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

Abstract

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 Ca 2؉ 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.

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly

“…It is not surprising to see increased levels of several ion transporters in germinated spore samples considering the observation of a large efflux of cations during the early stages of germination in Bacillus species (12). Mutagenesis studies of several ion transporters have also shown that ion transport plays an important role in spore germination (10,50). It is possible that several of the identified transporters, such as the cation efflux superfamily protein CPR_0983, the cation-transporting ATPase CPR_2026, and the ion compound ABC transporter CPR_1115, serve as channels or gates for cation efflux during spore germination, especially the protein encoded by CPR_2026, which shares 47% identity with the calcium pump ATPase YloB from B. subtilis.…”

“…It is not known if the reduced detection is due to degradation or reduced association with the membrane during purification. It was reported previously that mutation of a putative Na ϩ / H ϩ -K ϩ antiporter, GerO (CPR_0227), containing 10 potential membrane-spanning domains, negatively affected spore germination and outgrowth (10). However, translation of GerO was observed only in the mother cell compartment and not in the forespore (10).…”

“…However, the signal transduction involved in this process is poorly understood. During ion flux, monovalent cations such as K ϩ , Na ϩ , and H ϩ are released from the spore core to the environment, along with a large amount of calcium dipicolinic acid (Ca-DPA) (10,12,13). At least two ion channel proteins and transporters have been found to play a role in this stage, the spore inner membrane SpoVA proteins and the putative spore outer membrane Na ϩ /H ϩ -K ϩ antiporters (10, 14).…”

“…If ingested in high numbers, vegetative cells sporulate in the intestines and produce the C. perfringens enterotoxin (CPE), which is released upon lysis of the mother cell after sporulation is complete (5). While several studies have focused on the germination of C. perfringens spores (6)(7)(8)(9)(10), spore germination has been most intensively studied in Bacillus species. Based on the current model, the spore germination cascade is triggered by the interaction between the germinant and germinant receptors, and these germinant receptors are located in the spore inner membrane (11).…”

“…During ion flux, monovalent cations such as K ϩ , Na ϩ , and H ϩ are released from the spore core to the environment, along with a large amount of calcium dipicolinic acid (Ca-DPA) (10,12,13). At least two ion channel proteins and transporters have been found to play a role in this stage, the spore inner membrane SpoVA proteins and the putative spore outer membrane Na ϩ /H ϩ -K ϩ antiporters (10,14). Later events in spore germination include the degradation of the spore cortex by germination-specific cortex-lytic enzymes and the degradation of core proteins by proteases, followed by full rehydration of the spore core and resumption of metabolism (15).…”