Cyclic‐di‐AMPsynthesis by the diadenylate cyclaseCdaAis modulated by the peptidoglycan biosynthesis enzymeGlmMinLactococcus lactis

Abstract

SummaryThe second messenger cyclic-di-adenosine monophosphate (c-di-AMP) plays important roles in growth, virulence, cell wall homeostasis, potassium transport and affects resistance to antibiotics, heat and osmotic stress. Most Firmicutes contain only one c-di-AMP synthesizing diadenylate cyclase (CdaA); however, little is known about signals and effectors controlling CdaA activity and c-di-AMP levels. In this study, a genetic screen was employed to identify components which affect the c-di-AMP level in Lactococcus. We characterized suppressor mutations that restored osmoresistance to spontaneous c-di-AMP phosphodiesterase gdpP mutants, which contain high c-di-AMP levels. Loss-of-function and gain-of-function mutations were identified in the cdaA and gdpP genes, respectively, which led to lower c-di-AMP levels. A mutation was also identified in the phosphoglucosamine mutase gene glmM, which is commonly located within the cdaA operon in bacteria. The glmM I154F mutation resulted in a lowering of the c-di-AMP level and a reduction in the key peptidoglycan precursor UDP-N-acetylglucosamine in L. lactis. C-di-AMP synthesis by CdaA was shown to be inhibited by GlmM I154F more than GlmM and GlmM I154F was found to bind more strongly to CdaA than GlmM. These findings identify GlmM as a c-di-AMP level modulating protein and provide a direct connection between c-di-AMP synthesis and peptidoglycan biosynthesis.

Toxin ζ Triggers a Survival Response to Cope with Stress and Persistence

“…Downregulation of GlmS contributes indirectly to reducing the pool of UNAG synthesis, and a small UNAG pool increases levels of the essential cyclic 3,5-diadenosine monophosphate (c-di-AMP) second messenger (Witte et al, 2008; Zhu et al, 2016). Changes in the intracellular level of c-di-AMP, which play an essential role in K + transport and cell wall homeostasis (Gundlach et al, 2017), indirectly increase the intracellular (p)ppGpp pool (Rao et al, 2010; Corrigan et al, 2015).…”

Coping with an Essential Poison: a Genetic Suppressor Analysis Corroborates a Key Function of c-di-AMP in Controlling Potassium Ion Homeostasis in Gram-Positive Bacteria

“…Taken together, these results suggested that CdaA is the major diadenylate cyclase in B. subtilis and that c-di-AMP produced by this enzyme is the cause of c-di-AMP toxicity. Zhu et al have isolated suppressor mutants that overcome the salt sensitivity of Lactococcus lactis strains lacking the single c-di-AMPdegrading phosphodiesterase (28). With an impressive set of more than 60 distinct mutations affecting CdaA, and thus reduced levels of c-di-AMP, this study convincingly reinforced the conclusion that c-di-AMP accumulation causes osmosensitivity in L. lactis (28).…”

“…The suppressor mutants that are more resistant to heat stress carry mutations, and two distinct mutations affecting the single diadenylate cyclase CdaA are involved in this resistance: a V76G substitution at the outer side of the third transmembrane helix, and a frameshift at the C terminus-encoding end of the cdaA gene that results in the addition of a 32-amino-acid tail. Interestingly, a mutation corresponding to the V76G mutation was also found in the L. lactis suppressor screen, and mutations affecting the C terminus of the protein were detected in both B. subtilis and L. lactis (9,28). Both mutations resulted in a severe reduction of the c-di-AMP concentrations and restored the wild-type levels of the nucleotide.…”

“…Zhu et al have isolated suppressor mutants that overcome the salt sensitivity of Lactococcus lactis strains lacking the single c-di-AMPdegrading phosphodiesterase (28). With an impressive set of more than 60 distinct mutations affecting CdaA, and thus reduced levels of c-di-AMP, this study convincingly reinforced the conclusion that c-di-AMP accumulation causes osmosensitivity in L. lactis (28). The current study by Zarrella et al (31) adds another flavor to the adaptation of c-di-AMP-accumulating bacteria to stress conditions ( Fig.…”

“…For instance, elevated cellular concentrations of c-di-AMP due to the loss of the phosphodiesterase GdpP in L. monocytogenes, L. lactis, and B. subtilis reduced the susceptibility of the bacteria to ␤-lactams (2,28,35). A Streptococcus suis mutant tolerating large amounts of lysozyme had acquired a mutation in the cdaA diadenylate cyclase gene, which probably alters the cellular c-di-AMP concentration (36).…”