FipA constitutes a new family of FlhF interaction partners.

Volcanoplots representing Log-ratios versus significance values of proteins enriched in (A) FlhF-sfGFP or (B) FipA-sfGFP purifications using shotgun proteomics and liquid chromatography-mass spectrometry; sfGFP was used as control. The full list of pulled-down proteins can be found in the Supplementary Tables S1 and S2. C) Organization of the flagellar/chemotaxis gene region encoding FlhF and FipA in V. parahaemolyticus. D) Domain organization of FipA (TM, transmembrane region; DUF, domain of unknown function). E) Bacterial two-hybrid confirming the interaction between FipA and FlhF from V. parahaemolyticus. The indicated proteins (FipA, FlhF) were fused N- or C-terminally to the T18- or T25-fragment of the Bordetella pertussis adenylate cyclase. In vivo interaction of the fusion proteins in Escherichia coli is indicated by blue color. The corresponding assay in P. putida and S. putrefaciens is displayed in Supplementary Figure 2. F) Dendrogram of γ-proteobacteria, indicating the presence of FlhF or FipA homologues and the corresponding flagellation pattern. An extended version and sources are available in Supplementary Table 2.

FipA is required for correct flagellum formation.

(A, C) Representative soft-agar swimming assay of V. parahaemolyticus (A) or P. putida (C) strains (left panels) and the corresponding quantification (right panels). For the latter, the halo diameter measurements were normalized to the halo of the wild type on each plate. Data presented are from six (A) or three (C) independent replicates, asterisks represent a p-value <0.05 (according to ANOVA + Tukey tests). (B) Single-cell tracking of V. parahaemolyticus. Shown are representative swimming trajectories and quantification of swimming speed, total displacement and reversal rate. N indicates number of cells tracked among 3 biological replicates (ANOVA + Tukey test). (D) Representative electron micrographs of the indicated V. parahaemolyticus strains stained with uranyl acetate. (E) Quantification of flagellation pattern in the populations of the indicated V. parahaemolyticus strains. (F) Flagellum stain of indicated P. putida strains with Alexa Fluor 488-C5-maleimide and (G) quantification of the corresponding flagellation in the population. N indicates the number of cells counted among 3 biological replicates. For S. putrefaciens, see Supplementary Figure 3.

Localization of FlhF depends on FipA and HubP.

(A) Representative micrographs of the indicated strains of V. parahaemoloyticus expressing FlhF-sfGFP from its native promoter. Upper panel shows the DIC image, the lower panel the corresponding fluorescence image. Fluorescent foci are highlighted by white arrows. Scale bar = 2 μm. For an enlargment see Supplementary Fig. 6. (B) Demographs displaying FlhF-sfGFP fluorescence intensity along the cell length within the experiments shown in (A). (C) Quantification of localization patterns and (D) foci fluorescence intensity of the fluorescence microscopy experiment presented in (A). The data was combined from the given number (N) of cells combined from three biological replicates. (E) Representative micrographs of the indicated P. putida strains expressing FlhF-sfGFP from its native promoter. The upper panels show the DIC and the lower panels the corresponding fluorescence images. Fluorescent foci are marked by small white arrows. The scale bar equals 5 µm. The low intensity of the foci did not allow a quantitative analysis of foci intensities or the generation of demographs. For an enlargement of the micrographs see Supplementary Fig. 7. (F) Quantification of FlhF-sfGFP localization patterns in the corresponding strains of P. putida from the experiments shown in (E). Corresponding data on the localization of FlhF in S. putrefaciens is displayed in Supplementary Fig. 8.

Activity of FipA depends on FlhF and on its transmembrane domain.

(A) Micrographs of E. coli cells expressing FipA-sfGFP from V. parahaemolyticus, and a truncated version lacking the transmembrane domain (ΔTM). The left panels display the DIC and the right panels the corresponding fluorescence images. The scale bar equals 5 μm. (B) Electron micrographs of V. parahaemolyticus wild-type and mutant cells lacking the transmembrane domain of fipA, respectively. The corresponding quantification of the flagellation pattern is shown to the left of the micrographs. Note that the data for the wild-type cells is the same as in Fig. 2D, E. (C) Spreading behavior of the indicated P. putida strains (left) with the corresponding quantification (right). Loss of the FipA TM region phenocopies a complete fipA deletion.

Conserved residues in the domain of unknown function of FipA are essential for interaction with FlhF.

(A) Weight-based consensus sequence of the conserved region of DUF2802 as obtained from 481 species. The residues targeted in the FipA orthologs of V. parahaemolyticus, P. putida and S. putrefaciens are indicated along with their appropriate residue position. (B, C) Bacterial two-hybrid assay of FipA variants of V. parahaemolyticus (B) or P. putida (C) with an alanine substitution in the conserved residues indicated in (A). The constructs were tested for self-interaction and interaction with FlhF. In vivo interaction of the fusions in E. coli is indicated by blue coloration of the colonies. (D) Quantification from single-cell tracking of swimming V. parahaemolyticus cells expressing FipA bearing the indicated substitution in the DUF2802 domain (see Figure 2B for wild-type behavior). Asterisks indicate a p-value <0.05 (ANOVA + Tukey test) (F) Localization of VpFlhF in the absence of FipA or in cells with substitutions in the DUF2802 domain. Left: Micrographs showing the localization of FipA-sfGFP in the indicated strains; the upper panels display the DIC and the lower panels the corresponding fluorescence images (for an enlargement see Supplementary Fig. 9A). The scale bar equals 5 µm. Right: the corresponding quantification of the FlhF-sfGFP patterns in the indicated strains. (E) Soft-agar spreading assays of P. putida wild-type and indicated mutant strains, asterisks display a p-value of 0.05 (*) or 0.01 (**) (ANOVA). (G) Localization of P. putida FlhF in strains bearing substitutions in the DUF2802 interaction site. Left: micrographs displaying the localization of FlhF-mCherry in the indicated strains. Upper panels show the DIC and lower panels the corresponding fluorescence images. The scale bar equals 5 µm (for an enlargement see Supplementary Fig. 9B). Right: Corresponding quantification of the FlhF localization pattern in the indicated strains. Data for S. putrefaciens is displayed in Supplementary Fig. 10.

The localization pattern of FipA.

(A, B) Localization pattern of fluorescently labeled FipA in V. parahaemolyticus and P. putida. (A) Representative micrographs of V. parahaemolyticus expressing FipA-sfGFP from its native promoter. Scale bar = 2 μm. The upper panel shows the DIC and the lower panel the corresponding fluorescence channel. To the right the localization was quantified accordingly. (B) The same analysis for P. putida. (C, D) Time lapse analysis of FipA-sfGFP localization over a cell cycle in V. parahaemolyticus (C) and P. putida (D). The numbers in the upper DIC micrographs show the minutes after start of the experiment. The scale bars equal 1 µm (C) and 5 µm (D).

Normal localization of FipA depends on interaction with FlhF.

(A, B) Localization pattern of V. parahaemolyticus FipA-sfGFP in the indicated wild-type and mutant strains. The upper panels display the DIC micrographs, the middle panel the corresponding fluorescence imaging (scale bar equals 5 µm), and the lower panel the corresponding demograph showing the fluorescence of FipA-sfGFP along the cell length. (C) Quantification of the cell localization pattern from the experiment shown in (A, B) as combined from three biological replicates. (D, E, F) The same analysis for the corresponding P. putida strains as indicated. The scale bar equals 5 µm. The data for S. putrefaciens is displayed in Supplementary Figure 15.