillus species, although not with all strains and/or species and not with all germinants (3,29,34,35). The major questions provoked by these observations are how GerO and, likely to a lesser degree, GerQ func- tion in spore germination and/or outgrowth and whether the effects of these proteins are direct or indirect.…”

“…It is not clear whether the role in spore outgrowth is a direct or indirect one, since it is not clear that either of these proteins is actually present in dormant spores (see below). However, a requirement for the likely cation transporter, GerT, in spore outgrowth has been observed with B. cereus spores (29). It is also known that germinating and outgrowing spores of at least Bacillus species are resistant to very high salt concentrations and are much more resistant than are vegetative cells (36).…”

“…Similarly, in Bacillus cereus ATCC 10876, a GrmA-type homologue, GerN, is essential for spore germination with inosine but not L-alanine (35), and studies with everted vesicles have shown that GerN possesses electrogenic Na ϩ /H ϩ -K ϩ antiporter activity (32). The GerN homolog, GerT, also plays a minor role in B. cereus spore germination with inosine, as well as a major role in spore outgrowth under some conditions (29). However, in contrast to these latter results, GrmA-like antiporters appear to have no role in the germination of spores of B. megaterium QM B1551 and Bacillus subtilis (3).…”

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

“…However, when there is a large concentration of L-alanine in the environment, the racemase cannot convert enough alanine to prevent germination. These germinants and anti-germinants act either alone or in combinations and signal through five functional germination receptors, gerH, gerL, gerK, gerS, and gerX, on the spore coat [63,64,67,68]. The need for several germination receptors may not only reflect redundant mechanisms for sensing germinants, but also may be a mechanism for B. anthracis to sense different sites in the host for germination.…”