c-di-GMP regulates bacterial NAD biosynthesis via targeting the transcriptional repressor NadR

ABSTRACT As a near-ubiquitous bacterial second messenger, cyclic di-GMP (c-di-GMP) regulates a multitude of important biological processes. The regulatory effects of c-di-GMP on bacterial physiological processes are mediated through its interaction with various effector molecules, including mRNA riboswitches and proteins. Although c-di-GMP effector proteins have been widely reported, yet unknown c-di-GMP effectors in bacteria wait to be discovered, and the physiological roles of this second messenger still remain to be explored. In a c-di-GMP/transcription factor binding screen, we identified NadR, a repressor of nicotinamide adenine dinucleotide (NAD) synthesis and salvage, as a c-di-GMP-responsive transcription factor in Salmonella enterica serovar Typhimurium. c-di-GMP was found to bind to NadR with high affinity. c-di-GMP binding inhibits the binding of NadR to its target DNA, thus upregulating the expression of NadR-repressed genes involved in NAD synthesis and salvage. c-di-GMP also stimulates the nicotinamide mononucleotide adenylyltransferase and ribosylnicotinamide kinase activities of NadR. As a result, elevated intracellular c-di-GMP levels lead to increased NAD synthesis and enhanced resistance to DNA damage in S . Typhimurium. NadR proteins from three other species belonging to Enterobacterales are capable of sensing c-di-GMP, suggesting that c-di-GMP-mediated modulation of intracellular NAD homeostasis is a conserved mechanism employed by members of Enterobacterales. IMPORTANCE Cyclic di-GMP (c-di-GMP) functions as a highly versatile signaling molecule in bacteria, orchestrating diverse physiological processes critical for survival and adaptation. While nicotinamide adenine dinucleotide (NAD) plays pivotal roles in numerous cellular processes and functions, bacteria have been shown to modulate its biosynthetic and recycling pathways through a variety of regulatory mechanisms. However, a connection between c-di-GMP signaling and NAD metabolism in bacteria has never been revealed before. Here, we identify NadR, a transcriptional repressor of NAD synthesis and salvage, as a c-di-GMP effector, and show that c-di-GMP upregulates NAD biosynthesis by activating NadR-repressed genes, thus enhancing the defense of Salmonella against DNA damage. Our study reveals a previously unrecognized regulatory mechanism in bacterial NAD metabolism and expands the understanding of the physiological roles of c-di-GMP in bacteria.