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

Abstract

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...

New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus

“…YbbR homologs have been shown to directly interact with DacA homologs (also referred to as CdaA) in other bacteria (21–23). In addition, an interaction between CdaA and GlmM has been reported, leading to the hypothesis that DacA, YbbR, and GlmM form a three-protein complex (22, 32) (Fig.…”

“…This product is subsequently used for the production of an early peptidoglycan synthesis intermediate. The second gene in the operon, ybbR , encodes a predicted membrane protein that has been shown to interact with, and influence the activity of DacA (21–23). However, there are conflicting reports as to whether YbbR stimulates or inhibits the activity of the cyclase.…”

A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci

“…Increased intracellular c-di-AMP levels in bacteria have been associated with increased tolerance to a wide range of environmental challenges, including cell wall damage, high temperatures, starvation, and acid stress, as well as some antibiotics, including lysostaphin, oxacillin, and penicillin G (4,11,12). While many genes regulated by c-di-AMP have been reported (10), the signal transduction pathways that convert environment stresses to c-di-AMP signals have not been identified, though it has been suggested that membrane-associated enzymes such as c-di-AMP synthase A (CdaA, also referred as YbbP) and its regulator CdaR (c-di-AMP synthase A regulator, also referred to as YybR) may sense damage directly (2,7,13). The cyclic dinucleotide signaling pathway and its potential role in stress response have not been characterized in enterococci.…”

Coping with an Essential Poison: a Genetic Suppressor Analysis Corroborates a Key Function of c-di-AMP in Controlling Potassium Ion Homeostasis in Gram-Positive Bacteria

“…A Streptococcus suis mutant tolerating large amounts of lysozyme had acquired a mutation in the cdaA diadenylate cyclase gene, which probably alters the cellular c-di-AMP concentration (36). In contrast, a decrease of the cellular c-di-AMP levels in B. subtilis and L. monocytogenes caused cell lysis (2,7,17). Moreover, strains that synthesize smaller amounts of c-di-AMP are more susceptible to cell wall-targeting antibiotics (15).…”

“…Moreover, strains that synthesize smaller amounts of c-di-AMP are more susceptible to cell wall-targeting antibiotics (15). Therefore, it has been concluded that c-di-AMP is essential for proper cell envelope synthesis (2,7,35). However, today we know that the nucleotide rather indirectly affects the integrity of the cell envelope by modulating the cellular turgor, a physical variable that has to be tightly adjusted depending on the osmotic pressure of the environment (15).…”