Mutational analysis of the chemoreceptor-coupling domain of the Escherichia coli chemotaxis signaling kinase CheA

Abstract

During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and protein stability tests and in vitro assays with purified mutant proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3' interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA.

FIG. 1.

(A) CheA domain structure and genetic tools used in this study. Transducing phages λfla2 and λfla3Δ30 carry adjacent segments of the cheA gene, delineated by an EcoRI site. Thin lines denote cheA coding material carried in the phages; thick lines indicate deleted material. (B) Chemotaxis defects of CheA P5 missense mutants isolated in this study. Mutant derivatives of plasmid pPA113 were tested in strain RP9535 (ΔcheA) for ability to support chemotaxis on tryptone soft agar plates. The plates contained 0.4 μM sodium salicylate and 12.5 μg/ml chloramphenicol and were photographed after 8 h of incubation at 32.5°C. The L526P and L552R mutants are group 1 mutants, the K616E and G629D mutants are group 2 mutants, and the R555Q and I581V mutants are group 3 mutants. WT, wild type.

FIG. 2.

In vivo test of CheA autophosphorylation. Mutant CheA plasmids (CheA*) were tested over a range of inducer concentrations for ability to produce pseudotactic changes in the colony size of strain RP9540 on tryptone soft agar. (A) Components of the chemotaxis signaling pathway present in RP9540 that can be used by autophosphorylation (auto-P)-proficient CheA molecules to induce CW flagellar rotation. (B) Examples of different test results and their relative auto-P scores used in compiling Table 1 (see the text). Plates were incubated at 32.5°C for 17 h.

FIG. 3.

An in vivo CheA autophosphorylation (auto-P) test with sensitivity enhanced compared to the one shown in Fig. 2. In this test, mutant plasmids (CheA*) were examined for pseudotactic effects in strain RP9543, which also lacks CheZ. (A) Phosphorylation reactions and motor rotation effects expected of a partially active CheA. (B) Examples of different test results and their relative auto-P scores used to compile Table 1 (see the text). Plates were incubated at 32.5°C for 17 h.

FIG. 4.

In vivo test for CheA receptor-coupled activation. Mutant CheA plasmids (CheA*) were tested in strain RP9542. (A) Mutant CheA proteins able to form active ternary complexes with the receptor and CheW proteins of RP9542 should cause much higher levels of phospho-CheY and CW flagellar rotation than activation-incompetent CheA proteins. (B) Examples of different test results and their relative activation scores used to compile Table 1 (see the text). Plates were incubated at 32.5°C for 12 h.

FIG. 5.

Degradation rates of mutant CheA proteins. Cells containing plasmid-borne cheA mutations were grown with optimal induction to mid-log phase and then blocked for protein synthesis, as detailed in Materials and Methods. The cellular content of full-length CheA molecules was followed over time by quantitative immunoblotting. The lines represent least-squares best fits of the data points to a single-exponential-decay process. The examples presented here cover the range of decay rates observed with CheA P5* mutants.

FIG. 6.

Structure-function relationships of CheA P5 mutants. (A) Alpha-carbon backbone of the P5 domain of E. coli CheA, modeled from the X-ray coordinates of the T. maritima P3-P4-P5 dimer (see text). The four groups of P5* mutations described in this study mainly define different regions in the P5 structure. The alpha-carbons of the mutant residues are shown in space-filled representation: white, group 1; light gray, group 3; dark gray, group 4; and black, group 2. Two previously proposed CheA-binding sites are indicated by light-gray (5) and black (41) lines near the left end of P5, distal to the N terminus (N). (B) P5 structure from panel A, rotated 90° to show the two folding subdomains and the approximate location of the adjoining P3 and P4 domains. (C) Modeled structure of the P3-P4-P5 dimer of E. coli CheA. The P3 and P4 domains are shown in ribbon representations; both P5 domains are shown in space-filled representation with different shading for the two subdomains. Residues defined by group 2 mutations (CheW binding defective) are shown in black, and group 4 mutation sites (deactivation defective) are shown in dark gray. The group 1 and group 3 sites are not shown in this panel.