Cyclic di-AMP, a multifaceted regulator of central metabolism and osmolyte homeostasis in Listeria monocytogenes.

Cyclic di-AMP is an emerging second messenger that is synthesized by many archaea and bacteria, including the Gram-positive pathogenic bacterium Listeria monocytogenes. Listeria monocytogenes played a crucial role in elucidating the essential function of c-di-AMP, thereby becoming a model system for studying c-di-AMP metabolism and the influence of the nucleotide on cell physiology. c-di-AMP is synthesized by a diadenylate cyclase and degraded by two phosphodiesterases. To date, eight c-di-AMP receptor proteins have been identified in L. monocytogenes, including one that indirectly controls the uptake of osmotically active peptides and thus the cellular turgor. The functions of two c-di-AMP-receptor proteins still need to be elucidated. Here, we provide an overview of c-di-AMP signalling in L. monocytogenes and highlight the main differences compared to the other established model systems in which c-di-AMP metabolism is investigated. Moreover, we discuss the most important questions that need to be answered to fully understand the role of c-di-AMP in osmoregulation and in the control of central metabolism.

Adaptation of Listeria monocytogenes to perturbation of c-di-AMP metabolism underpins its role in osmoadaptation and identifies a fosfomycin uptake system.

The human pathogen Listeria monocytogenes synthesizes and degrades c-di-AMP using the diadenylate cyclase CdaA and the phosphodiesterases PdeA and PgpH respectively. c-di-AMP is essential because it prevents the uncontrolled uptake of osmolytes. Here, we studied the phenotypes of cdaA, pdeA, pgpH and pdeA pgpH mutants with defects in c-di-AMP metabolism and characterized suppressor mutants restoring their growth defects. The characterization of the pdeA pgpH mutant revealed that the bacteria show growth defects in defined medium, a phenotype that is invariably suppressed by mutations in cdaA. The previously reported growth defect of the cdaA mutant in rich medium is suppressed by mutations that osmotically stabilize the c-di-AMP-free strain. We also found that the cdaA mutant has an increased sensitivity against isoleucine. The isoleucine-dependent growth inhibition of the cdaA mutant is suppressed by codY mutations that likely reduce the DNA-binding activity of encoded CodY variants. Moreover, the characterization of the cdaA suppressor mutants revealed that the Opp oligopeptide transport system is involved in the uptake of the antibiotic fosfomycin. In conclusion, the suppressor analysis corroborates a key function of c-di-AMP in controlling osmolyte homeostasis in L. monocytogenes.

Molecular mechanisms underlying glyphosate resistance in bacteria.

Glyphosate is a nonselective herbicide that kills weeds and other plants competing with crops. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, thereby depleting the cell of EPSP serving as a precursor for biosynthesis of aromatic amino acids. Glyphosate is considered to be toxicologically safe for animals and humans. Therefore, it became the most-important herbicide in agriculture. However, its intensive application in agriculture is a serious environmental issue because it may negatively affect the biodiversity. A few years after the discovery of the mode of action of glyphosate, it has been observed that bacteria evolve glyphosate resistance by acquiring mutations in the EPSP synthase gene, rendering the encoded enzyme less sensitive to the herbicide. The identification of glyphosate-resistant EPSP synthase variants paved the way for engineering crops tolerating increased amounts of the herbicide. This review intends to summarize the molecular mechanisms underlying glyphosate resistance in bacteria. Bacteria can evolve glyphosate resistance by (i) reducing glyphosate sensitivity or elevating production of the EPSP synthase, by (ii) degrading or (iii) detoxifying glyphosate and by (iv) decreasing the uptake or increasing the export of the herbicide. The variety of glyphosate resistance mechanisms illustrates the adaptability of bacteria to anthropogenic substances due to genomic alterations.

An extracytoplasmic protein and a moonlighting enzyme modulate synthesis of c-di-AMP in Listeria monocytogenes.

The second messenger cyclic di-AMP (c-di-AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c-di-AMP can regulate the synthesis of potassium uptake systems in some bacteria and it also directly inhibits and activates potassium import and export systems, respectively. Therefore, c-di-AMP production and degradation have to be tightly regulated depending on the environmental osmolarity. The Gram-positive pathogen Listeria monocytogenes relies on the membrane-bound diadenylate cyclase CdaA for c-di-AMP production and degrades the nucleotide with two phosphodiesterases. While the enzymes producing and degrading the dinucleotide have been reasonably well examined, the regulation of c-di-AMP production is not well understood yet. Here we demonstrate that the extracytoplasmic regulator CdaR interacts with CdaA via its transmembrane helix to modulate c-di-AMP production. Moreover, we show that the phosphoglucosamine mutase GlmM forms a complex with CdaA and inhibits the diadenylate cyclase activity in vitro. We also found that GlmM inhibits c-di-AMP production in L. monocytogenes when the bacteria encounter osmotic stress. Thus, GlmM is the major factor controlling the activity of CdaA in vivo. GlmM can be assigned to the class of moonlighting proteins because it is active in metabolism and adjusts the cellular turgor depending on environmental osmolarity.

c-di-AMP assists osmoadaptation by regulating the Listeria monocytogenes potassium transporters KimA and KtrCD.

Many bacteria and some archaea produce the second messenger cyclic diadenosine monophosphate (c-di-AMP). c-di-AMP controls the uptake of osmolytes in Firmicutes, including the human pathogen Listeria monocytogenes, making it essential for growth. c-di-AMP is known to directly regulate several potassium channels involved in osmolyte transport in species such as Bacillus subtilis and Streptococcus pneumoniae, but whether this same mechanism is involved in L. monocytogenes, or even whether similar ion channels were present, was not known. Here, we have identified and characterized the putative L. monocytogenes' potassium transporters KimA, KtrCD, and KdpABC. We demonstrate that Escherichia coli expressing KimA and KtrCD, but not KdpABC, transport potassium into the cell, and both KimA and KtrCD are inhibited by c-di-AMP in vivo For KimA, c-di-AMP-dependent regulation requires the C-terminal domain. In vitro assays demonstrated that the dinucleotide binds to the cytoplasmic regulatory subunit KtrC and to the KdpD sensor kinase of the KdpDE two-component system, which in Staphylococcus aureus regulates the corresponding KdpABC transporter. Finally, we also show that S. aureus contains a homolog of KimA, which mediates potassium transport. Thus, the c-di-AMP-dependent control of systems involved in potassium homeostasis seems to be conserved in phylogenetically related bacteria. Surprisingly, the growth of an L. monocytogenes mutant lacking the c-di-AMP-synthesizing enzyme cdaA is only weakly inhibited by potassium. Thus, the physiological impact of the c-di-AMP-dependent control of potassium uptake seems to be less pronounced in L. monocytogenes than in other Firmicutes.

The KupA and KupB Proteins of Lactococcus lactis IL1403 Are Novel c-di-AMP Receptor Proteins Responsible for Potassium Uptake.

Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic and heat resistance. In Lactococcus lactis, a lactic acid bacterium which is used in the dairy industry and as a cell factory in biotechnological processes, the only reported interaction partners of c-di-AMP are the pyruvate carboxylase and BusR, the transcription regulator of the busAB operon for glycine betaine uptake. However, recent studies uncovered a major role of c-di-AMP in the control of potassium homeostasis, and potassium is the signal that triggers c-di-AMP synthesis. In this study, we have identified KupA and KupB, which belong to the Kup/HAK/KT family, as novel c-di-AMP binding proteins. Both proteins are high-affinity potassium transporters, and their transport activities are inhibited by binding of c-di-AMP. Thus, in addition to the well-studied Ktr/Trk potassium channels, KupA and KupB represent a second class of potassium transporters that are subject to inhibition by c-di-AMP.IMPORTANCE Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis, none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.

Identification of the first glyphosate transporter by genomic adaptation.

The soil bacterium Bacillus subtilis can get into contact with growth-inhibiting substances, which may be of anthropogenic origin. Glyphosate is such a substance serving as a nonselective herbicide. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which generates an essential precursor for de novo synthesis of aromatic amino acids in plants, fungi, bacteria and archaea. Inhibition of the EPSP synthase by glyphosate results in depletion of the cellular levels of aromatic amino acids unless the environment provides them. Here, we have assessed the potential of B. subtilis to adapt to glyphosate at the genome level. In contrast to Escherichia coli, which evolves glyphosate resistance by elevating the production and decreasing the glyphosate sensitivity of the EPSP synthase, B. subtilis primarily inactivates the gltT gene encoding the high-affinity glutamate/aspartate symporter GltT. Further adaptation of the gltT mutants to glyphosate led to the inactivation of the gltP gene encoding the glutamate transporter GltP. Metabolome analyses confirmed that GltT is the major entryway of glyphosate into B. subtilis. GltP, the GltT homologue of E. coli also transports glyphosate into B. subtilis. Finally, we found that GltT is involved in uptake of the herbicide glufosinate, which inhibits the glutamine synthetase.

Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment.

Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multidrug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild-type cells were not known. Using RNA-Seq and promoter-lacZ fusions, we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil.

A Delicate Connection: c-di-AMP Affects Cell Integrity by Controlling Osmolyte Transport.

Bacteria use second-messenger molecules to adapt to their environment. Several second messengers, among them cyclic di-AMP (c-di-AMP), have been discovered and intensively studied. Interestingly, c-di-AMP is essential for growth of Gram-positive bacteria such as Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus. Many studies demonstrated that perturbation of c-di-AMP metabolism affects the integrity of the bacterial cell envelope. Therefore, it has been assumed that the nucleotide is essential for proper cell envelope synthesis. In this Opinion paper, we propose that the cell envelope phenotypes caused by perturbations of c-di-AMP metabolism can be interpreted differently: c-di-AMP might indirectly control cell envelope integrity by modulating the turgor, a physical variable that needs to be tightly adjusted. We also discuss open questions related to c-di-AMP metabolism that need to be urgently addressed by future studies.

Control of potassium homeostasis is an essential function of the second messenger cyclic di-AMP in Bacillus subtilis.

The second messenger cyclic di-adenosine monophosphate (c-di-AMP) is essential in the Gram-positive model organism Bacillus subtilis and in related pathogenic bacteria. It controls the activity of the conserved ydaO riboswitch and of several proteins involved in potassium (K+) uptake. We found that the YdaO protein was conserved among several different bacteria and provide evidence that YdaO functions as a K+ transporter. Thus, we renamed the gene and protein KimA (K+ importer A). Reporter activity assays indicated that expression beyond the c-di-AMP-responsive riboswitch of the kimA upstream regulatory region occurred only in bacteria grown in medium containing low K+ concentrations. Furthermore, mass spectrometry analysis indicated that c-di-AMP accumulated in bacteria grown in the presence of high K+ concentrations but not in low concentrations. A bacterial strain lacking all genes encoding c-di-AMP-synthesizing enzymes was viable when grown in medium containing low K+ concentrations, but not at higher K+ concentrations unless it acquired suppressor mutations in the gene encoding the cation exporter NhaK. Thus, our results indicated that the control of potassium homeostasis is an essential function of c-di-AMP.

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

UNLABELLED: 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.

Hydrogen sulfide is a novel potential virulence factor of Mycoplasma pneumoniae: characterization of the unusual cysteine desulfurase/desulfhydrase HapE.

Mycoplasma pneumoniae is a human pathogen causing atypical pneumonia with a minimalized and highly streamlined genome. So far, hydrogen peroxide production, cytadherence, and the ADP-ribosylating CARDS toxin have been identified as pathogenicity determinants. We have studied haemolysis caused by M. pneumoniae, and discovered that hydrogen peroxide is responsible for the oxidation of heme, but not for lysis of erythrocytes. This feature could be attributed to hydrogen sulfide, a compound that has previously not been identified as virulence factor in lung pathogens. Indeed, we observed hydrogen sulfide production by M. pneumoniae. The search for a hydrogen sulfide-producing enzyme identified HapE, a protein with similarity to cysteine desulfurases. In contrast to typical cysteine desulfurases, HapE is a bifunctional enzyme: it has both the cysteine desulfurase activity to produce alanine and the cysteine desulfhydrase activity to produce pyruvate and hydrogen sulfide. Experiments with purified HapE showed that the enzymatic activity of the protein is responsible for haemolysis, demonstrating that HapE is a novel potential virulence factor of M. pneumoniae.