Interdomain linkers of homologous response regulators determine their mechanism of action

Abstract

OmpR and PhoB are response regulators that contain an N-terminal phosphorylation domain and a C-terminal DNA binding effector domain connected by a flexible interdomain linker. Phosphorylation of the N terminus results in an increase in affinity for specific DNA and the subsequent regulation of gene expression. Despite their sequence and structural similarity, OmpR and PhoB employ different mechanisms to regulate their effector domains. Phosphorylation of OmpR in the N terminus stimulates the DNA binding affinity of the C terminus, whereas phosphorylation of the PhoB N terminus relieves inhibition of the C terminus, enabling it to bind to DNA. Chimeras between OmpR and PhoB containing either interdomain linker were constructed to explore the basis of the differences in their activation mechanisms. Our results indicate that effector domain regulation by either N terminus requires its cognate interdomain linker. In addition, our findings suggest that the isolated C terminus of OmpR is not sufficient for a productive interaction with RNA polymerase.

FIG. 1.

Structures of the OmpR-PhoB chimeras. The PhoB N terminus, linker, and C terminus are depicted in black. The OmpR domains are shown in grey. The three-letter chimera designations, from left to right, refer to the sources of the N terminus, linker, and C terminus, respectively.

FIG. 2.

Transcriptional activation by the PhoB_N-OmpR_C chimeras. β-Galactosidase assays were performed as described in Materials and Methods. Activities of the ompF-lacZ strain MH513.101 and the ompC-lacZ strain MH225.101 are shown. The ompR⁺ strains are derivatives of MH225.101 and MH513.101 that contain a chromosomal copy of ompR. At least two independent assays were performed on each strain; the error bars indicate +1 standard deviation. ompR, ompR null.

FIG. 3.

Phosphorylation of the PhoB_N-OmpR_C chimeras by PhoR. The kinase assay is described in Materials and Methods. Lane 1 contains 2 μg of thioredoxin-tagged PhoR that has been autophosphorylated by [γ-³²P]ATP; the three bands correspond to the proteolytic cleavage products of PhoR. The response regulator (RR; 5.5 μg) in each reaction is indicated by the labels under the gel. The reactions were carried out for 30 min at 37°C and stopped by the addition of SDS-PAGE loading buffer.

FIG. 4.

Phosphorylation of the PhoB_N-OmpR_C chimeras by EnvZ. The kinase assay is described in Materials and Methods. Lane 1 contains 35 μg of EnvZ that has been autophosphorylated by [γ-³²P]ATP. The response regulator in each reaction (RR; 5.5 μg) is indicated by the labels under the gel. The reactions were carried out for 30 min at 37°C and stopped by the addition of SDS-PAGE loading buffer.

FIG. 5.

DNA binding of BRR and BBR to the F1 binding site. BRR (circles) or BBR (triangles) was titrated into the binding reaction in the presence of 3 nM fluorescein-labeled F1 oligonucleotide. The means from five separate measurements performed at each titration point were plotted. The curve illustrates the change in anisotropy (A), where (A−A_o)/A_o represents the difference in anisotropy in the presence of protein minus the anisotropy in the absence of protein divided by the anisotropy in the absence of protein. This value is plotted as a function of the total protein concentration.

FIG. 6.

Transcriptional activation by the OmpR_N-PhoB_C chimeras. Alkaline phosphatase assays were performed as described in Materials and Methods. The activities of strains BW25115 (envZ⁺) and BW28669 (envZ null [envZ]) are indicated by the solid and open bars, respectively; the error bars indicate +1 standard deviation. Assays were performed in triplicate on three independent colonies. The control strain was grown with plasmid pARA-RRB without arabinose induction. RBB and RRB refer to strains containing plasmids pARA-RBB and pARA-RRB, respectively.

FIG. 7.

Phosphorylation of the OmpR_N-PhoB_C chimeras by EnvZ. The kinase assays are described in Materials and Methods. The phosphotransfer reaction was performed for 1 h at room temperature and stopped by the addition of SDS-PAGE loading buffer. Lane 1 contains 2 μg of EnvZ autophosphorylated by [γ-³²P]ATP. The response regulator (RR; 0.3 μg) in each reaction is indicated by the labels under the gel.