FlhF regulates the number and configuration of periplasmic flagella in Borrelia burgdorferi

Abstract

The Lyme disease bacterium Borrelia burgdorferi has 7-11 periplasmic flagella (PF) that arise from the cell poles and extend toward the midcell as a flat-ribbon, which is distinct from other bacteria. FlhF, a signal recognition particle (SRP)-like GTPase, has been found to regulate the flagellar number and polarity; however, its role in B. burgdorferi remains unknown. B. burgdorferi has an FlhF homolog (BB0270). Structural and biochemical analyses show that BB0270 has a similar structure and enzymatic activity as its counterparts from other bacteria. Genetics and cryo-electron tomography studies reveal that deletion of BB0270 leads to mutant cells that have less PF (4 ± 2 PF per cell tip) and fail to form a flat-ribbon, indicative of a role of BB0270 in the control of PF number and configuration. Mechanistically, we demonstrate that BB0270 localizes at the cell poles and controls the number and position of PF via regulating the flagellar protein stability and the polar localization of the MS-ring protein FliF. Our study not only provides the detailed characterizations of BB0270 and its profound impacts on flagellar assembly, morphology and motility in B. burgdorferi, but also unveils mechanistic insights into how spirochetes control their unique flagellar patterns.

Keywords: Borrelia burgdorferi; Lyme disease; motility; periplasmic flagella; signal recognition particle (SRP)-GTPase.

FIGURE 1

BB0270 is an FlhF-like GTPase. (a) Domain structure of BB0270 (FlhF_Bb). The positions of the N-terminal basic (B), the central (N) domains, the conserved nucleotide-binding elements (G1-G5) as well as the I-box in FlhF_Bb are indicated. (b) Multiple sequence alignments of different FlhF G-domains. The numbers represent the positions of aa in the FlhF proteins of B. burgdorferi (Bb), B. subtilis (Bs), V. cholerae (Vc) and P. aeruginosa (Pa). Red circles indicate two residues that are replaced by site-directed mutagenesis. The GenBank accession numbers for these proteins: FlhF_Bb (WP_002556869), BsFlhF (WP_003231945), VcFlhF (WP_001881782), and PaFlhF (WP_003114281). The alignment was carried out using the program MacVector 10.6. (c) Homology modeling analysis shows that FlhF_Bb shares a similar structural topology to BsFlhF. The figure was generated by homology modeling using BsFlhF (PDB ID: 2PX3) as a template. Green is FlhF_Bb and cyan is BsFlhF. (d) FlhF_Bb harbors a conserved GTP and Mg²⁺ binding site. This is a close up view of the GTP and Mg²⁺ binding site of FlhF_Bb. The map was generated by homology modeling using BsFlhF (PDB ID: 2PX3) (Bange, Petzold, Wild, Parlitz, et al., 2007) as a template. The conserved aa involved GTP and Mg²⁺ binding sites were as labeled. (e) SDS-PAGE analysis of GST-FlhF_Bb recombinant proteins expressed in E. coli. Lane 1, molecular marker; lane 2 and lane 3, E. coli whole cell lysates induced without or with IPTG; lane 4–6, purified wild type (rFlhF_Bb) and two mutated (rFlhF^K187A and rFlhF^R218G) recombinant proteins. (f) Michaelis-Menten plots show kinetic plots for the GTPase activity of purified rFlhF_Bb and two variant recombinant proteins. The final data were expressed as means ± standard deviations (SD) of triplicates from three independent assays. The detail enzymatic parameters are present in Table 1

FIGURE 2

Deletion of flhF_Bb affects the cell growth, shape and motility of B. burgdorferi. (a) The growth curves of WT, ΔflhF and ΔflhF^com strains. Cells were grown in BSK-II medium (pH 7.6) at 34°C for 8 days. Cell counting was repeated in triplicates in two independent experiments, and the results were expressed as means ± SD. (b) Dark-field microscopic analysis of WT and ΔflhF mutant. The cells were grown in BSK-II medium to the early stationary phase and then subjected to microscopic analysis under dark-field illumination at x 400 magnifications using a Zeiss Axiostar Plus microscope. (c) Swimming plate assays of WT, ΔflhF and ΔflhF^com strains. For this assay, B. burgdorferi cells (5 μl) were stab-inoculated into semisoft agar plates containing dPBS-diluted (10:1) BSK-II medium and 0.35% agarose, as previously described (Motaleb et al., 2000). ΔflaB, a nonmotile mutant, was used as a control to determine the initial inoculation size on the plates

FIGURE 3

Cryo-ET analysis reveals that the ΔflhF mutant has less PF. (a) Low magnification image of a ΔflhF cell showing the overview of the cell shape. Two boxed regions in the cell tip and body were imaged by cryo-ET. (b) A section of ΔflhF cell tomogram showing four PF originated from the cell tip. (c) A section of ΔflhF midcell tomogram showing the flagellar filaments are loosely distributed along the cell body. (d, e) 3D segmentations of the ΔflhF mutant cell tip and the cell body. (k, l) High magnification image of ΔflhF cell tip tomogram and 3D segmentation. (f) Low magnification image of a WT cell. (g) A section of WT cell tomogram showing eight PF originated from the cell tip. (h) A section of WT midcell tomogram showing the flagellar filaments from both cell ends are overlapped and tightly organized into a flat-ribbon. (i, j) 3D segmentations of a WT cell tip and the cell body. (m, n) High magnification image of WT cell tip tomogram and 3D segmentation. OM: outer membrane; IM: inner membrane

FIGURE 4

Whole cell tomography shows that deletion of flhF_Bb alters the configuration of PF. (a) A representative tomographic slice of the tip of a WT cell. (b) Surface view of the cell tip. The motors are colored in red. The scale bar is 100 nm. PF are colored in purple. (c) Surface views of a half of the cell. The scale bar is 250 nm. The PF from the other tip are colored in yellow. (d) Surface views of the full WT cell. The scale bar is 500 nm. (e) A representative tomographic slice of the tip of a ΔflhF cell. (f) Surface view of the mutant cell tip. The scale bar is 100 nm. (g) Surface views of a half of the mutant cell. The scale bar is 250 nm. (h) Surface views of a full ΔflhF cell. The scale bar is 500 nm

FIGURE 5

Deletion of flhF_Bb affects the level and stability of flagellar proteins in B. burgdorferi. (a) A diagram illustrating the overall structure of PF. Red arrows denote four flagellar proteins that represent the MS-ring (FliF), the rod (FlgG), the hook (FlgE) and the filament (FlaB). (b) Detection of FliF, FlgG, FlgE and FlaB by immunoblotting analyses. Similar amounts of WT, ΔflhF and ΔflhF^com whole-cell lysates were subjected to SDS-PAGE and then probed with specific antibodies against these four proteins respectively. DnaK was used as a loading control. Immunoblots were developed using horseradish peroxidase secondary antibody with an ECL luminol substrate as previously described (Zhang et al., 2019). (c) Detection of four flagellar gene transcripts by qRT-PCR. The levels of flaB, flgE, flgG and fliF transcripts were measured by qRT-PCR as previously described (Sal et al., 2008; Sze et al., 2011). The transcript of enolase gene (eno) was used as an internal control to normalize the qPCR data. The results were expressed as the mean threshold cycles (CT) of triplicate samples. (d) & (e) Protein turnover assays. For this assay, protein translation was arrested by adding spectinomycin (100 μg/ml) to the cultures of WT and ΔflhF mutant, and then samples were collected at the indicated time points and subjected to immunoblotting. Of note, compared to the wild type, the loading amount of the flhF mutant was increased about 3-folds in order to detect these flagellar proteins during a period of 12 hr

FIGURE 6

Polar localization of FlhF_Bb in B. burgdorferi. (a) & (b) Fluorescence images of cells expressing GFP alone (a) or FlhF_Bb-GFP (b). The micrographs were taken under a fluorescence microscope (magnification, × 620) with a fluorescein isothiocyanate emission filter. (c-e) Immunofluorescence microscopic analysis of the ΔflhF/flhF-gfp (c), WT (d), and ΔflhF mutant (e) cells. Bacterial cells were fixed with methanol, stained with anti-FlhF and counterstained with anti-rat Texas Red-conjugated antibody, as previously described (Zhang, Liu, et al., 2012). The micrographs were taken under differential interference contrast (DIC) light and a fluorescence microscope with a tetramethylrhodamine isothiocyanate (TRITC) emission filter, and the resultant images were merged

FIGURE 7

The polar localization of FliF is impaired in the ΔflhF mutant. Immunofluorescence images of the WT (a), ΔfliF (b) and ΔflhF mutant (c) cells. B. burgdorferi cells were fixed with methanol, stained with anti-FliF, and counterstained with anti-rat Texas Red-conjugated antibody, as previously described (Zhang, Liu, et al., 2012). The micrographs were taken under DIC light and a fluorescence microscope with a TRITC emission filter, and the resultant images were merged