The PAMP c-di-AMP Is Essential for Listeria monocytogenes Growth in Rich but Not Minimal Media due to a Toxic Increase in (p)ppGpp

Whiteley, Aaron T.; Pollock, Alex J.; Portnoy, Daniel A.

doi:10.1016/j.chom.2015.05.006

Cited by 138 publications

(168 citation statements)

References 61 publications

(107 reference statements)

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“…Interestingly, the growth defects associated with depletion of CdaA can be detected already after approximately three generations in culture medium without IPTG. This is in contrast to what we routinely observe with other strains allowing depletion of essential genes (such as secA), which usually need up to six generations to cause retardation of growth (39) and confirms that cdaA is an essential gene in L. monocytogenes during growth in rich medium (11,12). To conclude, inactivation of cdaR and cdaA has additive negative effects on L. monocytogenes growth.…”

Section: Cdar Does Not Affect Synthesis and Membrane Localization Ofcontrasting

confidence: 50%

“…That processes other than peptidoglycan biosynthesis are affected by depletion of CdaA is in good agreement with our observation that penicillin had no effect on autolysis of CdaA-depleted cells. A recent study revealed that c-di-AMP is essential only for the growth of L. monocytogenes on rich medium (12). It has been shown that the lack of c-di-AMP led to a decrease in intracellular GTP levels and consequently to the activation of several genes that are usually repressed by the pleiotropic transcription factor CodY (50).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to DisA and CdaS, which have relatively limited phylogenetic distributions, CdaA is more widespread in bacteria (1). Some bacteria, such as the human pathogen Listeria monocytogenes, possess only a single, CdaA-type, diadenylate cyclase (11), which was shown to be essential for growth on rich medium (12), and CdaA depletion leads to cessation of bacterial growth (13).…”

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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: Cdar Does Not Affect Synthesis and Membrane Localization Ofcontrasting

confidence: 50%

Section: Discussionmentioning

confidence: 99%

mentioning

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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“…This conclusion is underlined by the isolation of suppressor mutants of L. monocytogenes that are able to grow in the absence of c-di-AMP. The common theme of those mutants is a global regulatory defect in the CodY regulon that encompasses genes subject to nutritional signaling (25).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a study using L. monocytogenes identified conditions that allow growth of this bacterium in the absence of c-di-AMP: c-di-AMP production was dispensable if the bacterium grew in minimal medium or if the stringent response was inactivated due to a loss of ppGpp synthesis. This suggests that c-di-AMP is required for L. monocytogenes to cope with the potential toxicity of ppGpp (25).…”

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confidence: 99%

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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A Luminescence‐Based Coupled Enzyme Assay Enables High‐Throughput Quantification of the Bacterial Second Messenger 3’3’‐Cyclic‐Di‐AMP

Zaver

Pollock

Boradia³

et al. 2020

ChemBioChem

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Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in their environments. In recent years, the second messenger, cyclic di‐(3’,5’)‐adenosine monophosphate (c‐di‐AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. We and others have shown that defects in c‐di‐AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c‐di‐AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c‐di‐AMP levels. To address this limitation, we describe here the development of a luciferase‐based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high‐throughput quantification of 3’3’‐c‐di‐AMP. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide‐based anti‐phage signaling system (CBASS) effector, 3’3’‐cGAMP. These findings establish CDA‐Luc as a more affordable and sensitive alternative to conventional c‐di‐AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.

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The PAMP c-di-AMP Is Essential for Listeria monocytogenes Growth in Rich but Not Minimal Media due to a Toxic Increase in (p)ppGpp

Cited by 138 publications

References 61 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

A Luminescence‐Based Coupled Enzyme Assay Enables High‐Throughput Quantification of the Bacterial Second Messenger 3’3’‐Cyclic‐Di‐AMP

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