Cellular Stoichiometry of Methyl-Accepting Chemotaxis Proteins in Sinorhizobium meliloti

Abstract

The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli, the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilisIMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti is of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better understanding of the role of chemotaxis in these processes, detailed knowledge on the regulation and composition of the chemosensory machinery is essential. Here, we show that chemoreceptor gene expression in S. meliloti is controlled through the main transcriptional regulators of motility. Chemoreceptor abundance is much lower in S. meliloti than in Escherichia coli and Bacillus subtilis Moreover, the chemoreceptor-to-kinase CheA ratio is different from that of E. coli but similar to that of B. subtilis.

Keywords: alfalfa; chemoreceptors; flagellar motor; plant symbiosis; transcriptional control.

FIG 1

Representative immunoblots used to quantify transmembrane chemoreceptors. (A) McpV. Lane 1 (ΔmcpV) contains RU11/830 (mcpV deletion strain) cell lysate from 1 ml 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. ΔmcpV + McpV-LBD lanes contain purified McpV-LBD (7.5, 5.0, 4.0, 3.0, 2.0, and 1.0 ng) mixed with RU11/830 cell lysates. Representative immunoblots were used to quantify transmembrane chemoreceptors. (B) McpZ. Lanes 1 to 3 contain RU11/001 (WT) cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25. Lane 4 (ΔmcpZ) contains RU11/818 (mcpZ deletion strain) cell lysate from 1 ml culture at an OD₆₀₀ of 0.25. ΔmcpZ + McpZ-LBD lanes contain purified McpZ-LBD (2.6, 1.3, 0.78, 0.52, and 0.26 ng) mixed with RU11/830 cell lysates. (C) McpU. Lanes 1 to 3 contain RU11/001 (WT) cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25. Lane 4 (ΔmcpU) contains RU11/828 (mcpU deletion strain) cell lysate from 1 ml culture at an OD₆₀₀ of 0.25. ΔmcpU + McpU-LBD lanes contain purified McpU-LBD (2.0, 1.0, 0.5, 0.1, and 0.05 ng) mixed with RU11/828 cell lysates. McpU-LBD exists in monomeric and dimeric forms, as indicated. (D) McpX. Lanes 1 to 3 contain RU11/001 (WT) cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25. Lane 4 (ΔmcpX) contains RU11/805 (mcpX deletion strain) cell lysate from 1 ml culture at an OD₆₀₀ of 0.25. ΔmcpX + McpX-LBD lanes contain purified McpX-LBD (1.0, 0.75, 0.5, and 0.25 ng) mixed with RU11/805 cell lysates. McpX-LBD exists in monomeric and dimeric forms, as indicated. (E) McpW. Lanes 1 and 3 contain RU13/143 (mcpW-egfp) cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25. Lane 2 contains RU11/001 (WT) cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25. Lane 4 (ΔmcpW) contains RU11/803 (mcpW deletion strain) cell lysate from 1 ml culture at an OD₆₀₀ of 0.25. ΔmcpW + McpW-LBD lanes contain purified McpW-LBD (0.5, 0.25, 0.1, 0.075, and 0.05 ng) mixed with RU11/803 cell lysates. The intensity of the McpW-GFP band in lanes 1 and 3 (arrows) is equal to the difference in intensities between the band in lane 2 (asterisk) and its corresponding nonspecific band in lane 4.

FIG 2

Representative immunoblot used to quantify cytosolic receptors and CheA. (A) McpY. Lanes 1 to 3 contain RU11/001 (WT) cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25. Lane 4 (ΔmcpY) contains RU11/804 (mcpY deletion strain) cell lysate from 1 ml culture at an OD₆₀₀ of 0.25. ΔmcpY + McpY lanes contain purified McpY (0.1, 0.05, 0.01, and 0.005 ng) mixed with RU11/804 cell lysates. The arrow indicates the McpY protein in the WT cell lysates. (B) IcpA. Lanes 1 to 3 contain RU11/001 (WT) cell lysates from 1 ml of culture at an OD₆₀₀ of 0.25. Lane 9 (ΔicpA) contains RU11/815 (icpA deletion) cell lysate from 1 ml culture at an OD₆₀₀ of 0.25. ΔicpA + IcpA lanes contain purified IcpA (1.28, 0.96, 0.64, 0.32, and 0.16 ng) mixed with RU11/815 cell lysates. (C) CheA. Lane 1 (ΔcheA) contains RU11/310 (cheA deletion strain) cell lysate from 1 ml 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. ΔcheA + CheA lanes contain purified CheA (1.0, 0.8, 0.6, 0.4, and 0.2 ng) mixed with RU11/310 cell lysates.

FIG 3

Localization of McpU, McpV, IcpA, and CheA fused to eGFP in S. meliloti cells by fluorescence microscopy. (A) McpU-eGFP; (B) McpV-eGFP; (C) IcpA-eGFP; (D) CheA-eGFP.

FIG 4

Sequence comparison of the 15 C-terminal amino acid residues in the NWETF motif-containing E. coli (E.c.) receptors Tar and Tsr and all eight S. meliloti (S.m.) chemoreceptors. The conserved pentapeptide sequence is marked in gray.