Gram-positive bacteria synthesize the second messenger cyclic di-AMP (c-di-AMP) to control cell wall and potassium homeostasis and to secure the integrity of their DNA. In the firmicutes, c-di-AMP is essential for growth. The model organism Bacillus subtilis encodes three diadenylate cyclases and two potential phosphodiesterases to produce and degrade c-di-AMP, respectively. Among the three cyclases, CdaA is conserved in nearly all firmicutes, and this enzyme seems to be responsible for the c-di-AMP that is required for cell wall homeostasis. Here, we demonstrate that CdaA localizes to the membrane and forms a complex with the regulatory protein CdaR and the glucosamine-6-phosphate mutase GlmM. Interestingly, cdaA, cdaR, and glmM form a gene cluster that is conserved throughout the firmicutes. This conserved arrangement and the observed interaction between the three proteins suggest a functional relationship. Our data suggest that GlmM and GlmS are involved in the control of c-di-AMP synthesis. These enzymes convert glutamine and fructose-6-phosphate to glutamate and glucosamine-1-phosphate. c-di-AMP synthesis is enhanced if the cells are grown in the presence of glutamate compared to that in glutamine-grown cells. Thus, the quality of the nitrogen source is an important signal for c-di-AMP production. In the analysis of c-di-AMP-degrading phosphodiesterases, we observed that both phosphodiesterases, GdpP and PgpH (previously known as YqfF), contribute to the degradation of the second messenger. Accumulation of c-di-AMP in a gdpP pgpH double mutant is toxic for the cells, and the cells respond to this accumulation by inactivation of the diadenylate cyclase CdaA. IMPORTANCEBacteria use second messengers for signal transduction. Cyclic di-AMP (c-di-AMP) is the only second messenger known so far that is essential for a large group of bacteria. We have studied the regulation of c-di-AMP synthesis and the role of the phosphodiesterases that degrade this second messenger. c-di-AMP synthesis strongly depends on the nitrogen source: glutamategrown cells produce more c-di-AMP than glutamine-grown cells. The accumulation of c-di-AMP in a strain lacking both phosphodiesterases is toxic and results in inactivation of the diadenylate cyclase CdaA. Our results suggest that CdaA is the critical diadenylate cyclase that produces the c-di-AMP that is both essential and toxic upon accumulation. In order to process environmental information in the cell, many organisms are capable of synthesizing so-called second messengers. These small molecules are formed in response to primary signals and perceived by cellular targets. Bacteria often use specific nucleotides as second messengers. These nucleotides include cyclic mononucleotides, such as cyclic AMP (cAMP) and cyclic GMP (cGMP), as well as cyclic dinucleotides, such as cyclic di-AMP (c-di-AMP), cyclic di-GMP (c-di-GMP), and (p) ppGpp (1-3).The investigation of c-di-AMP-mediated signaling has recently attracted much attention (2). This molecule is formed by many bacteria a...

Background: Cyclic di-AMP is an essential second messenger in eubacteria. Results: The c-di-AMP receptor DarA was identified in B. subtilis. The crystal structure and ITC data revealed the nucleotide specificity of DarA. Conclusion: DarA is a P II -like protein that undergoes conformational changes upon c-di-AMP binding. Significance: A novel P II -like protein is involved in c-di-AMP signaling.

SummarySIRT6 is a NAD + -dependent deacetylase that modulates chromatin structure and safeguards genomic stability. Until now, SIRT6 has been assigned to the nucleus and only nuclear targets of SIRT6 are known. Here, we demonstrate that in response to stress, C. elegans SIR-2.4 and its mammalian orthologue SIRT6 localize to cytoplasmic stress granules, interact with various stress granule components and induce their assembly. Loss of SIRT6 or inhibition of its catalytic activity in mouse embryonic fibroblasts impairs stress granule formation and delays disassembly during recovery, whereas deficiency of SIR-2.4 diminishes maintenance of P granules and decreases survival of C. elegans under stress conditions. Our findings uncover a novel, evolutionary conserved function of SIRT6 in the maintenance of stress granules in response to stress.

Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium Bacillus subtilis, this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that ymdB mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that B. subtilis cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.

In order to resist harmful environmental conditions, many bacteria form multicellular aggregates called biofilms. In these biofilms, they protect themselves in a self-produced matrix consisting of extracellular polysaccharides, proteins and DNA. In many bacteria, biofilm formation is stimulated in the presence of the second messenger cyclic di-GMP. In this issue of Environmental Microbiology Reports, Bedrunka and Graumann have studied matrix production by the proteins encoded in the Bacillus subtilis ydaJKLMN operon. For the first time, they were able to provide a link between c-di-GMP signalling and matrix production in this bacterium. The work demonstrates that the c-di-GMP receptor protein YdaK forms a membrane-bound complex with the YdaM and YdaN proteins, and that this interaction with YdaK is required for polysaccharide production by YdaL, YdaM and YdaN.

To identify cytosolic proteins that bind to cyclic di-AMP, a biotinylated analog of the nucleotide is used for protein pull-down experiments. In this approach, biotinylated c-di-AMP is coupled to Streptactin-covered beads. After protein separation using standard SDS-PAGE, the protein(s) of interest are identified by mass spectrometric analyses.

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