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

Abstract

Summary 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.

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

“…Structurally DacA consists of an N‐terminal transmembrane domain and a catalytic C‐terminal DAC domain. In many bacterial species, DacA is encoded in a three gene operon consisting of an extracytoplasmic regulatory protein, CdaR or YbbR, and the phosphoglucosamine mutase, GlmM, an enzyme involved in peptidoglycan biosynthesis, both of which have been shown to modulate the activity of DacA‐providing evidence for crosstalk between cell wall biosynthesis and c‐di‐AMP signaling networks [10,35,36] . In addition to synthesis, degradation of c‐di‐AMP must be dynamically regulated in order to allow bacteria to sense and respond to alterations in their environment.…”

Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM

“…Two genes, coding for the membrane-linked CdaA regulator CdaR (also named YbbR in some bacteria) and cytoplasmically-located peptidoglycan precursor synthesis enzyme GlmM, are found downstream and in an operon with cdaA (29,31). Through recent studies in B. subtilis, L. monocytogenes, Lactococcus lactis and S. aureus , it is has become apparent that these three genes are not only co-transcribed but that the encoded proteins also from a complex and that CdaR and GlmM can regulate the activity of the c-di-AMP cyclase CdaA (29,32,33). While CdaR has been reported to function as both an activator and repressor of CdaA activity depending on the growth conditions, GlmM has been shown to be a potent inhibitor of the cyclase activity (17,29,31,32,34).…”

“…GlmM is a phosphoglucomutase enzyme catalysing the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is subsequently used to produce the essential peptidoglycan precursor UDP-N-acetyl-glucosamine (35). In B. subtilis, L. monocytogenes and L. lactis a protein-protein interaction between CdaA and GlmM has been detected using bacterial two-hybrid assays performed in Escherichia coli (29,31,33). In B. subtilis this interaction has been further confirmed by in vivo protein cross-linking and pulldown assays (29) and in L. monocytogenes by the co-elution of purified proteins (33).…”

The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology

“…synthase A regulator (CdaR) and the phosphoglucosamine mutase, GlmM (48,65,[123][124][125][126]. In these bacteria, the N-terminal domains anchor both CdaA and CdaR to the membrane, and the C-terminal domains of CdaA and CdaR are located intracellularly and extracellularly, respectively.…”

“…This study pUAMS233 pProEX-HTb::rpoS (120)(121)(122)(123)(124)(125)(126)(127)(128)(129)(130)(131) ; Amp r This study pUAMS4 pGEM-T Easy::PflgB-aacC1 (AscI-flanked); Gent r , Amp r (89) pUAMS248B…”