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. 2013 Dec 17;2:e01402. doi: 10.7554/eLife.01402

Figure 5. CheA forms a complex with Pch and relies on flhF for polar placement.

(A) Representative images (10.8 × 10.8 microns) of CheA-mTq and Pch-Yfp localization in wild-type (top) and ΔflhF (bottom) strains. In the phase/fluorescence channel overlays, CheA-mTq fluorescence is shown in blue and Pch-Yfp fluorescence is shown in red. (B) Histogram of the smallest distance between any two CheA-mTq and Pch-Yfp foci in wild-type and ΔflhF strain backgrounds. (C) Histogram of the CheA-mTq foci distance to the nearest cell pole in wild-type and ΔflhF strain backgrounds. In (B) and (C) error bars depict counting error. (D) Percent of cells exhibiting polar localization of CheA-mTq in wild type and ΔflhF strain backgrounds from three biological replicates. Strains contain empty vector (pVC) or a complementing plasmid. Error bars depict the standard deviation. (E) Co-immunoprecipitation of CheA-mTq and Pch-VSV-G. CheA-mTq of the input lysates and anti VSV-G agarose bead elution fractions was detected by western blot utilizing a monoclonal anti-GFP antibody. Lanes 1–4 show control isolates lacking a CheA-mTq fusion. Lanes 1, 2 and 5, 6 show protein complexes eluted from anti VSV-G agarose beads.

DOI: http://dx.doi.org/10.7554/eLife.01402.010

Figure 5.

Figure 5—figure supplement 1. CheA localizes to the same pole as FliM.

Figure 5—figure supplement 1.

(A) Representative images (width of 19.9 microns) of CheA-mTq and FliM-mKate2 subcellular localization. (B) FliM-mKate2 does not disrupt flagellar assembly. Representative images (13.1 × 13.1 microns) of FliM-mKate2 localization together with Alexa Fluor 488 labeling of surface exposed amine groups. The bottom left panel shows the overlay of FliM-mKate2 localization (in red) with the phase contrast image. The bottom right panel shows FliM-mKate2 and amine labeled cells (in green), confirming that the base of the flagellum corresponds to the same location as the FliM-mKate2 focus. The top left panel is of the FliM-mKate2 fluorescence image, and the top right panel is of surface amines labeled with Alexa Fluor 488. (C) Bulk motility assays of strains with FliM-mKate2 and CheA-mTq fusions confirm these strains exhibit normal motility and chemotaxis.
DOI: http://dx.doi.org/10.7554/eLife.01402.011
Figure 5—figure supplement 2. CheA-mTq colocalizes with Pch-mCherry but not with FliM-mKate2.

Figure 5—figure supplement 2.

A representative histogram of the minimum separation between CheA-mTq and FliM-mKate2 foci and of the minimum separation between CheA-mTq and Pch-mCherry foci. Identical imaging parameters were used for both sets of fluorophore fusions. Error bars depict the counting error.
DOI: http://dx.doi.org/10.7554/eLife.01402.012
Figure 5—figure supplement 3. FliM-mKate2 polar localization is not dependent upon CheA.

Figure 5—figure supplement 3.

Representative images (14.8 microns in width) of FliM-mKate2 localization in wild type and ΔcheA backgrounds. From three biological replicates, a mean of 40% (SD of 5%) of wild-type cells exhibit polar localization and 41% (SD of 4%) of ΔcheA cells exhibit polar localization.
DOI: http://dx.doi.org/10.7554/eLife.01402.013