Abstract
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 ΔdacA 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.
Highlights
Cyclic nucleotides are signalling molecules, commonly called second messengers, which regulate cellular processes by binding to targeted effectors [1,2,3]
Cyclic-di-AMP (c-diAMP) synthesis was originally assumed to be essential for bacterial growth
We confirmed that the only di-adenylate cyclase enzyme in the opportunistic pathogen Streptococcus agalactiae is essential in standard growth conditions
Summary
Cyclic nucleotides are signalling molecules, commonly called second messengers, which regulate cellular processes by binding to targeted effectors [1,2,3]. Genes encoding for essential proteins might be inactivated in specific conditions or their inactivation can be compensated by secondary mutations [13]. This is the case for c-di-AMP synthesis in Listeria monocytogenes, in which spontaneous mutations in genes involved in central metabolism and in adaptation to starvation allow growth without c-di-AMP [14]. In Bacillus subtilis, c-di-AMP synthesis is essential in rich media [18, 19], but the absence of c-di-AMP synthesis can be compensated by spontaneous mutations leading to an increased activity of the NhaK cation/proton antiporter allowing to overcome potassium toxicity [20]. In Staphylococcus aureus, c-di-AMP synthesis becomes dispensable when accumulating mutations in amino acid and osmolyte transporters, as well as through mutations in genes encoding for proteins required for respiration, linking c-di-AMP essentiality with osmoregulation and metabolism [21]
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