A distant homologue of the FlgT protein interacts with MotB and FliL and is essential for flagellar rotation in Rhodobacter sphaeroides

Abstract

In this work, we describe a periplasmic protein that is essential for flagellar rotation in Rhodobacter sphaeroides. This protein is encoded upstream of flgA, and its expression is dependent on the flagellar master regulator FleQ and on the class III flagellar activator FleT. Sequence comparisons suggest that this protein is a distant homologue of FlgT. We show evidence that in R. sphaeroides, FlgT interacts with the periplasmic regions of MotB and FliL and with the flagellar protein MotF, which was recently characterized as a membrane component of the flagellum in this bacterium. In addition, the localization of green fluorescent protein (GFP)-MotF is completely dependent on FlgT. The Mot(-) phenotype of flgT cells was weakly suppressed by point mutants of MotB that presumably keep the proton channel open and efficiently suppress the Mot(-) phenotype of motF and fliL cells, indicating that FlgT could play an additional role beyond the opening of the proton channel. The presence of FlgT in purified filament-hook-basal bodies of the wild-type strain was confirmed by Western blotting, and the observation of these structures under an electron microscope showed that the basal bodies from flgT cells had lost the ring that covers the LP ring in the wild-type structure. Moreover, MotF was detected by immunoblotting in the basal bodies obtained from the wild-type strain but not from flgT cells. From these results, we suggest that FlgT forms a ring around the LP ring, which anchors MotF and stabilizes the stator complex of the flagellar motor.

Fig 1

Gene context, phenotype of the SF3 (flgT::aadA) mutant strain, and tertiary structure comparison with FlgT from V. alginolyticus. (A) Gene arrangement of the flagellar operon containing RSP_6086 (flgT). The arrows indicate the direction of gene transcription. The black box indicates the regulatory region that contains the sigma 54-dependent flgAp promoter characterized previously (85). The arrow above the black box symbolizes transcription from this promoter. (B) Swimming plate inoculated with the indicated strains. (C) Electron micrograph of WS8 and SF3 cells showing the presence of flagellar filaments. Bar = 1 μm. (D) Supernatant and pellet fractions obtained after strong vortexing of WS8 and SF3. Samples were analyzed by immunoblotting using anti-FliC antibodies. (E) Tertiary structure prediction of FlgT_Rs (red line) superimposed with the structure of FlgT from V. alginolyticus (82) (blue line).

Fig 2

Subcellular localization and expression of FlgT in different strains. (A) The presence of FlgT in the periplasm of wild-type cells was analyzed by using a protease sensitivity assay. For this, spheroplasts from 15 ml of WS8 cells growing exponentially (OD₆₀₀ = 0.8) were obtained by treatment with lysozyme and EDTA, and spheroplasts were incubated in the presence of proteinase K (100 μg/ml) for 20 min (+) and 40 min (+*). A control without proteinase K was also included and incubated with Tris buffer for 40 min (−). The resulting samples containing 5 μg of protein were analyzed by Western blotting using specific antibodies. As a control, the same samples were tested for the presence of the cytoplasmic protein FliH. (B) Five micrograms of total cell extracts of the indicated strains was analyzed by Western blotting using anti-FlgT or anti-CheY3 antibodies.

Fig 3

FlgT interactions tested by pulldown. Shown are pulldowns of FlgT-His₆ with GST alone (29 kDa), GST-FliL (48 kDa), and GST-MotB (63 kDa). After coprecipitation, the sample was divided in two and probed with anti-FlgT and anti-GST antibodies by immunoblotting.

Fig 4

FlgT interactions tested by double-hybrid assays. AH109 yeast cells were transformed with the plasmids indicated on the left. The pair AD-T and BD-P53 and the pair AD-T and BD-Lam are the positive and negative controls, respectively. Serial dilutions of cultures of the transformant cells were inoculated onto plates containing the growth medium indicated at the bottom, which is synthetic medium (SD) lacking leucine (−Leu), tryptophan (−Trp), histidine (−His), or adenine (−Ade). Pictures were taken after 10 days of incubation at 30°C.

Fig 5

GFP-MotF and GFP-FliL localization in strain SF3. (A) Representative images of GFP-MotF and GFP-FliL in WS8 and SF3 cells. Bar = 1 μm. (B) Western blot analysis of GFP-FliL and GFP-MotF expressed in WS8 and SF3 strains. A total cell extract containing 5 μg of protein was subjected to SDS-PAGE and tested by Western blotting using anti-GFP antibodies.

Fig 6

Mutations in MotB promote the swimming of SF3. (A) Map of the coding region of MotB indicating relevant features. Black box, transmembrane domain (TM); open box with a middle black line, plug region; open box, OmpA-like domain. The changes in each suppressor are indicated. (B) Swimming plate inoculated with strains WS8, SF3, SF5, and SF6. (C) Swimming plates inoculated with WS8, SF4, and SF4 expressing the indicated mutant allele of motB.

Fig 7

Electron micrographs and immunoblot analysis of purified filament-hook-basal bodies from strains WS8 and SF3. (A) Images of purified HBBs from strains WS8 (top) and SF3 (bottom). A white arrow denotes the H ring. Bar = 20 nm. (B) Western blot analysis of the purified basal bodies from WS8 and SF3 samples. One-tenth of the total sample (approximately 10 μg of total protein) of purified HBBs was analyzed by immunoblotting using specific antibodies.