VanG- and D-Ala-D-Ser-dependent peptidoglycan synthesis and vancomycin resistance in Clostridioides difficile

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.