Direct visualization of Escherichia coli chemotaxis receptor arrays using cryo-electron microscopy - PubMed (original) (raw)

Direct visualization of Escherichia coli chemotaxis receptor arrays using cryo-electron microscopy

Peijun Zhang et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Signal transduction in bacterial chemotaxis is initiated by the binding of extracellular ligands to a specialized family of methyl-accepting chemoreceptor proteins. Chemoreceptors cluster at distinct regions of the cell and form stable ternary complexes with the histidine autokinase CheA and the adapter protein CheW. Here we report the direct visualization and spatial organization of chemoreceptor arrays in intact Escherichia coli cells by using cryo-electron tomography and biochemical techniques. In wild-type cells, ternary complexes are arranged as an extended lattice, which may or may not be ordered, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. At disproportionately high receptor levels, membrane invaginations containing nonfunctional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be reestablished by increasing cellular levels of CheA and CheW. Our results demonstrate that chemotaxis in E. coli requires the presence of chemoreceptor arrays and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Visualization and identification of chemoreceptor arrays. (a) Low-dose cryo-projection image of the polar region in a wild-type E. coli cell, with the chemoreceptor array shown in greater detail in Inset. (b) Immunolabeling of cryo-sections from the same culture shown in a with anti-Tsr (5 nm gold) and anti-CheA (10 nm gold) antisera. (c) Schematic representation of the polar region of wild-type E. coli cells illustrating the assembly and orientation of the chemotaxis receptor array, based on a and b. (d–g) A selection of projection images demonstrating variability in size and location of chemoreceptor arrays within a single population of cells from the same EM specimen.

Fig. 2.

3D architecture of chemoreceptor arrays. (a and b) Cryo-tomography of intact wild-type E. coli highlighting the polar receptor array as seen in a single 5-nm tomographic slice (a) and as a segmented 3D representation of the chemoreceptor array (b). Densities corresponding to the inner membrane and CheA/CheW were manually segmented from a 279-slice cryo-tomogram by using the Amira visualization suite (TGS, San Diego, CA). For the sake of clarity, only a random subset of the striated densities have been segmented. Also shown is an expanded view of a manually segmented 3D representation (c) and a schematic representation of the chemoreceptor array displaying its position relative to the pole of the cell and to putative ribosomes in the cytoplasm (Inset). CheA/CheW and chemoreceptors are colored in blue and red, respectively, with the inner membrane in yellow and putative ribosomes in gray.

Fig. 3.

Effects of CheA, CheW, and Tsr expression on chemoreceptor array formation and chemotaxis phenotype. (a) Cells with wild-type levels of Tsr, CheA, and CheW display classical swarming behavior, which is absent in cells lacking CheA and CheW. Progressive titration of CheA/W levels results in an increase in the extent of swarming, with optimal activity observed for induction at 0.5 μM Na-S concentration. (b) Correlation between the percentages of cells displaying detectable polar arrays and the observed chemotaxis function in _cheA_−/_cheW_− cells (UU1607) carrying the inducible plasmid pPM23. (c) In cells lacking chemoreceptors, swarming behavior is absent. However, progressive titration of Tsr levels results in an increase in the extent of swarming, with optimal activity observed for induction at 20 μM IPTG concentration. (d) Correlation between the percentages of cells displaying detectable polar arrays and the observed chemotaxis function in chemoreceptor− cells (UU1250) carrying the inducible plasmid pJC3.

Fig. 4.

Effects of Tsr overexpression on chemoreceptor array formation: immunogold labeling of Tsr on frozen thin sections of E. coli UU1250/pJC3 cells induced with 50 μM IPTG by using anti-Tsr antiserum, recognized by 10-nm gold-labeled protein A. Shown are representative cells from the same section demonstrating diffuse Tsr localization and Tsr receptor arrays (a) similar to those seen in wild-type cells (b) and radial (c) and axial (d) Tsr receptor assemblies, which represent nonfunctional receptor arrays.

Fig. 5.

Formation of chemoreceptor arrays in E. coli. (a) When chemoreceptors are expressed at wild-type levels in the absence of CheA and CheW, diffuse receptor localization is observed at the polar regions of the cell. (b) In wild-type cells, the expression of each component is regulated, and functional chemoreceptor arrays are observed. (c) When Tsr is overproduced, numerous membrane invaginations are formed in which receptors interact radially and axially, with receptor–receptor interactions substituting for interactions of receptor with CheA and CheW. (d) Compensating high receptor levels with concurrent increases in CheA and CheW levels restores the formation of extended chemoreceptor arrays.

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Direct visualization of Escherichia coli chemotaxis receptor arrays using cryo-electron microscopy - PubMed (original) (raw)