Chemotactic Signaling by an Escherichia coli CheA Mutant That Lacks the Binding Domain for Phosphoacceptor Partners
-
FIG. 1.CheA domain organization and signaling activities. The relative lengths of the CheA domains (P1 to P5) and linkers (L1 and L2) are proportional to the sizes of their primary structures. The N-terminal P1 domain accepts a phosphoryl group at His-48 via interaction with the ATP-binding P4 domain. The C-terminal P5 domain enables chemoreceptors, assisted by the CheW coupling protein, to control CheA autophosphorylation activity in response to attractant or repellent stimuli. The ensuing motor response and subsequent sensory adaptation are controlled by the levels of phosphorylated CheY (Y∼P), which enhances CW flagellar rotation, and phosphorylated CheB (B∼P), which hydrolyzes glutamyl-methyl esters in the signaling domains of the chemoreceptors. The phosphorylated forms of these response regulators turn over rapidly through self-catalyzed hydrolysis. The CheZ protein further enhances dephosphorylation of Y∼P (but not B∼P). CheY and CheB acquire their phosphoryl groups from phospho-CheA. The P2 domain of CheA is not essential for these phosphotransfer reactions but rather enhances their rate by reversibly binding the unphosphorylated forms of CheY and CheB to increase phosphoacceptor concentrations in the vicinity of the P1 phosphodonor.
-
FIG. 2.Strategy for constructing cheA genes with deletions of P2. (A) Features of cheA flanking the P2 coding segment. A SacII restriction site was introduced at codon 150 (pKJ9-1) or codon 160 (pKJ9-2) to permit excision of the P2 coding segment by double digestion with SacII and EagI enzymes. (B) The PA linker. The boxed sequence depicts the double-stranded linker made by annealing the complementary single-stranded oligonucleotides kj-1 and kj-2 (Table 1). The PA linker carried a SacII-compatible overhang at one end and an EagI-compatible overhang at the other for joining the P2-excised coding segments. An internal SacII site in the PA linker permitted additional excision-replacement cycles for creating tandem PA linker inserts.
-
FIG. 3.The phosphorelay signaling reactions of chemotaxis. Only the net forward rates are depicted: CheA autophosphorylation (which is subject to modulation in signaling complexes with chemoreceptors and CheW), phosphotransfer from phospho-CheA (P∼CheA) to CheB and CheY, and the subsequent dephosphorylation of phospho-CheB (P∼CheB) and phospho-CheY (P∼CheY). Note that dephosphorylation of P∼CheY (but not that of P∼CheB) is accelerated by CheZ.
-
FIG. 4.Receptor-uncoupled autophosphorylation activity ofCheAΔP2. Reaction mixtures contained purified CheA and CheAΔP2 (5 μM) and [γ-32P]ATP (1 mM). The data points were fitted to an exponential function to determine the pseudo-first-order rate constant and steady-state phosphorylation level of each reaction (10).
-
FIG. 5.Receptor-uncoupled phosphotransfer from phospho-CheAΔP2 to CheB (A) and CheY (B). Reaction mixtures contained CheB or CheY at the indicated concentrations and, in the case of CheY, 0.5 μM CheZ to augment turnover of phospho-CheY. Phosphotransfer was initiated by addition of 0.5 μM 32P-CheA or 32P-CheAΔP2, and the phosphodonor levels were monitored for 600 s (14 time points) for CheB and for 120 s (seven time points) for CheY to determine their half-lives (t1/2) at each phosphoacceptor concentration tested.
-
FIG. 6.Steady-state levels of phospho-CheB (A) and phospho-CheY (B) in chemoreceptor-activated reaction systems. Reaction mixtures contained 0.5 μM CheA and CheAΔP2, 4 μM membrane-embedded Tsr (serine chemoreceptor), 4 μM CheW, and variable amounts of CheB or CheY. The CheY reaction mixtures also contained 0.5 μM CheZ to augment phospho-CheY turnover. The phosphorylation cascade was initiated by addition of 1 mM [γ-32P]ATP, and the levels of phospho-CheB and phospho-CheY were measured after 10 and 20 s, when the reactions had reached steady-state.
-
FIG. 7.Chemotaxis phenotypes of UU1121 (cheAΔP2). (A) Colony morphology on semisolid agar. Colonies of UU1120 (cheA+) and UU1121 (cheAΔP2) were transferred with a toothpick to a semisolid tryptone agar plate and incubated at 35°C for 8 h. (B) Flagellar rotation pattern. Strains UU1120 (cheA+) and UU1121 (cheAΔP2) were analyzed by cell tethering as described in Materials and Methods. Rotating cells were assigned to five categories: exclusively CCW or CW, predominantly CCW or CW with some reversals (CCW-R and CW-R, respectively), or with frequent reversals and no significant directional bias (CCW/CW).
-
FIG. 8.Suppression of CheAΔP2 chemotaxis defects by free P1 domains. The chemotaxis phenotypes of strain UU1121 (cheAΔP2) carrying pAG3 (expressing CheA[1-149] under IPTG-inducible control) are compared to those of UU1121 carrying pTM30 (vector control). (A) Rates of colony expansion on semisolid tryptone agar containing various IPTG concentrations. The diameters of colonies incubated at 35°C were measured at three or more time points to calculate an expansion rate. (B) Colony sizes on semisolid tryptone agar containing 320 μM IPTG. The plate was incubated for 8 h at 35°C. (C) Flagellar rotation patterns. The rotation patterns of tethered UU1121/pAG3 cells, grown without inducer or with 320 μM IPTG, were classified as described in the legend to Fig. 7B.
-
FIG. 9.Model of chemotactic signaling in a CheAΔP2 mutant containing free P1 domains. CheAΔP2 autophosphorylates at essentially normal rates and responds normally to chemoreceptor coupling control. However, without the target-acquiring P2 domain, the reduced rate of phosphotransfer to CheB and CheY cannot support efficient chemotaxis. A stoichiometric excess of free P1 domains may enhance chemotactic ability by buffering the CheAΔP2-generated pool of signaling phosphates through reversible exchange reactions with B∼P and Y∼P. See text for additional details of the model.
- American Society for Microbiology
