The Divergent Key Residues of Two Agrobacterium fabrum (tumefaciens) CheY Paralogs Play a Key Role in Distinguishing Their Functions

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.

Chemosensory systems interact to shape relevant traits for bacterial plant pathogenesis

“…This reveals how CheA1 and CheY1 work independently regarding motility. In bacteria containing more than one CheY homolog ( 9 , 77 , 78 ), it is common that one CheY homolog is essential for the chemotaxis response while the remaining CheY homolog works as a phosphate sink. This is the case for Sinorhizobium meliloti and Azorhizobium caulinodans .…”

“…However, this does not seem to be the case in PsPto, as a Δ cheY1 mutant does not show any defect in swimming motility. The dual role of two CheY homologs was also assessed in Agrobacterium fabrum ( 78 ). In this case, CheY1 and CheY2 from A. fabrum were both discovered to contribute to chemotaxis by binding FliM with different affinities, which culminated in different rates of swimming reversals, but both were shown to interact with the same CheA ( 78 ).…”

“…The dual role of two CheY homologs was also assessed in Agrobacterium fabrum ( 78 ). In this case, CheY1 and CheY2 from A. fabrum were both discovered to contribute to chemotaxis by binding FliM with different affinities, which culminated in different rates of swimming reversals, but both were shown to interact with the same CheA ( 78 ). In PsPto, we have shown the interaction between CheY2 and FliM, although the interaction between CheY1 and FliM could not be detected.…”

Agrobacterium fabrum atu0526-Encoding Protein Is the Only Chemoreceptor That Regulates Chemoattraction toward the Broad Antibacterial Agent Formic Acid

“…The entire atu0526 gene was knocked out using the method described previously [ 39 ]. pEX18Km, which carries a kanamycin resistant gene for a positive selection and a suicide gene, sacB, for counter-selection, was used for constructing a gene-knockout mutant [ 40 ]. The fragments of atu0526 upstream (500 bp fragment upstream of start codon) and downstream (500 bp fragment downstream of stop codon) were fused and inserted into pEX18Km though restriction sites Hin d III and Bam H I.…”