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

The genome of the pathogen Clostridium perfringens encodes two proteins, GerO and GerQ, homologous to monovalent cation transporters suggested to have roles in the germination of spores of some Bacillus species. GerO and GerQ were able to transport monovalent cations (K⁺ and/or Na⁺) in Escherichia coli, and gerO and gerQ were expressed only in the mother cell compartment during C. perfringens sporulation. C. perfringens spores lacking GerO were defective in germination with a rich medium, KCl, l-asparagine, and a 1:1 chelate of Ca²⁺ and dipicolinic acid (DPA), but not with dodecylamine, and the defect was prior to DPA release in germination. All defects in gerO spores were complemented by ectopic expression of wild-type gerO. Loss of GerQ had much smaller effects on spore germination, and these effects were most evident in spores also lacking GerO. A modeled structure of GerO was similar to that of the E. coli Na⁺/H⁺ antiporter NhaA, and GerO, but not GerQ contained two adjacent Asp residues thought to be important in the function of this group of cation transporters. Replacement of these adjacent Asp residues in GerO with Asn reduced the protein''s ability to complement the germination defect in gerO spores but not the ability to restore cation transport to E. coli cells defective in K⁺ uptake. Together, these data suggest that monovalent cation transporters play some role in C. perfringens spore germination. However, it is not clear whether this role is directly in germination or perhaps in spore formation.Clostridium perfringens is a gram-positive, spore-forming anaerobic pathogen that causes diseases in animals and humans (13). C. perfringens spores are metabolically dormant, are resistant to many environmental insults, and can survive for long periods. Once conditions are favorable, these spores can germinate, outgrow, return to vegetative growth, and then release toxins and cause disease (14).Bacterial spores initiate germination when they sense a variety of compounds termed germinants, which include nutrients, a 1:1 chelate of Ca²⁺ and pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) (Ca-DPA) and cationic surfactants (21, 31). In spores of Bacillus species, nutrient germinants are sensed by specific germinant receptors located in the spore''s inner membrane, each generally encoded by tricistronic operons of the gerA family. In Bacillus megaterium spores, the interaction of nutrient germinants with their cognate receptors leads to an energy independent efflux of ∼80% of the spore''s depot of Na⁺ and K⁺, as well as much H⁺ efflux causing a rise of the spore core''s pH, all within the first 5 min of germination; this efflux is followed by reuptake of K⁺ by an energy-dependent system (33). The spores'' large depot of Ca-DPA is also released shortly after monovalent cation release. The mechanism of release of monovalent cations during spore germination is not known, but monovalent cation antiporters could be involved somehow in this event. Indeed, a member of the CPA-2 monovalent cation-proton antiporter family of membrane transport proteins (27), GrmA, is essential for germination of B. megaterium ATCC 12872 spores (34), since grmA inactivation makes spores unable to release their DPA and complete germination with a variety of germinants. 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⁺/H⁺-K⁺ 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).In C. perfringens, there is no intact tricistronic gerA-like operon, and the only locus that encodes the three proteins (A, B, and C) of a likely germinant receptor is the gerK locus, comprising a bicistronic gerKA-gerKC operon, and a gerKB gene located just upstream of gerKA-gerKC but in the opposite orientation (16). However, GerKA and GerKC appear able to function in spore germination in the absence of GerKB (23). The lack of a classical GerA-type germinant receptor and the fact that C. perfringens spores germinate with K⁺ ions alone (21), raises the possibility that GrmA-like antiporters might also play some role in C. perfringens spore germination. The genome of C. perfringens strain SM101 has two genes encoding putative GrmA-like antiporters (see Fig. S1 in the supplemental material) that we have termed gerO (CPR0227) and gerQ (CPR1038). Orthologs of the gerO and gerQ genes are also present in the genomes of nine additional C. perfringens strains (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). In present study we have constructed gerO, gerQ, and gerO gerQ strains of C. perfringens and have examined the roles of GerO and GerQ in spore germination. The results show that GerO is essential for normal germination of C. perfringens spores, whereas GerQ plays at most only a minor role.