Identification of the cAMP phosphodiesterase CpdA as novel key player in cAMP‐dependent regulation in Corynebacterium glutamicum

Abstract

The second messenger cyclic AMP (cAMP) plays an important role in the metabolism of Corynebacterium glutamicum, as the global transcriptional regulator GlxR requires complex formation with cAMP to become active. Whereas a membrane-bound adenylate cyclase, CyaB, was shown to be involved in cAMP synthesis, enzymes catalyzing cAMP degradation have not been described yet. In this study we identified a class II cAMP phosphodiesterase named CpdA (Cg2761), homologs of which are present in many Actinobacteria. The purified enzyme has a Kmapp value of 2.5 ± 0.3 mM for cAMP and a Vmaxapp of 33.6 ± 4.3 µmol min mg . A ΔcpdA mutant showed a twofold increased cAMP level on glucose and reduced growth rates on all carbon sources tested. A transcriptome comparison revealed 247 genes with a more than twofold altered mRNA level in the ΔcpdA mutant, 82 of which are known GlxR targets. Expression of cpdA was positively regulated by GlxR, thereby creating a negative feedback loop allowing to counteract high cAMP levels. The results show that CpdA plays a key role in the control of the cellular cAMP concentration and GlxR activity and is crucial for optimal metabolism and growth of C. glutamicum.

Molecular Basis of Growth Inhibition by Acetate of an Adenylate Cyclase-Deficient Mutant of Corynebacterium glutamicum

“…cAMP Levels in C. glutamicum WT and Mutants Lacking cyaB or cpdA In order to confirm previous studies with the C. glutamicum mutant strain CgYA lacking approximately 200 bp coding region of the catalytic domain of the adenylate cyclase CyaB (Cha et al, 2010), we constructed another cyaB mutant lacking almost the entire coding region (see section "Materials and Methods"). The cAMP level of this mutant was compared with that of the WT and the cpdA mutant lacking the cAMP phosphodiesterase (Schulte et al, 2017b) using an enzyme-linked immunosorbent assay (ELISA). The strains were grown in CGXII glucose medium to an OD 600 of about 5 (exponential growth phase).…”

“…Cell extracts were prepared as described previously (Schulte et al, 2017b) and the cAMP concentration was measured without dilution with the direct cAMP ELISA kit (Enzo Life Sciences GmbH, Lörrach, Germany) following the manufacturer's instructions. Amounts of cAMP were related to the protein content of the supernatant of the cell extract.…”

“…Since the activities of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were even higher in the mutant than in the wild type (WT) during growth on LB-acetate, the authors speculated that the acetate uptake carrier might play a role in the growth defect of the CgYA mutant (Cha et al, 2010). Degradation of cAMP in C. glutamicum is catalyzed by the recently identified phosphodiesterase CpdA (Cg2761) (Schulte et al, 2017b). This enzyme belongs to the class II phosphodiesterases and deletion of the cpdA gene led to an increase of the intracellular cAMP concentration (Schulte et al, 2017b).…”

“…Degradation of cAMP in C. glutamicum is catalyzed by the recently identified phosphodiesterase CpdA (Cg2761) (Schulte et al, 2017b). This enzyme belongs to the class II phosphodiesterases and deletion of the cpdA gene led to an increase of the intracellular cAMP concentration (Schulte et al, 2017b). The cpdA mutant exhibited slower growth and a prolonged lag-phase on all tested carbon sources, including glucose, gluconate, citrate, acetate and ethanol (Schulte et al, 2017b).…”

“…This enzyme belongs to the class II phosphodiesterases and deletion of the cpdA gene led to an increase of the intracellular cAMP concentration (Schulte et al, 2017b). The cpdA mutant exhibited slower growth and a prolonged lag-phase on all tested carbon sources, including glucose, gluconate, citrate, acetate and ethanol (Schulte et al, 2017b). The growth defects could partially be complemented by overexpression of genes that are normally repressed by the cAMP-GlxR complex, such as ptsI-ptsG or citH, and that are involved in uptake or metabolism of the respective carbon source.…”