The Vibrio H-Ring Facilitates the Outer Membrane Penetration of the Polar Sheathed Flagellum

Abstract

The bacterial flagellum has evolved as one of the most remarkable nanomachines in nature. It provides swimming and swarming motilities that are often essential for the bacterial life cycle and pathogenesis. Many bacteria such as Salmonella and Vibrio species use flagella as an external propeller to move to favorable environments, whereas spirochetes utilize internal periplasmic flagella to drive a serpentine movement of the cell bodies through tissues. Here, we use cryo-electron tomography to visualize the polar sheathed flagellum of Vibrio alginolyticus with particular focus on a Vibrio-specific feature, the H-ring. We characterized the H-ring by identifying its two components FlgT and FlgO. We found that the majority of flagella are located within the periplasmic space in the absence of the H-ring, which are different from those of external flagella in wild-type cells. Our results not only indicate the H-ring has a novel function in facilitating the penetration of the outer membrane and the assembly of the external sheathed flagella but also are consistent with the notion that the flagella have evolved to adapt highly diverse needs by receiving or removing accessary genes.IMPORTANCE Flagellum is the major organelle for motility in many bacterial species. While most bacteria possess external flagella, such as the multiple peritrichous flagella found in Escherichia coli and Salmonella enterica or the single polar sheathed flagellum in Vibrio spp., spirochetes uniquely assemble periplasmic flagella, which are embedded between their inner and outer membranes. Here, we show for the first time that the external flagella in Vibrio alginolyticus can be changed as periplasmic flagella by deleting two flagellar genes. The discovery here may provide new insights into the molecular basis underlying assembly, diversity, and evolution of flagella.

Keywords: flagellar assembly; flagellar evolution; membrane penetration; periplasmic flagella.

FIG 1

Lack of FlgO results in reduced motility. (A) Motility of cells in soft agar. Two-microliter aliquots of overnight cultures of each strain was spotted onto a 0.25% soft agar VPG plate containing 2.5 μg/ml chloramphenicol and 0.02% (wt/vol) l-arabinose, and the plate was incubated at 30°C for 7 h. Deletion of flgO from the strain KK148 resulted in reduced motility, and ectopic expression of FlgO fused with a hexa-histidine tag at the C terminus (FlgO-His₆) from the arabinose-inducible plasmid pTSK128 restored motility (protein expression was confirmed [B]). VIO5 is the wild-type strain for polar flagellar motility; KK148 is multipolar flagellar strain and the parent of NMB337. The plasmid pBAD33 was used as the empty vector control. (B) Immunoblot analysis. Whole-cell lysates were separated by SDS-PAGE and transferred onto the PVDF membrane, and His-tagged proteins were detected by an anti-His tag antibody. The FlgO-His₆ protein was detected at the size equivalent to its mature form (indicated as the filled arrow). Experiments were conducted 3 times, and the typical results are shown here.

FIG 2

Characterization of the ΔflgO flagellum in situ by cryo-ET. (A to C) A representative slice of a 3D reconstruction of the V. alginolyticus ΔflgO strain KK148 with multiple polar flagella. (D to F) Zoom-in views of the slices that are shown in panels A to C. (G) A slice of a subtomogram average of the flagellar motor. (H) A slice of a subtomogram average of the flagellar motor in KK148. The structural difference between panels G and H is indicated with arrows. (I) Schematic model of the Vibrio motor. (J) 3D surface renderings of the image from panel G. (K, L) 3D surface renderings of the image from panel H. The H-ring is labeled in orange and pink, separately; the T-ring is colored yellow. OM, outer membrane; IM, inner membrane.

FIG 3

Characterization of the ΔflgT flagellum in situ by cryo-ET. (A, B) Representative slices of tomograms from KK148 ΔflgT cells. The motor is visible beneath of outer membrane. The motor is indicated in cyan. (C, D) Representative slices of from KK148 ΔflgT cells. The flagellar filament is visible in the periplasmic space and labeled in red. (E) The flagellar filament is extended in the periplasmic space and penetrates the outer membrane without a sheath. (F) The flagellar filament penetrates from the periplasm and is sheathed.

FIG 4

Cryo-ET reconstructions of the whole cells from KK148 and KK148 ΔflgT exhibiting dramatic differences in flagellar structures. (A, B) Tomographic slice of KK148 ΔflgT shown in different layers of the tomogram. (C, D) Images from panels A and B in a 3D segmentation to show the periplasmic flagella. (E) Representative tomogram slice of a KK148 whole cell shows multiple polar sheathed flagella. (F) 3D segmentation of the image from panel E.

FIG 5

Model of polar sheathed flagellar assembly. Vibrio species have a single polar sheathed flagellum. The H-ring labeled in red is required in the assembly of the polar sheathed flagellum, in addition to flagellar stabilization. The dysfunction of FlgT causes the loss of the H-ring and, consequently, changes the polar flagellum to a periplasmic flagellum. OM, outer membrane; PG, peptidoglycan layer; IM, inner membrane.