LAUSR

A Clostridioides difficile strain deficient in the ddl gene is unable to synthesize the dipeptide D‐Ala‐D‐Ala, an essential component of peptidoglycan and the target of vancomycin. We isolated spontaneous suppressors of a ∆ddl mutation that allowed cell growth in the absence of D‐Ala‐D‐Ala. The mutations caused constitutive or partly constitutive expression of the vancomycin‐inducible vanG operon responsible for the synthesis of D‐Ala‐D‐Ser, which can replace D‐Ala‐D‐Ala in peptidoglycan. The mutations mapped to the vanS or vanR genes, which regulate expression of the vanG operon. The constitutive level of vanG expression was about 10‐fold above that obtained by vancomycin induction. The incorporation of D‐Ala‐D‐Ser into peptidoglycan due to high expression of the vanG operon conferred only low‐level resistance to vancomycin, but VanG was found to synthesize D‐Ala‐D‐Ala in addition to D‐Ala‐D‐Ser. However, the same, low resistance to vancomycin was also observed in cells completely unable to synthesize D‐Ala‐D‐Ala and grown in the presence of D‐Ala‐D‐Ser. D‐Ala‐D‐Ala presence was required for efficient vancomycin induction of the vanG operon showing that vancomycin is not by itself able to activate VanS. D‐Ala‐D‐Ser, similar to D‐Ala‐D‐Ala, served as an anti‐activator of DdlR, the positive regulator of the ddl gene, thereby coupling vanG and ddl expression.