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Cyclic Di-AMP Homeostasis in Bacillus subtilis

BOTH LACK AND HIGH LEVEL ACCUMULATION OF THE NUCLEOTIDE ARE DETRIMENTAL FOR CELL GROWTH*
  • Felix M.P. Mehne
    Affiliations
    From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University Göttingen, D-37077 Göttingen, Germany
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  • Katrin Gunka
    Affiliations
    From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University Göttingen, D-37077 Göttingen, Germany
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  • Hinnerk Eilers
    Affiliations
    From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University Göttingen, D-37077 Göttingen, Germany
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  • Christina Herzberg
    Affiliations
    From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University Göttingen, D-37077 Göttingen, Germany
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  • Volkhard Kaever
    Affiliations
    Research Core Unit for Mass Spectrometry-Metabolomics and Institute of Pharmacology, Hannover Medical School, 30625 Hannover, Germany
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  • Jörg Stülke
    Correspondence
    To whom correspondence should be addressed: Dept. of General Microbiology, Inst. of Microbiology and Genetics, Georg-August University Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany. Tel.: 49-551-393781; Fax: 49-551-393808;
    Affiliations
    From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University Göttingen, D-37077 Göttingen, Germany
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  • Author Footnotes
    * This work was supported by grants from the Deutsche Forschungsgemeinschaft (to J. S.).
    This article contains supplemental Tables S1 and S2.
Open AccessPublished:November 28, 2012DOI:https://doi.org/10.1074/jbc.M112.395491
      The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.
      Background: Bacillus subtilis encodes three diadenylate cyclases.
      Results: Cyclic di-AMP is essential for the viability of B. subtilis; however, excess c-di-AMP also harms the cells. The activity of the cyclases is subject to regulation.
      Conclusion: The control of c-di-AMP homeostasis is crucial for B. subtilis.
      Significance: c-di-AMP is the first essential signaling nucleotide in bacteria.

      Introduction

      To respond appropriately to changing environments, bacteria have evolved a variety of signaling strategies. Many of these strategies involve changes of gene expression programs. However, quick responses may often be important for the bacterial cell. For this purpose, protein activities have to be modulated. This can occur by either covalent modification of the proteins (e.g. by phosphorylation or acetylation), degradation of the proteins, or non-covalent interaction with other proteins or low molecular weight effectors. The low molecular weight effectors may be either metabolites that are part of the normal metabolism or dedicated signaling molecules that are produced by the cell for the purpose of signal transduction (
      • Petsko G.A.
      • Ringe D.
      ).
      Among the best studied signaling molecules produced by bacteria are the so-called autoinducers, either acylated homoserine lactones or peptides in Gram-negative and Gram-positive bacteria, respectively (
      • Thoendel M.
      • Horswill A.R.
      Biosynthesis of peptide signals in gram-positive bacteria.
      ). In addition, specific signaling nucleotides have been discovered in all bacteria in which they have been searched (
      • Gomelsky M.
      cAMP, c-di-GMP, c-di-AMP and now cGMP: bacteria use them all!.
      ). Although the autoinducers serve mainly for purposes of cell-cell communication (quorum sensing), the signaling nucleotides are used for intracellular signaling. In addition to cyclic AMP and (p)ppGpp that are involved in carbon catabolite repression and the stringent response, respectively, many bacteria also synthesize cyclic dinucleotides such as cyclic di-AMP (c-di-AMP)
      The abbreviations used are: c-di-AMP
      cyclic di-AMP
      c-di-GMP
      cyclic di-GMP
      DAC
      diadenylate cyclase
      CdaA
      cyclic di-AMP synthase A
      CdaS
      cyclic di-AMP synthase S, sporulation-specific
      CdaR
      cyclic di-AMP synthase A regulator
      SRM
      selected reaction monitoring.
      and cyclic di-GMP (c-di-GMP). These nucleotides are often involved in the control of motility and biofilm formation i.e. in the switch between motile and sessile lifestyles (
      • Hengge R.
      Principles of c-di-GMP signaling in bacteria.
      ). Their synthesis is catalyzed by dedicated diadenylate or diguanylate cyclases. These proteins always contain conserved catalytic domains that may be combined with additional domains for signal input and output. In addition, bacteria that produce cyclic dinucleotides also contain nucleotide-specific phosphodiesterases for the degradation of the molecules (
      • Römling U.
      Great times for small molecules: c-di-AMP, a second messenger candidate in bacteria and archaea.
      ,
      • Schirmer T.
      • Jenal U.
      Structural and mechanistic determinants of c-di-GMP signalling.
      ).
      The synthesis, mode of action, and degradation of c-di-GMP have been studied in detail in many bacteria (for a review, see Ref.
      • Hengge R.
      Principles of c-di-GMP signaling in bacteria.
      ). In contrast, much less is known about the metabolism and physiological function of c-di-AMP. Diadenylate cyclase activity was first described for the DisA protein of Bacillus subtilis (
      • Witte G.
      • Hartung S.
      • Büttner K.
      • Hopfner K.P.
      Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.
      ). This octameric protein has two interdependent activities. (i) It binds DNA via its RuvA-like C-terminal DNA-binding domain and scans its integrity. (ii) It synthesizes c-di-AMP. If the protein arrives at branched DNA molecules present in Holliday junctions, then the catalytic activity is inhibited, and this reduction in c-di-AMP concentration results in the delay of sporulation (
      • Witte G.
      • Hartung S.
      • Büttner K.
      • Hopfner K.P.
      Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.
      ,
      • Oppenheimer-Shaanan Y.
      • Wexselblatt E.
      • Katzhendler J.
      • Yavin E.
      • Ben-Yehuda S.
      c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis.
      ). DisA contains a catalytic domain, called the diadenylate cyclase (DAC) domain (previously referred to as domain of unknown function, DUF147) (
      • Witte G.
      • Hartung S.
      • Büttner K.
      • Hopfner K.P.
      Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.
      ). The discovery of this c-di-AMP-producing enzyme revealed that proteins with similar DAC domains are present in many bacteria, both Gram-positive and Gram-negative, and archaea (
      • Römling U.
      Great times for small molecules: c-di-AMP, a second messenger candidate in bacteria and archaea.
      ). This observation suggests that c-di-AMP might be a widespread signaling nucleotide. Moreover, DAC domains are coupled not only to RuvA-like DNA-binding domains but also to a wide variety of different domains of unknown function. These domains may control the signal in- and/or output of the proteins (
      • Römling U.
      Great times for small molecules: c-di-AMP, a second messenger candidate in bacteria and archaea.
      ). The presence of DAC domains in so many different organisms and in varying domain arrangements indicates that c-di-AMP levels may respond to a number of distinct stimuli and that c-di-AMP may play an important role in the control of different cellular activities.
      We are interested in the molecular biology of the Gram-positive model bacterium B. subtilis. Signaling in this bacterium involves a variety of transcription factors, alternative σ factors, RNA-mediated regulation via RNA-binding proteins or riboswitches, and protein phosphorylation. Moreover, signal transduction via small molecules plays an important role in B. subtilis. In this bacterium, small peptides control the initiation of sporulation and the induction of genetic competence (
      • Pottathil M.
      • Lazazzera B.A.
      The extracellular Phr peptide-Rap phosphatase signaling circuit of Bacillus subtilis.
      ). Moreover, ppGpp mediates the stringent response by inhibiting GTP synthesis and thus by differential control of transcription of mRNAs that use an A or a G as the first nucleotide (
      • Kriel A.
      • Bittner A.N.
      • Kim S.H.
      • Liu K.
      • Tehranchi A.K.
      • Zou W.Y.
      • Rendon S.
      • Chen R.
      • Tu B.P.
      • Wang J.D.
      Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.
      ,
      • Krásný L.
      • Tiserová H.
      • Jonák J.
      • Rejman D.
      • Sanderová H.
      The identity of the transcription +1 position is crucial for changes in gene expression in response to amino acid starvation in Bacillus subtilis.
      ,
      • Tojo S.
      • Kumamoto K.
      • Hirooka K.
      • Fujita Y.
      Heavy involvement of stringent transcription control depending on the adenine or guanine species of the transcription initiation site in glucose and pyruvate metabolism in Bacillus subtilis.
      ). The genome of B. subtilis encodes several potential diguanylate cyclases and potential c-di-GMP-specific phosphodiesterases (
      • Mäder U.
      • Schmeisky A.G.
      • Flórez L.A.
      • Stülke J.
      SubtiWiki—a comprehensive community resource for the model organism Bacillus subtilis.
      ,
      • Chen Y.
      • Chai Y.
      • Guo J.H.
      • Losick R.
      Evidence for cyclic di-GMP-mediated signaling in Bacillus subtilis.
      ). Very recently, c-di-GMP was found to control motility by binding the YpfA protein, which in turn interacts with and inhibits the motor protein MotA in B. subtilis (
      • Chen Y.
      • Chai Y.
      • Guo J.H.
      • Losick R.
      Evidence for cyclic di-GMP-mediated signaling in Bacillus subtilis.
      ). In addition to these well established signaling nucleotides, c-di-AMP synthesis by the DisA protein was recently discovered for the first time (
      • Witte G.
      • Hartung S.
      • Büttner K.
      • Hopfner K.P.
      Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.
      ). In contrast to most other bacteria that contain only one diadenylate cyclase, B. subtilis encodes two additional proteins with DAC domains that may possibly be involved in c-di-AMP synthesis. Finally, the c-di-AMP-specific phosphodiesterase GdpP (previously referred to as YybT (
      • Luo Y.
      • Helmann J.D.
      Analysis of the role of Bacillus subtilis σM in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis.
      )) degrades cyclic di-AMP (
      • Rao F.
      • See R.Y.
      • Zhang D.
      • Toh D.C.
      • Ji Q.
      • Liang Z.X.
      YybT is a signaling protein that contains a cyclic dinucleotide phosphodiesterase domain and a GGDEF domain with ATPase activity.
      ). Recently, c-di-AMP was implicated in the control of cell wall homeostasis in B. subtilis, and it was demonstrated that this signaling nucleotide is essential for the growth of the bacterium (
      • Luo Y.
      • Helmann J.D.
      Analysis of the role of Bacillus subtilis σM in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis.
      ).
      In this work, we have analyzed the activity of the so far unknown diadenylate cyclases of B. subtilis, YbbP and YojJ. Our results indicate that both proteins have enzymatic activity. Moreover, we demonstrate that the activity of YbbP is modulated by a protein-protein interaction with the modulator protein YbbR. The activity of the sporulation-specific diadenylate cyclase YojJ is self-restricted. Full YojJ activity and accumulation of c-di-AMP due to the inactivation of the phosphodiesterase gene gdpP result in impaired growth. Thus, the cells need a certain level of c-di-AMP, and strongly reduced or increased amounts of the nucleotide seem to be deleterious for the cell. Based on our results, YbbP, YojJ, and YbbR were renamed cyclic di-AMP synthase A (CdaA), cyclic di-AMP synthase S, sporulation-specific (CdaS), and cyclic di-AMP synthase A regulator (CdaR), respectively. These designations will be used hereafter.

      DISCUSSION

      In this work, we have demonstrated that the intracellular concentration of the signaling nucleotide c-di-AMP has to be adjusted to a certain level. Amounts of the nucleotide that are too low or too high are disadvantageous for the cell.
      The genetic evidence presented here and in a previous study (
      • Luo Y.
      • Helmann J.D.
      Analysis of the role of Bacillus subtilis σM in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis.
      ) demonstrates that the three diadenylate cyclases of B. subtilis can replace each other in the generation of a c-di-AMP pool sufficient for growth. However, the three enzymes seem to have distinct functions. DisA scans the DNA and stops c-di-AMP production if it encounters problems with DNA integrity such as Holliday junctions. The reduced DisA-mediated c-di-AMP synthesis results in a delay of sporulation (
      • Oppenheimer-Shaanan Y.
      • Wexselblatt E.
      • Katzhendler J.
      • Yavin E.
      • Ben-Yehuda S.
      c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis.
      ,
      • Bejerano-Sagie M.
      • Oppenheimer-Shaanan Y.
      • Berlatzky I.
      • Rouvinski A.
      • Meyerovich M.
      • Ben-Yehuda S.
      A checkpoint protein that scans the chromosome for damage at the start of sporulation in Bacillus subtilis.
      ). This specific function is rather unlikely to be involved in the essential role of c-di-AMP. The diadenylate cyclase CdaS is specifically expressed during sporulation most likely by RNA polymerase containing the late forespore-specific σ factor σG (
      • Nicolas P.
      • Mäder U.
      • Dervyn E.
      • Rochat T.
      • Leduc A.
      • Pigeonneau N.
      • Bidnenko E.
      • Marchadier E.
      • Hoebeke M.
      • Aymerich S.
      • Becher D.
      • Bisicchia P.
      • Botella E.
      • Delumeau O.
      • Doherty G.
      • Denham E.L.
      • Fogg M.J.
      • Fromion V.
      • Goelzer A.
      • Hansen A.
      • Härtig E.
      • Harwood C.R.
      • Homuth G.
      • Jarmer H.
      • Jules M.
      • Klipp E.
      • Le Chat L.
      • Lecointe F.
      • Lewis P.
      • Liebermeister W.
      • March A.
      • Mars R.A.
      • Nannapaneni P.
      • Noone D.
      • Pohl S.
      • Rinn B.
      • Rügheimer F.
      • Sappa P.K.
      • Samson F.
      • Schaffer M.
      • Schwikowski B.
      • Steil L.
      • Stülke J.
      • Wiegert T.
      • Devine K.M.
      • Wilkinson A.J.
      • van Dijl J.M.
      • Hecker M.
      • Völker U.
      • Bessières P.
      • Noirot P.
      The condition-dependent whole-transcriptome reveals high-level regulatory architecture in bacteria.
      ). In agreement with this observation, CdaS was unable to provide the cell with sufficient c-di-AMP in the absence of DisA and CdaA unless it was expressed from a regulated promoter that is also active during exponential growth. Thus, the function of CdaS seems to be limited to the spore.
      Several lines of evidence suggest that CdaA is implicated in the control of cell wall biosynthesis. First, CdaA is encoded in an operon with proteins involved in cell wall biosynthesis. Interestingly, the genetic clustering of CdaA-related diadenylate cyclase genes with the essential glm genes involved in the generation of glucosamine 1-phosphate, a key precursor for cell wall biosynthesis, is conserved in most δ-proteobacteria and firmicutes (with the notable exception of cell wall-less mollicutes). Second, a link of at least one of the diadenylate cyclases to cell wall metabolism is also supported by the recent study of Luo and Helmann (
      • Luo Y.
      • Helmann J.D.
      Analysis of the role of Bacillus subtilis σM in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis.
      ). These authors report that accumulation of c-di-AMP due to the inactivation of the gdpP gene encoding the specific phosphodiesterase results in increased resistance to cell wall antibiotics such as β-lactams and conclude that c-di-AMP plays an essential role in peptidoglycan homeostasis. Third, this conclusion is supported by our observation that severe overproduction of c-di-AMP interferes with cell morphology in a Mg2+-dependent manner (see Fig. 6). This is reminiscent of the phenotypes of other mutations that affect cell wall synthesis (
      • Murray T.
      • Popham D.L.
      • Setlow P.
      Bacillus subtilis cells lacking penicillin-binding protein 1 require increased level of divalent cations for growth.
      ,
      • Görke B.
      • Foulquier E.
      • Galinier A.
      YvcK of Bacillus subtilis is required for a normal cell shape and for growth on Krebs cycle intermediates and substrates of the pentose phosphate pathway.
      ,
      • Formstone A.
      • Errington J.
      A magnesium-dependent mreB null mutant: implications for the role of MreB in Bacillus subtilis.
      ,
      • Kawai Y.
      • Daniel R.A.
      • Errington J.
      Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix.
      ).
      The obvious functional specialization of the three diadenylate cyclases suggests that the c-di-AMP synthesized by the individual proteins is active in a time- and compartment-specific manner. This is rather obvious for CdaS, which is expressed only in the forespore, suggesting that c-di-AMP produced by CdaS has a spore-specific function. In the vegetative cell, DisA is associated to the DNA, whereas CdaA contains three transmembrane domains and is associated to the membrane (
      • Hahne H.
      • Wolff S.
      • Hecker M.
      • Becher D.
      From complementarity to comprehensiveness—targeting the membrane proteome of growing Bacillus subtilis by divergent approaches.
      ). Thus, the enzymes seem to form distinct c-di-AMP pools in a temporally and spatially ordered manner. In this way, the c-di-AMP may also be close to its potential target proteins. A similar hypothesis has been proposed for c-di-GMP, which can be produced by more than a dozen different proteins in a single bacterial cell (
      • Seshasayee A.S.
      • Fraser G.M.
      • Luscombe N.M.
      Comparative genomics of cyclic-di-GMP signaling in bacteria: post-translational regulation and catalytic activity.
      ).
      The rather specialized functions of DisA and CdaS suggest that CdaA is the major player in c-di-AMP production in B. subtilis and is key for the essential function of the nucleotide. In this context, the conserved genetic linkage with the essential glmM and glmS genes is noteworthy. The idea of a crucial function for CdaA is further supported by the observation that the single diadenylate cyclases of the Gram-positive pathogens Listeria monocytogenes, Staphylococcus aureus, and Streptococcus pneumoniae are highly similar to CdaA (including the domain organization) and that the encoding genes are essential (
      • Chaudhuri R.R.
      • Allen A.G.
      • Owen P.J.
      • Shalom G.
      • Stone K.
      • Harrison M.
      • Burgis T.A.
      • Lockyer M.
      • Garcia-Lara J.
      • Foster S.J.
      • Pleasance S.J.
      • Peters S.E.
      • Maskell D.J.
      • Charles I.G.
      Comprehensive identification of essential Staphylococcus aureus genes using transposon-mediated differential hybridization (TMDH).
      ,
      • Song J.H.
      • Ko K.S.
      • Lee J.Y.
      • Baek J.Y.
      • Oh W.S.
      • Yoon H.S.
      • Jeong J.Y.
      • Chun J.
      Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis.
      ,
      • Woodward J.J.
      • Iavarone A.T.
      • Portnoy D.A.
      c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response.
      ). Moreover, the genes homologous to cdaA and cdaR are also linked to glmM and glmS in those organisms. The intimate genetic, transcriptional, and functional linkage of the cdaAR and glmMS genes suggests that they form a conserved module, the cda-glm module.
      As mentioned above, the c-di-AMP concentration has to be tightly controlled to ensure that it does not fall below or exceed a certain physiological concentration. In the case of CdaA, the enzyme has a basal activity, and this activity can be increased by a specific regulatory interaction with the CdaR protein. CdaR proteins are widespread in bacteria; however, this is the first report of their function. These proteins consist of a repeated conserved domain (the YbbR domain); B. subtilis CdaR contains four such domains. The structural analysis of YbbR domains from Desulfitobacterium hafniense (
      • Barb A.W.
      • Cort J.R.
      • Seetharaman J.
      • Lew S.
      • Lee H.W.
      • Acton T.
      • Xiao R.
      • Kennedy M.A.
      • Tong L.
      • Montelione G.T.
      • Prestegard J.H.
      Structures of domains I and IV from YbbR are representative of a widely distributed protein family.
      ) revealed a striking similarity to the ribosomal protein L25. Because L25 proteins bind the 5 S ribosomal RNA (
      • Gongadze G.M.
      • Meshcheryakov V.A.
      • Serganov A.A.
      • Fomenkova N.P.
      • Mudrik E.S.
      • Jonsson B.H.
      • Liljas A.
      • Nikonov S.V.
      • Garber M.B.
      N-terminal domain, residues 1–91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the rRNA containing loop E.
      ,
      • Schmalisch M.
      • Langbein I.
      • Stülke J.
      The general stress protein Ctc of Bacillus subtilis is a ribosomal protein.
      ), it is tempting to speculate that CdaR might also bind RNA and that this binding might in turn control the interaction with and activation of CdaA.
      The activity of CdaS seems to be limited to keep the c-di-AMP levels at a physiologically acceptable level. Interestingly, a single amino acid substitution is sufficient to increase the activity of the protein by a factor of 100. The structure of CdaS can be inferred by modeling based on the known structure of the homologous protein from Bacillus cereus (see Fig. 7). The protein consists of three identical subunits, and each monomer contains two long α-helices at the N terminus that are followed by the rather globular DAC domain. Interestingly, the mutation resulting in the hyperactive CdaS protein is located in the loop that connects the two N-terminal helices. This might result in a repositioning of the two helices with respect to each other and the catalytic DAC domain. Based on this finding, it is tempting to speculate that the N-terminal helices are involved in the control of the activity of the DAC domain and thus in the spore-specific synthesis of c-di-AMP. Similarly, inhibitory protein domains have been found in the B. subtilis transcription factor RocR and the alternative σ factor σ54 (
      • Gardan R.
      • Rapoport G.
      • Débarbouillé M.
      Role of the transcriptional activator RocR in the arginine-degradation pathway of Bacillus subtilis.
      ,
      • Cannon W.
      • Gallegos M.T.
      • Casaz P.
      • Buck M.
      Amino-terminal sequences of σN (σ54) inhibit RNA polymerase isomerisation.
      ).
      Figure thumbnail gr7
      FIGURE 7A model of the three-dimensional structure of CdaS based on the known structure of the corresponding YojJ protein of B. cereus (Protein Data Bank code 2FB5) using the SWISS-MODEL homology-modeling server (
      • Kiefer F.
      • Arnold K.
      • Künzli M.
      • Bordoli L.
      • Schwede T.
      The SWISS-MODEL repository and associated resources.
      ). The DAC domain and the N-terminal helices are shown in light blue and dark blue, respectively. The position of the amino acid exchange (L44F) resulting in the hyperactive CdaS protein is highlighted in red.
      Future studies will focus on the distinct molecular mechanisms controlling the c-di-AMP levels in B. subtilis. Moreover, we will search for the receptors of c-di-AMP and thus for the precise cellular functions of this fascinating signaling molecule.

      Acknowledgments

      We thank Annette Garbe for excellent technical assistance and Carina Gross and Victoria Keidel for help with some experiments. We are grateful to Sigal Ben-Yehuda for providing strains YA5 and YA188. We acknowledge the contributions of Christine Diethmaier for the initiation of this project. We are grateful to Richard Daniel and Sven Halbedel for helpful discussions.

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