Control of direction of flagellar rotation in bacterial chemotaxis

Abstract

The motile behavior of the bacterium Escherichia coli depends on the direction of rotation of its flagellar motors. Binding of the phosphorylated signaling molecule CheY to a motor component FliM is known to enhance clockwise rotation. It is difficult to study this interaction in vivo, because the dynamics of phosphorylation of CheY by its kinase CheA and the hydrolysis of CheY (accelerated by CheZ) are not under direct experimental control. Here, we examine instead the interaction with the flagellar motor of a double mutant CheY13DK106YW that is active without phosphorylation. The behavioral assays were carried out on tethered cells lacking CheA and CheZ. The effects of variation in intracellular concentration of the mutant protein were highly nonlinear. However, they can be explained by a thermal isomerization model in which the free energies of clockwise and counterclockwise states depend linearly on the amount of CheY bound.

Figure 1

The segment of DNA (Lower) crossed into the chromosome of a cheA deletion strain (Upper) to yield strain HCB900, drawn to the same scale. The shaded regions are regions of homology used in the cross-in. cheY was replaced by cheY^13DK106YW and most of cheZ was deleted. Base pairs are numbered and fragments are labeled as in Table 2. Arrows, transcription units; arrowheads, hybridization sites for primers; open boxes and lines, genomic DNA; solid boxes, lines, and circle, plasmid DNA.

Figure 2

Induction of CheY**. At least six immunoblots (24 samples) were analyzed for each induction level. The error bars are standard deviations of the means of each immunoblot. The line is a polynomial fit. Ng of CheY** were determined at each IPTG level and the molecules per cell were computed from the molecular mass (14.3 kDa) and the measured cell density (6.2 × 10⁸ cells per ml). Corresponding concentrations were computed from the measured dry weight/ml cell culture (0.26 mg) and the cytoplasmic volume/mg dry weight (1.4 μl, ref. 32).

Figure 3

Behavioral effects of induction of CheY**. The circle values were obtained by measuring the bias and reversal rate for each cell, computing k⁺ and k⁻ for each cell, and taking the means (and standard errors) over the cell population (at least 30 cells at each level of induction). The × values were obtained from exponential fits to interval distributions (where possible), computing the bias and reversal frequency for each cell, and taking the means over the cell population (with fewer than 30 cells at low and high bias, where reversal frequencies were infrequent). The dashed line values were obtained from fits of the model mechanism to k⁺ and k⁻ (Fig. 4). The solid line values were obtained from fits of the model mechanism to CW bias and reversal frequency. See the text. (Inset) A segment of an angular speed record spanning 10 sec; CCW is +.

Figure 4

Fit to Eq. 3 for ΔG_o = 14.4 kT, yielding Mr = 23.1 kT, and K_D = 9.1 μM. (Inset) The free-energy diagram.