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.IMPORTANCEAzorhizobium 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.

Keywords: Azorhizobium caulinodans; chemotaxis; response regulator CheY; symbiosis.

FIG 1

Organization of chemotaxis response regulator genes cheY1 and cheY2 in genome of A. caulinodans ORS571. The arrows indicate the direction of transcription of open reading frames and are drawn relative to scale.

FIG 2

Chemotaxis behavior of A. caulinodans ORS571 and ΔcheY1, ΔcheY2, and ΔcheY1 ΔcheY2 mutants. (A) Swim tests of the wild-type (WT), mutants, and complemented strains (ΔcheY1C and ΔcheY2C) on soft agar plates. The ΔcheA and ΔfliM mutants were used as control mutant strains. The soft agar plates contained 10 mM succinate, sodium lactate, or proline as the carbon source and with 10 mM ammonium chloride as the nitrogen source. The plates with succinate as sole carbon source were used as representative plates. (B) The chemotactic ring diameters were measured for each strain. Average diameters are expressed as percentages relative to that of the WT (defined as 100%). *, P < 0.05 versus the WT strain.

FIG 3

Competitive quantitative capillary assays. Statistical analysis of the WT, ΔcheY1, ΔcheY2, and ΔcheY1 ΔcheY2 cell ratios in the capillary filled with buffer (left), succinate (middle), or sodium lactate (right) as the carbon source. The ΔfliM mutant was used as a control mutant strain. The error bars represent the standard deviations (SDs) of data from three independent experiments. *, P < 0.05 versus the WT strain; **, P < 0.01 versus the WT strain.

FIG 4

Paths of swimming cells of A. caulinodans ORS571 (WT), ΔcheY1, and ΔcheY2 strains grown in TY medium. Tracks of free-swimming cells were recorded by an Olympus DP73 digital microscope camera. Computerized motion analysis was performed using ICY software. Representative tracks are shown. Each of these trajectories represents 2 s of swimming behavior. Each strain was tested in at least five biological replicates.

FIG 5

Quantization analysis of extracellular polysaccharide (EPS) production of the wild type and ΔcheY1, ΔcheY2, ΔcheY1 ΔcheY2, and Δeps1 mutants on plates with 10 mM indicated carbon sources (sodium lactate, succinate, or proline) and with 10 mM ammonium chloride as a nitrogen source. The error bars indicate SDs from the means of three independent experiments. *, P < 0.05 versus the WT strain; **, P < 0.01 versus the WT strain.

FIG 6

Analysis of colonization and nodulation phenotypes. (A) Competitive colonization level on the root surface. Bacteria were reisolated from seedlings that were inoculated by the mixed cultures (WT and mutant or complemented strains at a ratio of approximately 1:1) and were detected by PCR. *, P < 0.05 versus the WT strain; **, P < 0.01. (B) Root nodules induced by WT, ΔcheY1, and ΔcheY2 strains 30 days after inoculation. (C) Stem nodules induced by WT, ΔcheY1, and ΔcheY2 strains 30 days after inoculation. Leghemoglobin of stem nodules shows characteristic orange-brown color. (D) Competitive nodulation tests. Bacteria were reisolated from roots or stem nodules that had been inoculated by the mixed cultures (WT and mutant or complemented strains at a ratio of approximately 1:1) and were detected by PCR. The error bars represent the standard deviations from three independent experiments. *, P < 0.05 versus the WT strain; **, P < 0.01.