Abstract
The chemotactic response regulator CheY, when phosphorylated by the phosphoryl group from phosphorylated CheA, can bind to the motor switch complex to control the flagellar motor rotation. Agrobacterium fabrum (previous name: Agrobacterium tumefaciens), a phytopathogen, carries two paralogous cheY genes, cheY1 and cheY2. The functional difference of two paralogous CheYs remains unclear. Three cheY-deletion mutants were constructed to test the effects of two CheYs on the chemotaxis of A. fabrum. Phenotypes of three cheY-deletion mutants show that deletion of each cheY significantly affects the chemotactic response, but cheY2-deletion possesses more prominent effects on the chemotactic migration and swimming pattern of A. fabrum than does cheY1-deletion. CheA-dependent cellular localization of two CheY paralogs and in vitro pull-down of two CheY paralogs by FliM demonstrate that the distinct roles of two CheY paralogs arise mainly from the differentiation of their binding affinities for the motor switch component FliM, agreeing with the divergence of the key residues on the motor-binding surface involved in the interaction with FliM. The single respective replacements of key residues R93 and A109 on the motor-binding surface of CheY2 by alanine (A) and valine (V), the corresponding residues of CheY1, significantly enhanced the function of CheY2 in regulating the chemotactic response of A. fabrum CheY-deficient mutant Δy to nutrient substances and host attractants. These results conclude that the divergence of the key residues in the functional subdomain is the decisive factor of functional differentiation of these two CheY homologs and protein function may be improved by the substitution of the divergent key residues in the functional domain for the corresponding residues of its paralogs. This finding will help us to better understand how paralogous proteins sub-functionalize. In addition, the acquirement of two CheY2 variants, whose chemotactic response functions are significantly improved, will be very useful for us to further explore the mechanism of CheY to bind and regulate the flagellar motor and the role of chemotaxis in the pathogenicity of A. fabrum.
Highlights
IntroductionChemotaxis allows the motile microbes to move towards more favorable environments via sensing and tracking the gradients of attractants and repellents in their surroundings
The genome of A. fabrum carries two paralogous cheY genes, cheY1 and cheY2. Both are in the unique chemotaxis operon of A. fabrum [9]
Since the affinities of CheY1 and CheY2 for FliM are different, we emphasized the difference between CheY1 and CheY2 in the key residues involved in the interaction with
Summary
Chemotaxis allows the motile microbes to move towards more favorable environments via sensing and tracking the gradients of attractants and repellents in their surroundings. Chemotaxis signal is transduced by a two-component system, which typically consists of a central histidine kinase CheA with a sensor domain and a phosphorylatable response regulator CheY [1,2,3,4]. Chemotaxis signal transduction begins with the recognition of chemoeffectors by chemoreceptors ( called methyl-accepting chemotaxis protein, MCP) that form a ternary core complex with the coupling protein, CheW, and the sensor kinase, CheA [5,6]. E. coli, the MCP-CheW-CheA ternary core complexes are arrayed in cell poles. When chemoeffectors bind to the ligand binding do-
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