Clostridium perfringens type A isolates produce the spore-specific enterotoxin, CPE, upon sporulation in the gastrointestinal tract of humans and animals. Spo0A is a response regulator representing the major transcription factor for sporulation initiation in C. perfringens. In Bacillus subtilis, a multicomponent phosphorelay involving five histidine kinases (KinA-KinE), the intermediate response regulator, Spo0F, and the phosphotransferase, Spo0B, leads to the activation of a similar Spo0A response regulator by phosphate transfer to its N-terminal domain. The phosphodonor for Spo0A in C. perfringens remains unknown. Genetic comparison with the B. subtilis genome revealed multiple histidine kinase orthologues, but no similar intermediate phosphorelay components were detected. The lack of intermediate phosphate messengers suggests that Spo0A in C. perfringens may be directly phosphorylated by one or more histidine kinases. This study investigated the potential for involvement of two histidine kinases (CPE 0213, CPE 1754) in the activation of Spo0A. Transcriptional activity of the kinase genes in sporulating cell cultures was confirmed by reverse-transcription PCR. The genes were inactivated by single-crossover vector insertions, and the resulting sporulation frequencies were found to be 27% and 33% of wild type level for cpe0213 and cpe1754 mutants, respectively. Reintroduction of the functional genes, via a multicopy plasmid construct, failed to restore sporulation frequency to wild type levels.
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