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We isolated spontaneous suppressors of a \u2206<jats:italic>ddl<\/jats:italic> mutation that allowed cell growth in the absence of D\u2010Ala\u2010D\u2010Ala. The mutations caused constitutive or partly constitutive expression of the vancomycin\u2010inducible <jats:italic>vanG<\/jats:italic> operon responsible for the synthesis of D\u2010Ala\u2010D\u2010Ser, which can replace D\u2010Ala\u2010D\u2010Ala in peptidoglycan. The mutations mapped to the <jats:italic>vanS<\/jats:italic> or <jats:italic>vanR<\/jats:italic> genes, which regulate expression of the <jats:italic>vanG<\/jats:italic> operon. The constitutive level of <jats:italic>vanG<\/jats:italic> expression was about 10\u2010fold above that obtained by vancomycin induction. The incorporation of D\u2010Ala\u2010D\u2010Ser into peptidoglycan due to high expression of the <jats:italic>vanG<\/jats:italic> operon conferred only low\u2010level resistance to vancomycin, but VanG was found to synthesize D\u2010Ala\u2010D\u2010Ala in addition to D\u2010Ala\u2010D\u2010Ser. However, the same, low resistance to vancomycin was also observed in cells completely unable to synthesize D\u2010Ala\u2010D\u2010Ala and grown in the presence of D\u2010Ala\u2010D\u2010Ser. D\u2010Ala\u2010D\u2010Ala presence was required for efficient vancomycin induction of the <jats:italic>vanG<\/jats:italic> operon showing that vancomycin is not by itself able to activate VanS. D\u2010Ala\u2010D\u2010Ser, similar to D\u2010Ala\u2010D\u2010Ala, served as an anti\u2010activator of DdlR, the positive regulator of the <jats:italic>ddl<\/jats:italic> gene, thereby coupling <jats:italic>vanG<\/jats:italic> and <jats:italic>ddl<\/jats:italic> expression.<\/jats:p>","DOI":"10.1111\/mmi.14980","type":"journal-article","created":{"date-parts":[[2022,9,6]],"date-time":"2022-09-06T08:03:27Z","timestamp":1662451407000},"page":"526-540","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["<scp>VanG<\/scp>\u2010 and <scp>D\u2010Ala\u2010D\u2010Ser<\/scp>\u2010dependent peptidoglycan synthesis and vancomycin resistance in <i>Clostridioides difficile<\/i>"],"prefix":"10.1111","volume":"118","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0171-3128","authenticated-orcid":false,"given":"Boris R.","family":"Belitsky","sequence":"first","affiliation":[{"name":"Department of Molecular Biology and Microbiology 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