Amplification of signaling activity of the arc two-component system of Escherichia coli by anaerobic metabolites. An in vitro study with different protein modules

In Escherichia coli, changes in redox condition of growth are sensed and signaled by the Arc two-component system. This system consists of ArcB as the membrane-associated sensor kinase and ArcA as the cytoplasmic response regulator. ArcB is a tripartite kinase, possessing a primary transmitter, a receiver, and a secondary transmitter domain that catalyzes the phosphorylation of ArcA via a His --> Asp --> His --> Asp phosphorelay, as well as the dephosphorylation of ArcA-P by a reverse phosphorelay. When ArcA and ArcB were incubated with ATP, the peak levels of phosphorylated proteins increased in the presence of the fermentation metabolites D-lactate, acetate, or pyruvate. In this study, we report that these effectors accelerate the autophosphorylation activity of ArcB and enhance the transphosphorylation of ArcA, but have no effect on the dephosphorylation of ArcA-P. Moreover, the presence of the receiver domain of ArcB is essential for the effectors to influence the autophosphorylation rate of the primary transmitter domain of ArcB.     

Evidence against the physiological role of acetyl phosphate in the phosphorylation of the ArcA response regulator in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The Arc two-component signal transduction system of Escherichia coli comprises the ArcB sensor kinase and the ArcA response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphos-phorylates ArcA, which, in turn, represses or activates its target operons. ArcA has been shown to be able to autophosphorylate in vitro at the expense of acetyl-P. Here, the in vivo effect of acetyl phosphate on the redox signal transduction by the Arc system was assessed. Our results indicate that acetyl phosphate can modulate the expression of ArcA-P target genes only in the absence of ArcB. Therefore, the acetyl phosphate dependent ArcA phosphorylation route does not seem to play a significant role under physiological conditions.     

Measurement of DNA-protein equilibria using gel chromatography: application to the HinfI restriction endonuclease

A method is described for measuring equilibrium constants of DNA-protein interactions using gel chromatography. This technique has been used to study the sequence-specific interaction of the HinfI restriction endonuclease with DNA. HinfI has a monomeric molecular weight of 31000 and exists as a dimer in its active form. The protein binds to supercoiled DNA molecules containing its recognition site with an apparent free energy of -13.9 kcal/mol of sites. This interaction is highly salt sensitive and causes a release of 3.4 ion pairs. The affinity of the nuclease for its recognition site is largely independent of both pH (6.5-8.5) and temperature (7-35 degrees C) and was not affected by variations in the degenerate middle position of the site. Linear DNA fragments containing the HinfI recognition site were bound as tightly as supercoiled molecules. Binding to nonspecific DNA sites or to methylated DNA sites was approximately 6 orders of magnitude weaker. In general, enzyme activity and binding affinity paralleled each other.