Sinorhizobium meliloti chemoreceptor McpU mediates chemotaxis toward host plant exudates through direct proline sensing

Abstract

Bacterial chemotaxis is an important attribute that aids in establishing symbiosis between rhizobia and their legume hosts. Plant roots and seeds exude a spectrum of molecules into the soil to attract their bacterial symbionts. The alfalfa symbiont Sinorhizobium meliloti possesses eight chemoreceptors to sense its environment and mediate chemotaxis toward its host. The methyl accepting chemotaxis protein McpU is one of the more abundant S. meliloti chemoreceptors and an important sensor for the potent attractant proline. We established a dominant role of McpU in sensing molecules exuded by alfalfa seeds. Mass spectrometry analysis determined that a single germinating seed exudes 3.72 nmol of proline, producing a millimolar concentration near the seed surface which can be detected by the chemosensory system of S. meliloti. Complementation analysis of the mcpU deletion strain verified McpU as the key proline sensor. A structure-based homology search identified tandem Cache (calcium channels and chemotaxis receptors) domains in the periplasmic region of McpU. Conserved residues Asp-155 and Asp-182 of the N-terminal Cache domain were determined to be important for proline sensing by evaluating mutant strains in capillary and swim plate assays. Differential scanning fluorimetry revealed interaction of the isolated periplasmic region of McpU (McpU40-284) with proline and the importance of Asp-182 in this interaction. Using isothermal titration calorimetry, we determined that proline binds with a Kd (dissociation constant) of 104 μM to McpU40-284, while binding was abolished when Asp-182 was substituted by Glu. Our results show that McpU is mediating chemotaxis toward host plants by direct proline sensing.

FIG 1

Chemotactic responses of S. meliloti wild type (WT, RU11/001), mcpU deletion (ΔmcpU, RU11/828) and che strain (RU13/149, ΔmcpS ΔmcpT ΔmcpU ΔmcpV ΔmcpW ΔmcpX ΔmcpY ΔmcpZ ΔicpA [Δ9]) toward alfalfa seed exudate. Strains from the early log phase in RB were tested with agarose capillaries containing 0.15 mg of alfalfa seed exudate/ml in RB solidified with 1% agarose. Photographs were taken at ×100 magnification under dark-field microscopy after 10 min for the wild type and after 20 min for the mcpU and che deletion strains, respectively.

FIG 2

Complementation of the chemotactic response of the S. meliloti mcpU deletion strain to proline. (A) Quantitative proline swim plate assay with increasing amounts of IPTG. Strains RU11/828 (▲) and RU11/828 with pBS1053 (●) were pipetted onto RB plates containing 10⁻⁴ M proline with various concentrations of IPTG to induce expression of mcpU under the control of the lac promoter from plasmid pBS1053. The percentages of the wild-type swim diameter on 0.27% agar are the means of three replicates, each performed in duplicate. Error bars represent the standard deviations from the mean. (B) Quantitative capillary assay of the mcpU deletion strain (ΔmcpU, RU11/828), the complemented strain (ΔmcpU/mcpU, RU11/828 with pBS1053) induced with 500 μM IPTG, and the chemotaxis-negative strain (che, RU13/149) with 10 mM proline. The results for each strain are the means of three experiments, each in triplicate. The means of each strain were normalized to the number of wild-type cells per capillary. Error bars represent the standard deviations from the mean.

FIG 3

Identification of McpU residues involved in proline sensing using the structure of the Vibrio cholerae McpN as a model. The structure of the periplasmic amino-terminal domain of the McpN dimer (Protein Data Bank code 3C8C) is oriented to display the tandem Cache domains of V. cholerae McpN. The view on the right provides a close up of the alanine ligand in the binding pocket of the N-terminal Cache domain on the monomer on the right. Two aspartate residues (Asp172 and Asp201) likely coordinate the ligand. The corresponding residues in S. meliloti McpU, Asp155 and Asp182, were chosen for further studies.

FIG 4

Chemotactic responses of S. meliloti mcpU mutant strains toward proline in hydrogel capillaries. (A) Wild type (RU11/001); (B) che deletion strain (RU13/149); (C) ΔmcpU strain (RU11/828); (D) McpU^D155A strain (BS184); (E) McpU^D182A strain (BS182); (F) McpU^D182E strain (BS187). Strains from early log phase in RB were tested with hydrogel capillaries containing 1 mM proline. Photographs were taken at ×100 magnification under dark-field microscopy after 20 min.

FIG 5

Chemotactic responses of S. meliloti mcpU mutant strains toward proline in a quantitative swim plate assay compared to the wild-type strain. The mcpU deletion strain (ΔmcpU, RU11/828), McpU^D155A (D155A, BS184), McpU^D182A (D182A, BS182), and McpU^D182E (D182E, BS187) strains were pipetted onto RB plates containing 10⁻⁴ M proline. The percentages of the wild-type swim diameter on 0.27% agar are the means of three replicates, each performed in duplicate. Error bars represent the standard deviations from the mean.

FIG 6

Immunoblot analysis of McpU and McpU-variant EGFP fusions in S. meliloti membrane fractions. S. meliloti cells were fractionated, and equal volumes of membrane fractions were electrophoretically separated, blotted onto nitrocellulose, and detected with anti-GFP monoclonal antibody. Lane 1, BS183 (McpU^D182A-EGFP); lane 2, RU13/301 (McpU-EGFP); lane 3, BS185 (McpU^D155A-EGFP); lane 4, RU11/001 (WT).

FIG 7

DSF profiles for binding of proline to McpU-PR and McpU-PR variants. Protein stability was monitored as a function of fluorescence intensity. Proteins were tested at a concentration of 10 μM with or without 10 mM proline. McpU-PR (black squares), McpU-PR with proline (gray squares), McpU-PR^D182A (black circles), McpU-PR^D182A with proline (gray circles) McpU-PR^D182E (black triangles), and McpU-PR^D182E with proline (gray triangles).

FIG 8

ITC of McpU-PR and McpU-PR^D182E with proline. Titration of McpU-PR (A) and McpU-PR^D182E (B) was carried out at a protein concentration of 812 μM with 10-μl injections of 9.8 mM proline. Reference data were produced by titrating buffer with proline and subtracting the resulting heat of ligand dilution from the experimental curves shown. Upper panels show the raw titration data, and lower panels show the normalized and dilution corrected peak areas of the raw titration data. The data were fitted with the One-Set-of-Sites model of the MicroCal version of Origin7.