Cellular localization of predicted transmembrane and soluble chemoreceptors in Sinorhizobium meliloti

Abstract

Bacterial chemoreceptors primarily locate in clusters at the cell pole, where they form large sensory complexes which recruit cytoplasmic components of the signaling pathway. The genome of the soil bacterium Sinorhizobium meliloti encodes seven transmembrane and two soluble chemoreceptors. We have investigated the localization of all nine chemoreceptors in vivo using genome-encoded fusions to a variant of the enhanced green fluorescent protein and to monomeric red fluorescent protein. Six of the transmembrane (McpT to McpX and McpZ) and both soluble (McpY and IcpA) receptors localize to the cell pole. Only McpS, encoded from the symbiotic plasmid pSymA, is evenly distributed in the cell. While the synthesis of all polar localized receptors is confined to exponential growth correlating with the motility phase of cells, McpS is only weakly expressed throughout cell culture growth. Therefore, motile S. meliloti cells form one major chemotaxis cluster that harbors all chemoreceptors except for McpS. Colocalization and deletion analysis demonstrated that formation of polar foci by the majority of receptors is dependent on other chemoreceptors and that receptor clusters are stabilized by the presence of the chemotaxis proteins CheA and CheW. The transmembrane McpV and the soluble IcpA localize to the pole independently of CheA and CheW. However, in mutant strains McpV formed delocalized polar caps that spread throughout the cell membrane while IcpA exhibited increased bipolarity. Immunoblotting of fractionated cells revealed that IcpA, which lacks any hydrophobic domains, nevertheless is associated to the cell membrane.

FIG. 1.

Localization of receptor-EGFP fusions in S. meliloti cells by fluorescence microscopy. Receptors fused to EGFP were monitored in a wild-type background. The following cells were grown to mid-log phase McpS-EGFP (RU13/299) (A), McpT-EGFP (RU13/300) (B), McpU-EGFP (RU13/301) (C), McpV-EGFP (RU13/212) (D), McpW-EGFP (RU13/143) (E) McpX-EGFP (RU13/144) (F), McpY-EGFP (RU13/302) (G), McpZ-EGFP (RU13/241) (H), IcpA-EGFP (RU13/303) (I), and McpS-EGFP expressed from the pBBR1MCS-2 derivative pRU2887 (Table 1) (18) (J) The following cells were grown to stationary phase: McpS-EGFP (RU13/299) (K) and McpZ-EGFP (RU13/241) (L). Differential interference contrast signals were faded. White bars correspond to 1 μm.

FIG. 2.

Timing of chemoreceptor gene expression in wild-type S. meliloti during growth over a period of 80 h. Averaged growth curves (_*-_*; OD₆₀₀) and promoter activities (in Miller units) monitored by lacZ reporter gene constructs are shown. (A) PmcpS (⋄), PmcpT (□), PmcpU (▾), and PmcpY (▪). (B) PmcpW (•), PmcpX (▵), PmcpZ (▴), and PicpA (○). Points represent the mean of three independent experiments, and bars represent the standard deviations. Arrows (bottom) define the period of sampling for Western blot analysis shown below. (C) Samples of 4.7 × 10⁸ cells of wild-type strain RU11/001 were taken at the intervals indicated at the bottom of the blot. Equal amounts of total cell protein were separated by SDS-polyacrylamide gel electrophoresis, blotted on nitrocellulose, and detected with anti-IcpA antibody (1:100).

FIG. 3.

Colocalization of fluorescently labeled chemotaxis proteins within the same bacterial cell. (A) McpU-EGFP and McpX-mRFP in strain RU13/315. (B) McpZ-EGFP and McpX-mRFP in strain RU13/317. (C) IcpA-EGFP and McpX-mRFP in strain RU13/318. (D) IcpA-EGFP and CheA-mRFP in strain RU13/314. Left panels show cells after excitation of EGFP, right panels show cells after excitation of mRFP, and merged images are shown in the middle. Differential interference contrast signals were faded. White scale bars correspond to 1 μm.

FIG. 4.

Localization of IcpA- and McpV-EGFP fusions expressed from pBBR1MCS-2 in S. meliloti wild-type (RU11/001) and ΔvisNR (RU11/814) cells by fluorescence microscopy. (A) McpV-EGFP in strain RU11/001. (B) McpV-EGFP in strain RU11/814. (C) IcpA-EGFP in strain RU11/001. (D) IcpA-EGFP in strain RU11/814. Differential interference contrast signals were faded. White scale bars correspond to 1 μm.

FIG. 5.

Immunoblot analysis of IcpA (A) and McpY (B) in subcellular fractions. S. meliloti wild-type cells were fractionated, and equal volumes were electrophoretically separated, blotted on nitrocellulose, and detected with anti-IcpA and anti-McpY antibodies. Lane 1, soluble fraction after French press passage; lane 2, soluble fraction after Triton X-100 treatment; lane 3, insoluble membrane fraction after Triton X-100 treatment.