Inhibition of Aspartate Chemotaxis of Escherichia coli by Site-Directed Sulfhydryl Modification of the Receptor

Abstract

Thr-154 of the chemoreceptor Tar in Escherichia coli is important for aspartate sensing. Taking advantage of the fact that Tar has no Cys residues, we have further investigated the role of Thr-154 by replacing it with Cys in order to subject it to SH modification. Tar-T154C retained the abilities of aspartate sensing and repellent sensing. However, when cells with Tar-T154C were treated with an SH-modifying reagent, 5, 5'-dithiobis-2-nitrobenzoic acid (DTNB), they specifically lost the ability to sense aspartate; the ability was restored by the reducing reagent, 1, 4-dithiothreitol. DTNB showed no detectable effect on the function of wild-type Tar or serine-replaced Tar, Tar-T154S. Thus, DTNB modifies Cys-154 of Tar-T154C in intact cells and causes a specific defect in the aspartate-sensing ability of Tar. The addition of 1 mM or higher concentrations of aspartate resulted in protection of Cys-154 from the modification; serine had no effect in this regard. These results suggest that not only is Thr-154 important for aspartate sensing but also, it may be located at the actual aspartate-binding site.