Cellular Stoichiometry of Chemotaxis Proteins in Sinorhizobium meliloti

Abstract

Chemotaxis systems enable microbes to sense their immediate environment, moving toward beneficial stimuli and away from those that are harmful. In an effort to better understand the chemotaxis system of Sinorhizobium meliloti, a symbiont of the legume alfalfa, the cellular stoichiometries of all ten chemotaxis proteins in S. meliloti were determined. A combination of quantitative immunoblot and mass spectrometry revealed that the protein stoichiometries in S. meliloti varied greatly from those in Escherichia coli and Bacillus subtilis To compare protein ratios to other systems, values were normalized to the central kinase CheA. All S. meliloti chemotaxis proteins exhibited increased ratios to various degrees. The 10-fold higher molar ratio of adaptor proteins CheW1 and CheW2 to CheA might result in the formation of rings in the chemotaxis array that consist of only CheW instead of CheA and CheW in a 1:1 ratio. We hypothesize that the higher ratio of CheA to the main response regulator CheY2 is a consequence of the speed-variable motor in S. meliloti, instead of a switch-type motor. Similarly, proteins involved in signal termination are far more abundant in S. meliloti, which utilizes a phosphate sink mechanism based on CheA retrophosphorylation to inactivate the motor response regulator versus CheZ-catalyzed dephosphorylation as in E. coli and B. subtilis Finally, the abundance of CheB and CheR, which regulate chemoreceptor methylation, was increased compared to CheA, indicative of variations in the adaptation system of S. meliloti Collectively, these results mark significant differences in the composition of bacterial chemotaxis systems.IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti contributes greatly to host-plant growth by fixing atmospheric nitrogen. The provision of nitrogen as ammonium by S. meliloti leads to increased biomass production of its legume host alfalfa and diminishes the use of environmentally harmful chemical fertilizers. To better understand the role of chemotaxis in host-microbe interaction, a comprehensive catalogue of the bacterial chemotaxis system is vital, including its composition, function, and regulation. The stoichiometry of chemotaxis proteins in S. meliloti has very few similarities to the systems in Escherichia coli and Bacillus subtilis In addition, total amounts of proteins are significantly lower. S. meliloti exhibits a chemotaxis system distinct from known models by incorporating new proteins as exemplified by the phosphate sink mechanism.

Keywords: chemoreceptor methylation; flagellar motility; plant symbiont; rhizosphere; two-component system.

FIG 1

Chemotaxis gene loci in the S. meliloti genome. Arrows depicting individual genes are drawn to scale.

FIG 2

Representative immunoblots used to quantify chemotaxis proteins. For each panel, lane 1 contains cell lysate of the respective deletion strain from 1 ml of culture at an OD₆₀₀ of 0.25, lanes 2 to 4 contain RU11/001 (WT) cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25, and lanes 5 to 9 contain different amounts of purified protein to be quantified mixed with cell lysates of the respective deletion strain, as indicated. (A) CheB (3, 2.5, 1.5, 1, and 0.5 ng) mixed with RU11/312 cell lysates. (B) CheD (16, 13, 9, 6, and 3 ng) mixed with RU11/312 cell lysates. (C) CheR (7.5, 6, 4.5, 3, and 1.5 ng) mixed with RU11/306 cell lysates. (D) CheT (0.3, 0.2, 0.1, 0.075, and 0.050 ng) mixed with RU11/319 cell lysates. (E) CheW1 (2, 1.5, 1, 0.5, and 0.25 ng) mixed with BS198 cell lysates. (F) CheW2 (0.9, 0.7, 0.5, 0.3, and 0.17 ng) mixed with RU11/811 cell lysates. (G) CheY1 (12, 10.5, 7.8, 6.4, and 5 ng) mixed with RU11/816 cell lysates. (H) CheY2 (12, 9.6, 6.4, 5.1, and 3.2 ng) mixed with RU11/307 cell lysates. One milliliter of culture at an OD₆₀₀ of 0.25 contained 2.56 × 10⁸ cells (53).

FIG 3

Representative immunoblot used to quantify 3×FLAG-CheS. Lane 1 (WT) contains RU11/001 cell lysate from 1 ml of culture at an OD₆₀₀ of 0.25. Lanes 2 to 4 contain BS265 cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25. WT+McpV-LBD 3×FLAG lanes contain purified McpV-LBD-3×FLAG (7, 5, 3, 1, and 0.5 ng) mixed with RU11/001 cell lysates. One milliliter of culture at an OD₆₀₀ of 0.25 contained 2.56 × 10⁸ cells (53).

FIG 4

Relationship analysis and sequence alignment of CheW proteins from various bacterial species. (A) Cladogram illustrating the relationship between E. coli K-12 CheW (NP_416401.1), A. tumefaciens CheW1 (WP_004430791.1) and CheW2 (WP_003504949.1), and S. meliloti CheW1 (WP_014528989.1), CheW2 (WP_003536237.1), and CheW3 (WP_003528560.1). (B) Multiple sequence alignment of E. coli CheW, A. tumefaciens CheW1 and CheW2, and S. meliloti CheW1, CheW2, and CheW3. Arrowheads denote residues involved in the interaction with the P5 domain of CheA using E. coli as a paradigm model.

FIG 5

Chemotactic responses of various S. meliloti cheW mutant strains in a quantitative swim plate assay compared to the wild-type strain. Strain designations: ΔW1, in-frame deletion of cheW1 (RU11/414); ΔW2, in-frame deletion of cheW2 (RU11/811); ΔW3, in-frame deletion of cheW3 (BS195); Δ(W1,W2), in-frame deletions of cheW1 and cheW2 (RU11/812); Δ(W1,W3), in-frame deletions of cheW1 and cheW3 (BS196); Δ(W2,W3), in-frame deletions of cheW2 and cheW3 (BS197); Δ(W1,W2,W3), in-frame deletions of cheW1, cheW2, and cheW3 (BS198). The percentages of the wild-type swim diameter on 0.3% Bromfield agar are the means from seven replicates. Error bars represent the standard deviations from the mean. Statistical significance was determined by a two-tailed Student t test (P < 0.05). Asterisks denotes no statistically significant difference from the wild type.

FIG 6

Diagram of in vivo stoichiometries in S. meliloti, B. subtilis, and E. coli signaling complexes. The cellular stoichiometries of the chemotaxis signaling proteins in this study, receptor ratios previously published for S. meliloti, and previously reported ratios from E. coli and B. subtilis (51–53) are shown. The models are normalized to show the cellular ratios to a CheA dimer in each species.