AbstractClostridium difficilespore germination is critical for the transmission of disease.C. difficilespores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the bile acid germinant receptor is known, the amino acid germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy used previously to identify the bile acid receptor, CspC. Surprisingly, we identified strains that do not require amino acids as co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations inyabG.InC. difficile, yabGexpression is required for the processing of CspBA to CspB and CspA and preproSleC to proSleC during spore formation. A definedyabGmutant exacerbated the EMS mutant phenotype. Moreover, we found that various mutations incspAcaused spores to germinate in the presence of TA alone without the requirement of an amino acid. Thus, our study provides evidence that apart from regulating the CspC levels in the spore, CspA is important for recognition of amino acids as co-germinants duringC. difficilespore germination and that two pseudoproteases (CspC and CspA) function as theC. difficilegerminant receptors.
The requirement for the amino acid co-germinant duringC. difficilespore germination is influenced by mutations inyabGandcspA
