A novel component of the Rhodobacter sphaeroides Fla1 flagellum is essential for motor rotation

Abstract

Here we describe a novel component essential for flagellar rotation in Rhodobacter sphaeroides. This protein is encoded by motF (RSP_0067), the first gene of a predicted transcriptional unit which contains two hypothetical genes. Sequence analysis indicated that MotF is a bitopic membrane-spanning protein. Protease sensitivity assays and green fluorescent protein (GFP) fusions confirmed this prediction and allowed us to conclude that the C terminus of MotF is located in the periplasmic space. Wild-type cells expressing a functional GFP-MotF fusion show a single fluorescent focus per cell. The localization of this protein in different genetic backgrounds allowed us to determine that normal localization of MotF depends on the presence of FliL and MotB. Characterization of a ΔmotF pseudorevertant strain revealed that a single nucleotide change in motB suppresses the Mot(-) phenotype of the motF mutant. Additionally, we show that MotF also becomes dispensable when other mutant alleles of motB previously isolated as second-site suppressors of ΔfliL were expressed in the motF mutant strain. These results show that MotF is a new component of the Fla1 flagellum, which together with FliL is required to promote flagellar rotation, possibly through MotB.

Fig 1

Role of MotF in the flagellar motility of R. sphaeroides. (A) Gene arrangement of the flagellar region that contains motF, including three genes of the fliO operon and two genes downstream of motF. The arrows indicate the direction of transcription. (B) Swimming plate inoculated with WS8 (1), the VR5 mutant strain carrying an insertion in RSP_6092 (2), the VR1 mutant strain carrying an insertion in RSP_0067 (motF::aadA) (3), and VR1 carrying pRK_motF (4). (C) Electron micrograph of VR1 cells showing the presence of flagella. Bar = 500 nm. (D) Western blot analysis of the pellet (P) and supernatant (SN) fractions obtained after strong vortexing of WS8 and VR1 cell cultures. A polyclonal anti-FliC antibody was used for detection.

Fig 2

Analysis of the region located upstream of motF. Shown is the nucleotide sequence of the intercistronic region between flhB and motF. Below the nucleotide sequence, on the left, four amino acids corresponding to the C-terminal end of FlhB are shown; the stop codon is indicated with an asterisk. To the right, the first five amino acids corresponding to the N-terminal region of MotF are shown. Above the nucleotide sequence, the “+1” indicates the first base of the ATG initiation codon of MotF. The conserved nucleotides that match the σ⁵⁴ consensus promoter are underlined. Below, in a box, the σ⁵⁴ consensus promoter sequence is shown.

Fig 3

MotF topology. (A) Representative images of WS8 cells expressing GFP-MotF or MotF-GFP. Bar = 2 μm. (B) Western blot analysis of spheroplasts from WS8 and VR1 cells. −, spheroplasts without proteinase K; +, spheroplasts treated with proteinase K for 10 min; +*, spheroplasts treated with proteinase K for 20 min. The protein recognized by the antibody is indicated on the left. (C) Swimming plate inoculated with WS8, VR1, VR1/pRK_motF-gfp, and VR1/pRK_gfp-motF. (D) Example of a characteristic WS8 wild-type cell expressing GFP-MotF. Cells were stained with DAPI to covisualize the flagellum and the signal from GFP-MotF. Bar = 1 μm.

Fig 4

GFP-MotF localization in different genetic backgrounds. (A) Representative images of different strains expressing GFP-MotF: VR1 (motF::aadA), SP12 (ΔfleT1::aadA), SP13 (ΔfleQ1::Kan), SP18 (flgC1::Kan), FS3 (ΔfliL3::aadA), FS4 (ΔmotB1::Kan), and LC3 (ΔmotB::Ω^Spc)/pMG_motB D40A. Bar = 2 μm. (B) Western blot of GFP-MotF expressed in different strains. Total cell extracts of each strain were tested by Western blotting using a mouse polyclonal anti-GFP antibody.

Fig 5

Swimming of second-site suppressor strains. (A) Swimming plate inoculated with WS8 (wild type), VR2/pRK_motB⁺, and VR2/pRK_motBsup9. (B) Swimming assay testing the suppression of the motF::aadA mutation by the motB alleles isolated as second-site suppressors of ΔfliL::aadA. SUP1, MotB A67E; SUP3, MotB A56E; SUP4, MotB F63L; SUP5, MotB A67D; SUP6, MotB A67T; SUP7, MotB F63L; SUP8, MotB A67G. Each motB allele was tested in the VR2 strain (ΔmotB::Kan motF::aadA). The percentages represent the averages of the swimming ring diameters from three different assays. Values are expressed as percentages of the value determined for the WS8 strain.

Fig 6

Effect of the MotF region located after the TM region in swimming and MotF localization. (A) Swimming plate inoculated with VR1/pRK_motF and VR1 carrying pRK_ΔmotF3 or pRK_ΔmotF4; (B) Western blot of VR1 cells expressing wild-type MotF (1), VR1 cells expressing MotF without plasmid (2), VR1 expressing MotFΔ_29–49 (3), and VR1 expressing MotFΔ_77–98 (4). (C) Representative image of VR1 cells expressing GFP-MotFΔ_77–98.