DOS(Ec), a heme-regulated phosphodiesterase, plays an important role in the regulation of the cyclic AMP level in Escherichia coli.
Heme-regulated phosphodiesterase from Escherichia coli (DOS(Ec)) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because DOS(Ec) was originally cloned from E. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of DOS(Ec) using cAMP as a substrate is only 0.15 min(-1), which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the DOS(Ec) gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOS(Ec) gene knockout strain (Deltados). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher than in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOS(Ec) is regulated according to environmental O2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOS(Ec) expression.
Yoshimura-Suzuki
Tokiko
T
Department of Molecular Cell Signalling, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. yoshimura_tokiko@hotmail.com
Sagami
Ikuko
I
Yokota
Nao
N
Kurokawa
Hirofumi
H
Shimizu
Toru
T
D000332:Aerobiosis; D000693:Anaerobiosis; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006418:Heme / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006418:Heme; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; C464427:dosP protein, E coli; D010100:Oxygen