DOS Ec , a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli

Abstract

ABSTRACT 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 (Δ dos ). 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 O 2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOS Ec expression.