A novel RofA-family transcriptional regulator, GadR, controls the development of acid resistance in Listeria monocytogenes

Stomach acid provides a significant innate barrier to the entry of the food-borne pathogen Listeria monocytogenes into the human gastrointestinal tract. A key determinant of acid resistance in this bacterium is the conserved glutamate decarboxylase system, GadD2 (encoded by the gadT2D2 operon), which helps to maintain the intracellular pH during exposure to gastric acid. In this study, we identified a premature stop codon in a gene located immediately downstream of the gadT2D2 operon that was highly linked to an acid-sensitive phenotype. When this open reading frame was restored through homologous recombination, an acid-resistant phenotype was restored. Through a series of genetic, transcriptomic, and survival experiments, we established that this gene, which we designated gadR, encodes a transcriptional regulator of the gadT2D2 operon. GadR belongs to the RofA family of regulators, primarily found in streptococci, where they are involved in regulating virulence. The data further showed that gadR plays a critical role in the development of acid resistance in response to mild acid exposure, a response that is known as the adaptive acid tolerance response (ATR). A deletion analysis of the gadT2D2 promoter region identified two 18-bp palindromic sequences that are required for the GadR-mediated induction of gadT2D2, suggesting that they act as binding sites for GadR. Overall, this study uncovers a new RofA-like regulator of acid resistance in L. monocytogenes, which plays a significant role in both growth phase-dependent acid resistance and ATR and accounts for previously observed strain-to-strain differences in survival at low pH.