A cheZ-Like Gene in Azorhizobium caulinodans Is a Key Gene in the Control of Chemotaxis and Colonization of the Host Plant

Abstract

Chemotaxis can provide bacteria with competitive advantages for survival in complex environments. The CheZ chemotaxis protein is a phosphatase, affecting the flagellar motor in Escherichia coli by dephosphorylating the response regulator phosphorylated CheY protein (CheY∼P) responsible for clockwise rotation. A cheZ gene has been found in Azorhizobium caulinodans ORS571, in contrast to other rhizobial species studied so far. The CheZ protein in strain ORS571 has a conserved motif similar to that corresponding to the phosphatase active site in E. coli The construction of a cheZ deletion mutant strain and of cheZ mutant strains carrying a mutation in residues of the putative phosphatase active site showed that strain ORS571 participates in chemotaxis and motility, causing a hyperreversal behavior. In addition, the properties of the cheZ deletion mutant revealed that ORS571 CheZ is involved in other physiological processes, since it displayed increased flocculation, biofilm formation, exopolysaccharide (EPS) production, and host root colonization. In particular, it was observed that the expression of several exp genes, involved in EPS synthesis, was upregulated in the cheZ mutant compared to that in the wild type, suggesting that CheZ negatively controls exp gene expression through an unknown mechanism. It is proposed that CheZ influences the Azorhizobium-plant association by negatively regulating early colonization via the regulation of EPS production. This report established that CheZ in A. caulinodans plays roles in chemotaxis and the symbiotic association with the host plant.IMPORTANCE Chemotaxis allows bacteria to swim toward plant roots and is beneficial to the establishment of various plant-microbe associations. The level of CheY phosphorylation (CheY∼P) is central to the chemotaxis signal transduction. The mechanism of the signal termination of CheY∼P remains poorly characterized among Alphaproteobacteria, except for Sinorhizobium meliloti, which does not contain CheZ but which controls CheY∼P dephosphorylation through a phosphate sink mechanism. Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata, has an orphan cheZ gene besides two cheY genes similar to those in S. meliloti In addition to controlling the chemotaxis response, the CheZ-like protein in strain ORS571 is playing a role by decreasing bacterial adhesion to the host plant, in contrast to the general situation where chemotaxis-associated proteins promote adhesion. In this study, we identified a CheZ-like protein among Alphaproteobacteria functioning in chemotaxis and the A. caulinodans-S. rostrata symbiosis.

Keywords: Azorhizobium caulinodans; CheZ; Sesbania rostrata; chemotaxis; colonization.

FIG 1

Comparison of the organization of the chemotaxis gene clusters in the chromosomes of S. meliloti, A. caulinodans, and E. coli. The genes encoding CheY and CheZ are indicated in black and red, respectively. In A. caulinodans, cheY and cheZ, which are distant from che cluster, are orphan genes.

FIG 2

Amino acid sequence alignment of CheZ proteins from A. caulinodans ORS571, E. coli, and S. Typhimurium. The numbers on the left and right show the positions of the residues in E. coli, S. Typhimurium, and A. caulinodans. Gaps indicated by dashes are introduced to maximize the alignments. The similarity between the homologous proteins is highlighted by different shading: black, all amino acids in a column are identical; gray, the amino acids in a column belong to a weak similarity group. The conserved phosphatase active-site motif in E. coli is boxed, and the two key amino residues in the motif, D and Q, are marked with asterisks.

FIG 3

A. caulinodans chemotaxis behavior. (A) Comparison of the chemotactic responses of the wild-type ORS571 and the cheZ deletion mutant, AC601. The chemotactic ring diameters were measured after 48 h for each strain in the presence of the desired chemoeffector, succinate or malate, in a soft agar plate assay with or without NH₄Cl as a nitrogen source. (B) Comparison of the chemotactic responses of ORS571(pBBR1MCS-2), AC601(pBBR1MCS-2), and AC602(pBBR2CheZ) complemented mutants and AC603(pBBR2CheZD165A) and AC604(pBBR2CheZQ169A) mutant strains obtained after site-directed mutagenesis of the putative phosphatase active site. Examples of the chemotaxis rings observed after 48 h in the presence of succinate and NH₄Cl by each counterpart strain are shown in panels A and B. (C) Competitive chemotactic responses of A. caulinodans strains using the quantitative capillary assay. Strains were mixed in equal ratios; the capillary contained either 10 mM succinate or PBS only as a control. (D) Chemotaxis ring formation of the E. coli wild type (RP437), the cheZ mutant (UU2685), and strain UU2685CheZ containing the ORS571 cheZ gene. Error bars in panels A, B, and C show standard deviations of the means from triplicates. The scale bars in panels A and B represent 10 mm.

FIG 4

Analysis of swimming behaviors of A. caulinodans wild-type ORS571, cheZ mutant AC601, and complemented strain AC602. (A) Sample trajectories. Individual motile cells were tracked over time and their motion was compiled into trajectories that trace their movement. To track motile cells, we used high frequency (10-ms interval) phase contrast imaging. Some swimming direction changes are labeled with arrows as examples. (B) Flagellar rotation frequency. Up to 50 cells were examined for each strain; the average frequency of direction changes for each strain is indicated by black solid lines. Dashed line indicates the baseline for smooth swimming in the wild-type strain, which was observed immediately after adding an attractant. Each strain was tested by using at least three biological replicates.

FIG 5

EPS staining and production and relative expression of exp genes in A. caulinodans. (A) Wild-type ORS571 (left) and cheZ deletion mutant AC601 (right) colony morphologies on Congo red plates. Photographs were taken after 3 days of growth. (B) EPS production by the wild type and the cheZ mutant. The EPS was extracted and quantified as described in Materials and Methods. Error bars show the standard deviations from the means. *, P < 0.05 versus the wild-type strain. (C) RT-qPCR analyses of exp genes AZC_1833, AZC_1834, and AZC_3326. The expression levels were assessed by normalization to the 16S rRNA level.

FIG 6

Comparative flocculation of wild-type ORS571 and cheZ mutant AC601 in L3 minimal medium containing low nitrogen. (A) Observation of floc formation in plate assays at 48 h. (B) Enlargements of boxes in panel A for flocs formed after 48 h. (C) Quantification of flocculation formation. There are obvious differences in the total flocculation formation between the wild type and strain AC601 after 24 h, 48 h, and 72 h. (D) Complementation assays of the cheZ mutant. Flocculation formation of strain AC602 was quantified after 24 h. Strains ORS571 and AC601 harboring the empty plasmid pBBR1MCS-2 were used as controls. *, P < 0.05 versus the wild-type strain.

FIG 7

Surface adhesion properties of the A. caulinodans wild type and the cheZ mutant. (A) Competitive adhesion to plant roots of S. rostrata between strain ORS571 and the cheZ deletion mutant AC601 and between the complemented strains AC602 and AC601. The pattern of competitive colonization of the strains at different ratios is expressed on the x axis and the ratios of the strains (ORS571 to AC601 or AC602 to AC601) are indicated below each column. (B) Biofilm formation of the cheZ mutant compared to the wild type. Cells were grown for 72 h in L3 medium with or without a nitrogen source. The biofilm is stained with CV, and the amount of CV staining was quantified as described in Materials and Methods. Error bars show standard errors of the means from at least three replicates.