Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae , the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA , the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a Δ dacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment. Author summary Nucleotide-based second messengers play central functions in bacterial physiology and host-pathogen interactions. Among these signalling nucleotides, cyclic-di-AMP (c-di-AMP) synthesis was originally assumed to be essential for bacterial growth. In this study, we confirmed that the only di-adenylate cyclase enzyme in the opportunistic pathogen Streptococcus agalactiae is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable by accumulating spontaneous mutations in genes involved in osmotic regulation. We identified that c-di-AMP binds directly to four proteins necessary to maintain osmotic homeostasis, including three osmolyte transporters and the BusR transcriptional factor. We demonstrated that BusR negatively controls the expression of the busAB operon and that it is the main component leading to growth inhibition in the absence of c-di-AMP synthesis if osmoprotectants are present in the environment. Overall, c-di-AMP is essential to maintain osmotic homeostasis by coordinating osmolyte uptake and thus bacteria have developed specific mechanisms to keep c-di-AMP as the central regulator of osmotic homeostasis.
【초록키워드】 Mutation, susceptibility, Transcription, Infection, Protein, pathogen, viability, Control, pathway, Meningitis, phenotype, Deletion, Bacteria, Potassium, mutant, expression, mechanism, homeostasis, function, binding, Bacterial, cellular, nucleotide, host-pathogen interactions, regulate, compensatory mutations, nucleotides, Neonatal, identification, leads, opportunistic pathogen, host cells, betaine, glycine, host cell, Streptococcus agalactiae, dacA, second messenger, loss-of-function mutation, glycine betaine, second messengers, Regulation, osmotic regulation, enzyme, growth, accumulation, medium, loss-of-function, etiological agent, cellular physiology, cyclic, monophosphate, osmolyte, osmoregulation, septicaemia, promoter, Streptococcus, Loss, transcriptional, bind, identify, conserved, involved, functional, absence, dependent on, demonstrated, maintain, predominant, conditions, assumed, accumulating, hyperosmotic, osmotic, secondary messenger, 【제목키워드】 group, homeostasis, Regulation, Streptococcus, osmotic,