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. 2015 Oct;197(20):3265-74.
doi: 10.1128/JB.00564-15. Epub 2015 Aug 3.

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

Jan Gundlach  1 , Felix M P Mehne  1 , Christina Herzberg  1 , Jan Kampf  1 , Oliver Valerius  2 , Volkhard Kaever  3 , Jörg Stülke  4
Affiliations

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

Jan Gundlach et al. J Bacteriol. 2015 Oct.

Abstract

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.

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Figures

FIG 1
FIG 1
Major components of c-di-AMP signaling in B. subtilis studied in this work. (A) Cyclic di-AMP signaling in B. subtilis. Transmembrane regions are depicted as barrels, with one barrel per transmembrane domain. The three diadenylate cyclases, DisA, CdaA, and CdaS, are shown in yellow. CdaR and GlmM are shown in green and red, respectively. The phosphodiesterases GdpP and PgpH are shown in black. Please note that the illustration is not shown to scale. (B) Genetic organization of the highly conserved cda-glm operon in firmicutes. The operon is expressed under the control of a constitutive promoter; in addition, glmS is controlled by the glucosamine 6-phosphate-responsive glmS ribozyme.
FIG 2
FIG 2
The diadenylate cyclase CdaA is a membrane-associated enzyme. A crude extract of B. subtilis GP1381 (cdaA-FLAG) was separated by ultracentrifugation to obtain cytosolic and membrane fractions. To check for successful separation, the cytosolic and membrane fractions were tested with specific antibodies recognizing CggR and Rny, respectively. CdaA-FLAG was detected using commercial antibodies. CE, cell extract.
FIG 3
FIG 3
Interactions between CdaA, CdaR, and GlmM. (A) Detection of in vivo interaction partners of CdaR. B. subtilis cells (GP1331) were grown in CSE minimal medium supplemented with glucose in the presence or absence of the cross-linker paraformaldehyde (PFA). The copurified proteins were analyzed in an SDS-polyacrylamide gel and visualized by silver staining. Proteins were identified by mass spectrometry. CE, cell extract; E −, elution fraction without cross-linking; E +, elution fraction after cross-linking with paraformaldehyde. (B) B2H screen. (C) β-Galactosidase activities in B2H screen.
FIG 4
FIG 4
The intracellular c-di-AMP concentration depends on the nitrogen source. Cells were grown in SM minimal medium supplemented with the indicated nitrogen sources (68 mM [each]). Data for three biological replicates are shown; error bars show standard deviations. E, glutamate; Q glutamine.
FIG 5
FIG 5
Identification of PgpH as the major c-di-AMP-hydrolyzing enzyme. Cells were grown in SM minimal medium supplemented with glutamine (gray) or glutamate (black). Data for three biological replicates are shown; error bars show standard deviations. wt, wild type.
FIG 6
FIG 6
Mutations affecting the diadenylate cyclase CdaA. TM, transmembrane region; CC, coiled-coil domain; DAC, diadenylate cyclase domain; #, DGA and RHR active site motifs; SD, Shine-Dalgarno sequence (the first G of the sequence is replaced by a T in the suppressor mutant GP2134); *, frameshift mutations (both mutations result in the production of inactive truncated proteins); △, C-terminal deletion.
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