Expression of the cpdA gene, encoding a 3',5'-cyclic AMP (cAMP) phosphodiesterase, is positively regulated by the cAMP-cAMP receptor protein complex.
The intracellular level of cyclic 3',5'-AMP (cAMP), a signaling molecule that mediates a variety of cellular processes, is finely modulated by the regulation of its synthesis, excretion, and degradation. In this study, cAMP phosphodiesterase (CpdA), an enzyme that catalyzes the conversion of cAMP to AMP, was characterized in a pathogenic bacterium, Vibrio vulnificus. The cpdA gene exists in an operon composed of mutT, yqiB, cpdA, and yqiA, the transcription of which was initiated at position -22 upstream of mutT. A cpdA-null mutant of V. vulnificus contained significantly higher levels of cAMP than the wild type but showed no detectable cAMP when a multicopy plasmid of the cpdA gene was provided in trans, suggesting that CpdA is responsible for cAMP degradation. Cellular contents of the CpdA protein decreased dramatically in both cya and crp mutants. In addition, levels of expression of the cpdA::luxAB transcription fusion decreased in cya and crp mutants. The level of expression of cpdA::luxAB in the cya mutant increased in a concentration-dependent manner upon the exogenous addition of cAMP. The cAMP-cAMP receptor protein (CRP) complex bound directly to the upstream region of mutT, which includes a putative CRP-binding sequence centered at position -95.5 relative to the transcription start site. Site-directed mutagenesis or the deletion of this sequence in the cpdA::luxAB transcription fusion resulted in the loss of regulation by cAMP and CRP. Thus, this study demonstrates that CpdA plays a crucial role in determining the intracellular cAMP level and shows for the first time that the expression of cpdA is activated by the cAMP-CRP complex via direct binding to the regulatory region.
Kim
Han-Suk
HS
Department of Environmental Science and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yongin, Kyunggi-Do 449-791, South Korea.
Kim
Sung-Min
SM
Lee
Hyun-Jung
HJ
Park
Soon-Jung
SJ
Lee
Kyu-Ho
KH
D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon / Q000235:genetics; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D041261:Vibrio vulnificus / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases