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

Gundlach, Jan; Mehne, Felix M.P.; Herzberg, Christina; Kampf, Jan; Valerius, Oliver; Kaever, Volkhard; Stülke, Jörg

doi:10.1128/jb.00564-15

Cited by 114 publications

(197 citation statements)

References 51 publications

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“…The nucleotides extracted were dissolved with 200 l of H 2 O. After repeated centrifugation and addition of the internal standard [ 13 C, 15 N]c-di-AMP, part of the extracts was analyzed by LC-MS/MS as described previously (10). The cell pellets for protein quantification were dissolved in 800 l 0.1 M NaOH and heated for 10 min at 98°C.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting supernatant was pooled with the previous one. The cell pellets harvested for protein quantification were treated as described previously (10), and the protein concentration was determined using the Bradford assay (35).…”

Section: Methodsmentioning

confidence: 99%

“…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-di-AMP production by a single diadenylate cyclase is sufficient for viability of B. subtilis, but the existence of three diadenylate cyclases in this organism suggests that c-di-AMP levels need to be fine-tuned during different lifestyle conditions (6,9).…”

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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%

Section: Methodsmentioning

confidence: 99%

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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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“…The precise reason for the essentiality of this nucleotide is unknown, but c-di-AMP has been implicated in cell division and cell wall biosynthesis (164,165). Interestingly, c-di-AMP is not only essential but also toxic if it accumulates (166). Therefore, CdaA was selected among the three diadenylate cyclases in B. subtilis, and GdpP was selected as one of two phosphodiesterases that degrade the second messenger c-di-AMP.…”

Section: Signaling In Cell Divisionmentioning

confidence: 99%

The Blueprint of a Minimal Cell: MiniBacillus

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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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“…YfnI is important for generation of the polyglycerolphosphate and GroP-Glc 2 -diacylglycerol (DAG) glycolipid components of lipoteichoic acid during cell stress (64), whereas the last gene in the yqfF operon, dgkA, may be involved in phosphorylating undecaprenol to generate undecaprenyl phosphate (Und-P) (65). yqfF itself encodes a protein involved in turnover of cyclic di-AMP, an essential molecule involved in cell envelope homeostasis in Gram-positive organisms (66). Intriguingly, the deletion of whiA (a cotranscribed gene downstream of yvcK) (67) in combination with the deletion of zapA (68) causes a synthetic cell division defect.…”

Section: Metabolism and Cell Elongationmentioning

confidence: 99%

Metabolism Shapes the Cell

Sperber

Herman

2017

J Bacteriol

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More than 5 decades of work support the idea that cell envelope synthesis, including the inward growth of cell division, is tightly coordinated with DNA replication and protein synthesis through central metabolism. Remarkably, no unifying model exists to account for how these fundamentally disparate processes are functionally coupled. Recent studies demonstrate that proteins involved in carbohydrate and nitrogen metabolism can moonlight as direct regulators of cell division, coordinate cell division and DNA replication, and even suppress defects in DNA replication. In this minireview, we focus on studies illustrating the intimate link between metabolism and regulation of peptidoglycan (PG) synthesis during growth and division, and we identify the following three recurring themes. (i) Nutrient availability, not growth rate, is the primary determinant of cell size. (ii) The degree of gluconeogenic flux is likely to have a profound impact on the metabolites available for cell envelope synthesis, so growth medium selection is a critical consideration when designing and interpreting experiments related to morphogenesis. (iii) Perturbations in pathways relying on commonly shared and limiting metabolites, like undecaprenyl phosphate (Und-P), can lead to pleotropic phenotypes in unrelated pathways.KEYWORDS FtsZ, MreB, UDP-glucose, cell division, gluconeogenesis, metabolism, morphogenesis, peptidoglycan, phosphoenolpyruvate, undecaprenyl phosphate T o accurately partition chromosomes and other cell contents during reproduction, cells must possess mechanisms to organize repeated cycles of cell growth, chromosome replication, and division. Eukaryotes orchestrate this coordination using the cell cycle and separate growth, DNA synthesis, and cytokinesis into distinct, temporally sequestered phases. Bacteria, by contrast, simultaneously increase in cell size and replicate DNA before (or concurrently with) cell division. Elucidating the molecular mechanisms prokaryotes employ to achieve spatiotemporal organization of these intertwined yet functionally disparate processes is of considerable interest to scientists seeking to understand bacterial reproduction, and many outstanding questions remain to be answered. For example, how is DNA replication kept in sync with changing growth and division rates? How are cell dimensions maintained or actively rearranged in response to environmental or developmental cues? What signals do cells sense to switch between increasing in cell size and dividing during the cell cycle? Relatedly, how are these signals transduced to activate/deactivate the distinct machineries required for each process?Perhaps one of the biggest mysteries remaining in bacterial cell biology relates to understanding the regulatory cross talk that must occur to integrate central metabolism with macromolecular biosynthesis. Nutrients are converted into stored energy and precursors used to synthesize macromolecules like DNA and peptidoglycan (PG), so it is no surprise that nutrient availability has a profound impac...

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

Cited by 114 publications

References 51 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

The Blueprint of a Minimal Cell: MiniBacillus

Metabolism Shapes the Cell

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