Homeostasis of second messenger cyclic-di-AMP is critical for cyanobacterial fitness and acclimation to abiotic stress

Second messengers are intracellular molecules regulated by external stimuli known as first messengers that are used for rapid organismal responses to dynamic environmental changes. Cyclic di-AMP (c-di-AMP) is a relatively newly discovered second messenger implicated in cell wall homeostasis in many pathogenic bacteria. C-di-AMP is synthesized from ATP by diadenylyl cyclases (DAC) and degraded by specific c-di-AMP phosphodiesterases (PDE). C-di-AMP DACs and PDEs are present in all sequenced cyanobacteria, suggesting roles for c-di-AMP in the physiology and/or development of these organisms. Despite conservation of these genes across numerous cyanobacteria, the functional roles of c-di-AMP in cyanobacteria have not been well-investigated. In a unique feature of cyanobacteria, phylogenetic analysis indicated that the broadly conserved DAC, related to CdaA/DacA, is always co-associated in an operon with genes critical for controlling cell wall synthesis. To investigate phenotypes regulated by c-di-AMP in cyanobacteria, we overexpressed native DAC (sll0505) and c-di-AMP PDE (slr0104) genes in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) to increase and decrease intracellular c-di-AMP levels, respectively. DAC- and PDE-overexpression strains, showed abnormal aggregation phenotypes, suggesting functional roles for regulating c-di-AMP homeostasis in vivo. As c-di-AMP may be implicated in osmotic responses in cyanobacteria, we tested whether sorbitol and NaCl stresses impacted expression of sll0505 and slr0104 or intracellular c-di-AMP levels in Synechocystis. Additionally, to determine the range of cyanobacteria in which c-di-AMP may function, we assessed c-di-AMP levels in two unicellular cyanobacteria, i.e., Synechocystis and Synechococcus elongatus PCC 7942, and two filamentous cyanobacteria, i.e., Fremyella diplosiphon and Anabaena sp. PCC 7120. C-di-AMP levels responded differently to abiotic stress signals in distinct cyanobacteria strains, whereas salt stress uniformly impacted another second messenger cyclic di-GMP in cyanobacteria. Together, these results suggest regulation of c-di-AMP homeostasis in cyanobacteria and implicate a role for the second messenger in maintaining cellular fitness in response to abiotic stress.