Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB

Abstract

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.

FIG. 1

Levels of mutant MotB proteins in cells. Membranes from cells expressing the mutant MotB proteins were collected, treated with loading buffer, and electrophoresed. Proteins were blotted and probed with polyclonal anti-MotB antibody, as described in Materials and Methods. Replacements of Asp 32 are indicated above the lanes according to the standard single-letter code for the amino acids. wt, wild type.

FIG. 2

Effect of mutations in Asp 32 of MotB on overexpression-linked growth impairment. MotA and a fusion protein that contains the first 60 residues of MotB (and thus residue 32 and the membrane-spanning segment) were overexpressed from plasmid pLW3 (46, 54), which is inducible with IAA. Growth rates of uninduced cultures and cultures induced with 100 μg of IAA per ml were measured as described in Materials and Methods. (A) Representative duplicate growth curves for the wild type (measured by A₆₀₀). Open symbols, uninduced; solid symbols, induced with IAA. (B) Representative duplicate growth curves for the D32N mutant. Symbols are as in panel A. (C) Summary of growth rates for the wild type (w.t.) and five Asp 32-replacement mutants. Values are means ± standard deviations (n = 6).