The flagellar protein FliL is essential for swimming in Rhodobacter sphaeroides

Abstract

In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion.

FIG. 1.

Characterization of the fliL mutant strain. (A) Swimming of FS3 (ΔfliL3::aadA) and FS3 carrying the wild-type fliL allele cloned in pRK451. WS8 was included as a positive control. (B) Electron micrograph of FS3 cells showing the presence of flagella. (C) Western blot analysis of the supernatant fraction obtained after strong vortexing of WS8, FS3, and FS3/pRK-fliL cultures. A polyclonal anti-FliC antibody was used for detection. Strain SP7 (ΔrpoN2::Kan) was included as a negative control.

FIG. 2.

Determination of FliL topology. (A) Representative images of WS8 expressing FliL-GFP or GFP-FliL. Strains WS8 and WS8 expressing GFP from pRK415 were used as controls. Bars, 3 μm. (B) Western blot analysis of WS8 cells expressing GFP, GFP-MotF, and GFP-FliL. MotF is an inner membrane protein related to flagellar rotation in R. sphaeroides that has a C-terminal domain in the periplasmic side of the IM (V. F. Ramirez-Cabrera and L. Camarena, unpublished results). Lanes: +, spheroplasts treated with proteinase K; −, spheroplasts without proteinase K; T, total cell extract. (C) Western blot analysis of WS8 wild-type, FS3 (ΔfliL3::aadA), and SP13 (ΔfleQ1::Kan) cells expressing GFP-FliL. Lanes: +, spheroplasts treated with proteinase K; −, spheroplasts without proteinase K; T, total cell extract. As a control, the FS3 strain was included.

FIG. 3.

Characterization of GFP-FliL localization. (A) Representative images of different strains expressing GFP-FliL: WS8 (wild type), FS3 (ΔfliL3::aadA), SP13 (ΔfleQ1::Kan), SP18 (flgC1::Kan), FS4 (ΔmotB1::Kan). Bars, 3 μm. (B) Western blot of GFP-FliL expressed in different strains. Total cell extracts of the indicated strains were analyzed by Western blotting using an anti-GFP antibody. (C) Example of a characteristic WS8 wild-type cell expressing GFP-FliL. Cells were stained with Alexa 594 to covisualize flagella and the signal from GFP-FliL. (D) Representative images of WS8 cells exemplifying the motion of the lateral foci of GFP-FliL. A moving lateral focus is indicated by an arrow. Bar, 1 μm. (E) Swimming plate of WS8 and FS3 strains expressing GFP-FliL.

FIG. 4.

Characterization of the second-site suppressors isolated from the FS3 strain. (A) Swimming of the different suppressor strains. WS8 and FS3 (ΔfliL3::aadA) were included as positive and negative controls, respectively. (B) Nucleotide and residue changes present in motB for each suppressor mutant.

FIG. 5.

Swimming characterization of different strains expressing the motBsup alleles. (A) Swimming assay of the control strains WS8, FS4 (ΔmotB1::Kan), and FS5 (ΔmotB1::Kan ΔfliL3::aadA). Also included are FS4 and FS5 expressing the motB wild-type allele from pRK415. (B) Swimming of the double mutant FS5 (ΔmotB1::Kan ΔfliL3::aadA) expressing the motBsup alleles from the lac promoter of pRK415. (C) Swimming of FS4 (ΔmotB1::Kan) expressing the motBsup alleles from pRK415. (D) Swimming of the suppressor mutants expressing the fliL wild-type allele from pRK415.

FIG. 6.

(A) Interaction of His₆-FliLp with FliLp. Coprecipitation of His₆-FliLp, using GST-FliLp, GST-FliLpΔ15, or GST alone as bait. After the coprecipitation assay, the sample was divided in two and probed with anti-His and anti-GST antibodies. The proteins observed with anti-GST confirm a proper concentration of the bait, whereas the presence of the His-tagged protein indicates a positive interaction. (B) Double-hybrid assay to test protein-protein interactions. Yeast cells were transformed with the plasmids indicated at the left margin; GAL4AD-T and GAL4BD-Lam and GAL4AD-T and GAL4BD-p53 plasmid pairs served as negative and positive controls, respectively. Transformant cells were seeded on the medium indicated at the top. To rule out spurious activation of the reporter gene (HIS3) mediated by FliLp, AH109 transformed with GAL4BD-FliLp was also tested (lower part of the figure). Pictures were taken after 4 days of incubation for culture plates lacking Trp and Leu and plates lacking Trp, Leu, and His or 10 days for plates lacking Trp, Leu, His, and Ade.