CheY1 and CheY2 of Azorhizobium caulinodans ORS571 Regulate Chemotaxis and Competitive Colonization with the Host Plant

Abstract

The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37 kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata. Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the ΔcheY1 mutant exhibited decreased frequency of direction reversal, whereas the ΔcheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ΔcheY1 and ΔcheY2 mutants was lower than that in the WT, but the ΔcheY2 mutant had more obvious EPS defects that were similar to those of the ΔcheY1 ΔcheY2 and Δeps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ΔcheY1 and ΔcheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ΔcheY2 mutant was severely impaired compared to that of the ΔcheY1 mutant. Taken together, the ΔcheY2 phenotypes are more severe than the ΔcheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ΔcheY2 single-mutant phenotype. These defects of ΔcheY1 and ΔcheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions. IMPORTANCE Azorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.

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

“…The data demonstrate that: (a) both CheYs are required for the full chemotactic response of A. fabrum to nutrient gradient; (b) CheY1 and CheY2 have significantly different effects on the chemotactic response of A. fabrum , with CheY2 being more important than CheY1 for the chemotactic response. These results are consistent with a previous study on A. fabrum [ 45 ] and the different effects of two CheYs on the chemotactic response of A. fabrum are similar to those of Rhizobium meliloti [ 17 ] and Azorhizobium caulinodans [ 18 ]. The complementation of CheY1 and CheY2 to the corresponding CheY-deficient mutants (Δy1 and Δy2) confirms that the mutants were constructed correctly and did not affect the normal expression of other genes.…”

“…Some bacteria have only one complete set of core chemotaxis proteins, but two or more CheYs. For example, both Rhizobium (now, Sinorhizobium or Ensifer ) meliloti [ 17 ] and Azorhizobium caulinodans [ 18 ] have only one complete set of core chemotaxis proteins, but two CheYs. The phenotypes of cheY -deletion mutants of both bacteria show that both CheYs are required for full tactic response with one CheY having a more prominent role than the other CheY.…”

Protein Residues and a Novel Motif Involved in the Cellular Localization of CheZ in Azorhizobium caulinodans ORS571

“…Consistent with our bioinformatics analysis, deletion of cheA does not alter the cellular localization of CheZ AC ( Figure 3 ). We then tested the localization pattern of CheZ AC in the following chemotaxis mutants, Δ cheY1 , Δ cheY2 , or Δ cheA-R clusters (including cheA , cheY2 , cheW , cheB , and cheR ) ( Liu W. et al, 2018 ; Liu et al, 2020 ). Interestingly, CheZ AC maintains polar localization in both the Δ cheY1 , Δ cheY2 , and Δ cheA-R mutants backgrounds ( Figure 3 ).…”

FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant

“…A 650-bp fragment was amplified with primers AcfRDF and AcfRDR (Table 2 ) and cloned into the pCM351::UF after restriction with ApaI and Age1. The positive plasmid (pCM351::UF::DF) was transformed into ORS571 by using pRK2013 as the helper plasmid [ 24 ], and the acfR gene deletion mutant was subsequently screened based on homologous double exchange as previously described [ 26 ]. The correct integration of the mutants was confirmed by PCR and named Δ acfR.…”

“…Motility assays were performed on 0.3% soft agar L3 plates according to previous publications [ 26 ]. The L3 plates contained a 10 mM carbon source (sodium lactate or glycine) and 10 mM NH 4 Cl.…”