Identification of c-di-AMP-Binding Proteins Using Magnetic Beads.

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.

An Essential Poison: Synthesis and Degradation of Cyclic Di-AMP in Bacillus subtilis.

UNLABELLED: 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. IMPORTANCE: Bacteria 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: glutamate-grown 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.

Identification, characterization, and structure analysis of the cyclic di-AMP-binding PII-like signal transduction protein DarA.

The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3'3'-cGAMP) but not c-di-GMP or 2'3'-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA.