CenIR, an essential BlaIR-family regulatory system in C. difficile

The CenIR regulatory system of Clostridioides difficile comprises a predicted transcriptional repressor, CenI, and a predicted membrane metalloprotease, CenR. The physiological role of CenIR and activating signal(s) are not known. CenIR belongs to the BlaIR family of regulators that mediate resistance to β-lactam antibiotics. In canonical BlaIR systems, binding of a β-lactam to the extracellular transpeptidase domain of BlaR triggers proteolysis of BlaI and, thus, induction of a closely linked β-lactamase gene. However, CenR lacks a β-lactam-binding domain and transposon mutagenesis indicated CenI is essential for viability even when β-lactams are not present. Here, we confirmed the essentiality of CenIR and determined its regulon contains ~12 genes, including an exported protein of unknown function (CDR_0474) that is induced about 500-fold and a peptidoglycan hydrolase (Cwp6) that is induced about 7-fold when cells are depleted of CenIR. There are no essential genes or β-lactamases in the regulon. Phenotypic characterization of CenIR-depletion strains revealed slower growth, mild elongation, and cell lysis. Deletion of cdr_0474 corrected all three defects, while deletion of cwp6 only rescued the lysis phenotype. It was possible to delete cenIR in either a Δcdr_0474 or Δcwp6 background. We propose that CenIR is essential because its absence leads to lysis due to Cwp6 overproduction. Bioinformatic analyses revealed the predicted extracellular sensing domains in annotated "BlaR" proteins are diverse. Thus, BlaIR systems are not dedicated to defense against β-lactams but probably enable bacteria to adapt to a variety of environmental stimuli.

Many of the regulatory systems for controlling cell envelope biogenesis and stress responses have yet to be studied. Here, we characterize a Clostridioides difficile BlaIR-like regulatory system that we have named CenIR for cell envelope. Unlike canonical BlaIR systems, which bind β-lactams and induce a β-lactamase, CenIR lacks a β-lactam binding domain and is essential for viability even in the absence of antibiotics. We identified the genes in the regulon and found that CenIR is essential because its absence leads to the overproduction of the Cwp6 peptidoglycan hydrolase. We also show that most annotated BlaIR-like systems lack a β-lactam-binding domain, from which we infer that these systems have much broader physiological roles than generally appreciated.