Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes

Rosenberg, Jonathan B.; Dickmanns, Achim; Neumann, Piotr; Gunka, Katrin; Arens, Johannes; Kaever, Volkhard; Stülke, Jörg; Ficner, Ralf; Commichau, Fabian M.

doi:10.1074/jbc.m114.630418

Cited by 68 publications

(129 citation statements)

References 69 publications

(78 reference statements)

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“…Plasmid pBP33 was used to overexpress the N-terminally Strep-tagged diadenylate cyclase CdaA from L. monocytogenes as described previously (23). The protein was purified with the Strep-tag II-Strep-Tactin purification system (IBA, Göttingen, Germany) and used for the generation of polyclonal antibodies in rabbits (Seqlab, Göttingen, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, an N-terminally truncated variant of the L. monocytogenes diadenylate cyclase CdaA lacking the membrane-binding domain and the adjacent CC motif was structurally and biochemically characterized (23). The functionality of the CdaA variant was demonstrated by expression of the truncated cdaA gene in Escherichia coli, an organism that does not produce c-di-AMP (6,23), and c-di-AMP production was dependent on the presence of divalent ions in vitro (23).…”

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confidence: 99%

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Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes

et al. 2016

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Cyclic diadenylate monophosphate (c-di-AMP) is a second messenger utilized by diverse bacteria. In many species, including the Gram-positive human pathogen Listeria monocytogenes, c-di-AMP is essential for growth. Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with its potential regulatory protein, CdaR, via the transmembrane protein domain. The presence of the CdaR protein is not required for the membrane localization and abundance of CdaA. We have also found that CdaR negatively influences CdaA activity in L. monocytogenes and that the role of CdaR is most evident at a high growth temperature. Interestingly, a cdaR mutant strain is less susceptible to lysozyme. Moreover, CdaA contributes to cell division, and cells depleted of CdaA are prone to lysis. The observation that the growth defect of a CdaA depletion strain can be partially restored by increasing the osmolarity of the growth medium suggests that c-di-AMP is important for maintaining the integrity of the protective cell envelope. Overall, this work provides new insights into the relationship between CdaA and CdaR. IMPORTANCE Cyclic diadenylate monophosphate (c-di-AMP) is a recently identified second messenger that is utilized by the Gram-positive human pathogen Listeria monocytogenes.Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with CdaR, its potential regulatory protein. We show that CdaR is not required for membrane localization or abundance of the diadenylate cyclase, but modulates its activity. Moreover, CdaA seems to contribute to cell division. Overall, this work provides new insights into the relationship between CdaA and CdaR and their involvement in cell growth. Bacteria from diverse phyla produce the cyclic dinucleotide cyclic diadenylate monophosphate (c-di-AMP) that is synthesized and degraded by specific diadenylate cyclases and phosphodiesterases, respectively (1). The DNA integrity scanning protein DisA from Thermotoga maritima was the first diadenylate cyclase structurally and biochemically characterized (2), and its characterization led to the discovery of c-di-AMP. Many bacteria possess only a single, DisA-type, diadenylate cyclase (1), which is involved in the maintenance of DNA integrity (3, 4, 5). The cyclase activity of DisA is modulated by unusual DNA recombination intermediates (2), but it is presently unclear how c-di-AMP signals the cell that the chromosome integrity is affected.In addition to DisA, two diadenylate cyclases, CdaA and CdaS, are synthesized in the Gram-positive model bacterium Bacillus subtilis (6). While cdaA is expressed during vegetative growth, the cdaS gene is expressed during sporulation or germination of spores (7). The cdaS inactivation decreases the germination efficiency of spores, indicating a germination-specific function for this enzyme (8). Recently, it has been shown that c-di-AMP is essential for the growth of B. subtilis (6, 9, 10). c-d...

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Section: Methodsmentioning

confidence: 99%

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Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes

et al. 2016

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“…All diadenylate cyclases are characterized by a conserved catalytically active domain, the so-called DAC (diadenylate cyclase) domain (11,12). B. subtilis encodes three diadenylate cyclases: DisA, CdaA, and CdaS (7) (Fig.…”

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

et al. 2015

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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...

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“…The genetic complement of MiniBacillus likely represents a set of highly important genes for all of these organisms, suggesting that this set is an excellent starting point to identify promising novel potential antimicrobial drug targets. Indeed, the conserved essential diadenylate cyclase was recently proposed to be a potential drug target, screening systems have been set up, and a first drug has been discovered (168)(169)(170).…”

Section: Minibacillus As a Starting Point To Discover And Study Novelmentioning

confidence: 99%

The Blueprint of a Minimal Cell: MiniBacillus

Reuß

Commichau

Gundlach

et al. 2016

Microbiol Mol Biol Rev

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SUMMARYBacillus subtilisis one of the best-studied organisms. Due to the broad knowledge and annotation and the well-developed genetic system, this bacterium is an excellent starting point for genome minimization with the aim of constructing a minimal cell. We have analyzed the genome ofB. subtilisand selected all genes that are required to allow life in complex medium at 37°C. This selection is based on the known information on essential genes and functions as well as on gene and protein expression data and gene conservation. The list presented here includes 523 and 119 genes coding for proteins and RNAs, respectively. These proteins and RNAs are required for the basic functions of life in information processing (replication and chromosome maintenance, transcription, translation, protein folding, and secretion), metabolism, cell division, and the integrity of the minimal cell. The completeness of the selected metabolic pathways, reactions, and enzymes was verified by the development of a model of metabolism of the minimal cell. A comparison of theMiniBacillusgenome to the recently reported designed minimal genome ofMycoplasma mycoidesJCVI-syn3.0 indicates excellent agreement in the information-processing pathways, whereas each species has a metabolism that reflects specific evolution and adaptation. The blueprint ofMiniBacilluspresented here serves as the starting point for a successive reduction of theB. subtilisgenome.

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Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes

Cited by 68 publications

References 69 publications

Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes

Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes

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

The Blueprint of a Minimal Cell: MiniBacillus

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