(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.