Clostridioides difficile para -cresol production is induced by the precursor para -hydroxyphenylacetate.

is an aetiological agent for antibiotic-associated diarrhoeal disease. produces a phenolic compound, -cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. decarboxylates -hydroxyphenylacetate (HPA) to produce -cresol by the action of the HpdBCA decarboxylase encoded by the operon. Herein, we investigate regulation of the operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase and alkaline phosphatase reporters to detect basal and inducible expression. We show expression of is upregulated in response to elevated HPA. analysis identified three putative promoters upstream of operon, P, P and Pσ only the P promoter was responsible for both basal and -HPA-inducible expression of We demonstrated that turnover of tyrosine, a precursor for HPA, is insufficient to induce expression of the operon above basal levels because it is inefficiently converted to -HPA in minimal media. We show that induction of the operon in response to -HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-n-CTTTTT) upstream of the start codon (ATG) that is essential for inducing transcription of the operon in response to -HPA, which drives production of -cresol. This provides insights into the regulatory control of -cresol production, which affords a competitive advantage for over other intestinal bacteria, promoting dysbiosis. infection results from antibiotic-associated dysbiosis. -cresol, a phenolic compound produced by , selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Herein, we demonstrate that expression of the operon, encoding the HpdBCA decarboxylase which converts -HPA to -cresol, is upregulated in response to elevated exogenous HPA, with induction occurring between >0.1 and ≤0.25 mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and -HPA-inducible expression. We identified turnover of tyrosine, a HPA precursor, does not induce expression above basal level, indicating that exogenous -HPA was required for -cresol production. Identifying regulatory controls of -cresol production will provide novel therapeutic targets to prevent -cresol production, reducing 's competitive advantage.