Genetic and biochemical studies of phosphatase activity of PhoR

Abstract

In Escherichia coli, PhoR is the histidine kinase of the phosphate regulon. It has been postulated that PhoR may function as a phospho-PhoB phosphatase. Experiments with four precise phoR deletion mutants supported this hypothesis and suggested that this activity resides within the histidine phosphorylation domain. This biochemical activity was confirmed by using a separately expressed histidine phosphorylation domain.

FIG. 1.

Domain structure of PhoR. The diagram at the top indicates the predicted protein domains of PhoR, including the membrane-spanning region, the charged region (CR), and the PAS, DHp, and CA domains. The phoR deletion mutations presented in this paper are shown below. The diagrams depict the PhoR coding capacity of each of the mutants.

FIG. 2.

PhoR∗, DHp, and CA domains form functional fusions. The various proteins were incubated in the presence of [γ-³²P]ATP and separated by SDS-PAGE. Lane 1 contains PhoR∗, lane 2 contains PhoB, lane 3 contains PhoR∗ and PhoB, lane 4 contains the CA domain, lane 5 contains the DHp domain, lane 6 contains the CA and DHp domains, and lanes 7 and 8 are the CA and DHp domains with PhoB added after a 1-min and a 5-min incubation, respectively. Panel A shows the Coomassie-stained gel, and panel B shows the autoradiogram. The concentration of PhoR∗ in the reaction was 0.01 mg/ml, whereas the CA and DHp protein concentrations were 0.24 mg/ml and the PhoB concentration was 0.15 mg/ml. The volume of each reaction mix was 10 μl, and the incubation temperature was 25°C. Incubations lasted from 5 min for reactions with PhoR∗ to 90 min for reactions with the CA and/or DHp fragment. When PhoB was added to a reaction mix, the reaction mixes were incubated for an additional 1 to 5 min. All reactions were stopped by adding 5 μl of loading buffer and placing the reaction mixes on ice; 5-μl aliquots were then loaded and run on an SDS-10% polyacrylamide gel. The gel was dried, exposed to X-ray film for 16 h, and developed.

FIG. 3.

Phosphatase assay. Phospho-PhoB was prepared as previously described (11). In each reaction, 35 μg of [³²P]phospho-PhoB was incubated with the CA or DHp fusion protein at 37°C in 20 mM Tris-HCl (pH 7.0)-50 mM NaCl-10 mM MgCl₂. At 2, 4, 6, 8, 10, 12, 14, and 16 min, 10 μl was removed from each reaction mix, mixed with 5 μl of loading buffer, and placed on ice. When all reactions were completed, 8 μl of each reaction mix was separated on an SDS-10% polyacrylamide gel. The dried gel was analyzed with a Molecular Imager FX system from Bio-Rad. (A) The full-length gel is shown for a reaction with the DHp domain. (B) Only the phospho-PhoB band is shown for reactions with PhoB by itself, with the DHp domain plus PhoB, and with the CA domain plus PhoB.