Figure 4. CleA localizes to the flagellated cell pole to interact with FliM.

Examples of the subcellular localization of CleA-mGFPmut3 are shown in (a) and quantified as shown in (b). Variants of CleA fused to mGFPmut3 are indicated on the left with the three domains highlighted as in Figures 2 and 3. Individual amino acid substitutions are indicated. CleA fusion proteins were expressed in a ΔcleA deletion strain and additional genetic alterations as indicated on the right of each panel. Cells were classified according to the localization patterns shown in (a): delocalized cytosolic (blue); foci at flagellated pole of PD cells (red); polar foci in small SW cells (green); other localization patterns (purple), including PD cells with foci at both poles, PD cells with foci at stalked pole, cells with no GFP signal. Number of cells analyzed (top to bottom): 3452, 1450, 637, 2357, 2603, 795, 1148, 1347. (c) CleA interacts with the FliM flagellar switch protein. Binding of CleA to the N-terminus of FliM was determined by bacterial two-hybrid analysis. Fusion proteins between the T25 and T18 fragments of adenylate cyclase (Karimova et al., 1998) and the N-terminal peptide of FliM or FliM_ID57WA (aa 2–65), or full-length CleA were constructed and expressed in the E. coli cya mutant strain AB1770. Strains harboring combinations of FliM and CleA fusions were spotted on McConkey agar plates to score for interaction. Red color indicates reporter gene expression from a cAMP-dependent promoter. The spotting order is indicated in the grids on the right. A positive control with the adenylate cyclase fragments fused to the leucine zipper region of the yeast GCN4 protein (zip) is shown (lower right spots).

Figure 4—figure supplement 1. CleD localizes to the flagellated cell pole to interact with FliM.

(a) Localization of CleD-eGFP wild type and mutant variants. Individual amino acid substitutions are indicated. CleD fusion proteins were expressed in a ΔcleD deletion strain and additional genetic alterations as indicated on the right of each panel. Cells were classified according to the localization patterns shown in Figure 4a: delocalized cytosolic (blue); foci at flagellated pole of PD cells (red); polar foci in small SW cells (green); other localization patterns (purple), including PD cells with foci at both poles, PD cells with foci at stalked pole, cells with no GFP signal. Number of cells analyzed (top to bottom): 226,>200, 287, 2194,>200,>200, 645,>200, 981, 709. (b) CleD interacts with the FliM flagellar switch protein. Pull-down experiments were carried out with a strain expressing a Flag-tagged copy of CleD. Anti-Flag antibodies were used for co-immunoprecipitation with cell extracts (input) and precipitated fractions (output) being analyzed by immunoblots with anti-Flag and anti-FliM antibodies as indicated. Extracts of C. crescentus wild-type or a ΔfliM mutant were used as indicated. The experiment was repeated three times with a representative example shown. (c) Direct interaction of CleD with a peptide spanning FliM residues 47–62. The ¹H-¹⁵N HSQC spectra of the CleD (red) and CleD in complex with the FliM peptide (blue) are shown. The spectrum of CleD with c-di-GMP and the FliM_ID57WA mutant peptide or with CleD and the wild-type FliM peptide without c-di-GMP was indistinguishable from the spectrum recorded for CleD alone.

Figure 4—figure supplement 2. The conserved CheY binding site of the C. crescentus FliM switch protein is required for motor reversal.

(a) Alignments of the α4-β5-α5 region of E. coli CheY and C. crescentus CleA and CleD (top) and of the N-terminal peptides of E. coli and C. crescentus FliM (bottom). Residues shown to be important for the interaction between FliM and CheY in E. coli are highlighted in yellow. Conserved FliM residues that were mutated in this study to break the interaction between C. crescentus FliM and CheY or Cle are marked by asterisks. (b) Crystal structure of E. coli CheY (green) interacting with the N-terminal helix of FliM (orange) (PDB 1F4V). (c) Reversal frequencies of C. crescentus wild type and of a ΔfliM deletion strain expressing a fliM wild-type allele (red) or the fliM_ID57WA mutant (green) from plasmid pSRK-Km. The distributions were calculated from more than 850 cells (WT = 6719 cells;DfliM pSRK-FliM = 887 cells;DfliM pSRK-FliM_ID57WA = 936 cells) combined from multiple independent experiments. Experiments were repeated twice except for WT, which was repeated seven times.