Evolution of a large periplasmic disk in Campylobacterota flagella enables both efficient motility and autoagglutination

Abstract

The flagellar motors of Campylobacter jejuni (C. jejuni) and related Campylobacterota (previously epsilonproteobacteria) feature 100-nm-wide periplasmic "basal disks" that have been implicated in scaffolding a wider ring of additional motor proteins to increase torque, but the size of these disks is excessive for a role solely in scaffolding motor proteins. Here, we show that the basal disk is a flange that braces the flagellar motor during disentanglement of its flagellar filament from interactions with the cell body and other filaments. We show that motor output is unaffected when we shrink or displace the basal disk, and suppressor mutations of debilitated motors occur in flagellar-filament or cell-surface glycosylation pathways, thus sidestepping the need for a flange to overcome the interactions between two flagellar filaments and between flagellar filaments and the cell body. Our results identify unanticipated co-dependencies in the evolution of flagellar motor structure and cell-surface properties in the Campylobacterota.

Keywords: Campylobacter jejuni; autoagglutination; bacterial flagellum; bacterial genetics; electron cryotomography; fluorescence microscopy; glycosylation.

Figure 1:. Engineered motors with small basal disks nevertheless incorporate stator complexes and rotate similarly to WT motors.

(A) The flagellar motor of Campylobacter jejuni has the same components as that of the model organsims, as well as extra embellishments such as the basal disk and stator scaffolding architecture. (B) Increasing the level of ATc in the growth medium corresponded to an increased level of flgP expression. FlgR antisera was used as an internal loading control. (C) Slices of individual tomograms (top) and subtomogram averages (bottom) (scale bars = 20 nm) of motors at different flgP expression levels and (dashed white lines indicate width measurement, stators (MotB) are incorporated into the motor even under low-induction conditions (red arrowheads)) (D) measurements of basal disk widths of individual motors in electron cryo-tomograms (disk widths are significantly different between WT, flgP^AAA, EJC168 at 25 ng/mL ATc and 50 ng/mL ATc, 1-way ANOVA and two-tailed t-tests, p = <0.0001 – 0.0385, bars represent mean +/− SEM). In the WT, all the motors imaged possessed basal disks, while in EJC168 lower concentrations of ATc corresponded to a lower proportion of motors with disks. Disk-less motors were excluded from the analysis in (D). (E) The flgP^{S69A E157A K159A} (flgP^AAA) mutant constructed small-diameter basal disks, and the distribution of basal-disk diameters clustered more tightly than EJC168 at all induction levels and never extended beyond ~80 nm (left: slice through a single tomogram; right: subtomogram average of FlgP^AAA motor (scale bars = 20 nm)). (F) Still frames of high-speed video of fluorescently-labelled WT and flgP^AAA cells (scale bars = 1 μm). The position of filaments relative to the cell body (white arrowheads) can be used to approximate rotation rate. For both the WT and flgP^AAA rotation rate was found to be −100 Hz (1 revolution/10 ms).

Figure 2:. Low FlgP expression and mutation of FlgP enhance autoagglutination

(A) A flagellar filament is required for autoagglutination, and a functional motor prevents excessive autoagglutination. The sedimentation rates (i.e. autoagglutination) of a suspension of WT cells paralyzed with CCCP or a paralyzed mutant (ΔmotB::cat) are significantly faster than the non-treated cell suspension (two-tailed t-test, p = 0.0062). (B) Low FlgP expression in EJC168 corresponded to a faster sedimentation rate. The flgP^AAA mutant sedimented significantly faster than EJC168 induced with 25 ng/mL ATc (two-way ANOVA, p = <0.0001, error bars are mean +/− SEM).

Figure 3:. Displacement of the basal disk from the P-ring reduces filament unwrapping

(A) A 55-residue segment from Salmonella LppA (a.k.a Lpp or Braun’s lipoprotein) was inserted after C17 in FlgP to make FlgP-Lpp⁵⁵. (B) Subtomogram averaging of the flgP-lpp⁵⁵ mutant’s motor revealed that the basal disk had been displaced from the P-ring by ~7 nm (P-ring: white dashed line, first ring of basal disk: red dashed line) and had a narrower first ring of FlgP subunits, but that stator recruitment was not impacted (red circles)(scale bars = 20 nm). (C) Following overnight incubation in soft-agar, the flgP-lpp⁵⁵ mutant formed small-diameter swarms in motility agar compared to WT, despite (D) the presence of a population of cells swimming at WT velocity when cell suspensions were observed by 3D-holographic-tracking microscopy (arrowheads). (E) Kymographs generated from high-speed-fluorescence-video show that the flgP-lpp⁵⁵ mutant is incapable of unwrapping its filament from the cell body during motor reversals and does not change swimming direction, as opposed to the characteristic darting motility of WT C. jejuni where unwrapping leads to a reversal of swimming direction. Single asterisk: unwrapping of leading filament and wrapping of lagging filament; double asterisk: failure to unwrap (scale bars 1 μm). (F and G) The velocity of individual flgP-lpp⁵⁵ cells traversing a microfluidic device with 1 μm-wide channels was found to be identical to WT (two-tailed t-test, p = 0.212, bars represent mean +/− SEM), confirming our observations from holographic tracking, although flgP-lpp⁵⁵ cells fail to reverse upon encountering obstacles (supplemental videos 8 and 9). (H) The flgP-lpp⁵⁵ mutant has a significant host colonization defect relative to WT, colonizing the chicken cecum as poorly as a non-motile ΔflgP mutant (one-way ANOVA, p = <0.0001, bars represent mean cfu/g cecal content).

Figure 4:. Suppression of the flgP-lpp⁵⁵ motility defect occurs by restoring the basal disk-P-ring register or reducing filament glycosylation

(A) The flgG^T54N allele allows the distal rod to grow longer in order to accommodate a second P-ring (arrowheads), restoring flanging of the motor by the basal disk in the flgP-lpp⁵⁵ background. (B and C) The second suppressor we isolated arose in pseG, a UDP-sugar hydrolase involved in O-glycosylation of the flagellar filament. The pseG^I142T allele has no effect on motility in an otherwise WT background but increases soft-agar motility of the flgP-lpp⁵⁵ mutant. (D) The decreased sedimentation rate caused by the pseG^I142T allele in both the WT and flgP-lpp⁵⁵ backgrounds indicates lower levels of O-glycosylation of the filament. Restoration of the P-ring/basal disk register in the flgGT54N background suppressed the autoagglutination defect of flgP-lpp⁵⁵ (E) Observing multiple motor switching events by high speed video microscopy revealed that The pseG^I142T allele suppresses the flgP-lpp⁵⁵ motility defect by enabling unwrapping of the filament from the cell body upon motor reversal. (31/42 (74%) switches led to unwrapping in WT, 2/45 (4%) in flgP-lpp⁵⁵, and 22/42 (52%) in flgP-lpp pseG^I142T). (F) The increased unwrapping conferred by the pseG^I142T suppressor is demonstrated at the population level by the restored aerotactic behaviour of the flgP-lpp⁵⁵ pseG^I142T double mutant toward a region of higher O₂ concentration.

Figure 5:. Motility in the absence of a basal disk requires de-glycosylation of the cell surface

(A) Four independent disk-less cultures were inoculated into soft agar and allowed to incubate for extended periods of time. Speckles emanating from the point of inoculation were picked, purified, stored and also used to inoculate a subsequent prolonged incubation in soft agar. After repeating four to five times for each independent lineage, all four lineages had evolved a non-bushy swarm phenotype in soft agar. (B) Generating pgl, kps and 0661 knockouts singly and in combination revealed that the sugarSNPs were loss-of-function mutations and that both loss 0661 and N-glycosylation (i.e. pgl) are required to suppress the ΔflgPQ motility defect. (C) Comparison of the subtomogram average structures of the ΔflgPQ and EJC103 mutants’ motors confirmed that motility in the evolved disk-less mutants was not due restoration of stator scaffolding in the motor. (D) Suppression of the non-motile phenotype in the absence of the basal disk corresponded with decreased sedimentation rate upon removal of pgl and kps surface polysaccharides in autoagglutination assays. (E) Comparison of the sedimentation rates of cells paralyzed by deletion of the stators (ΔmotAB::cat) in both the WT background and the Δ0661 ΔpglAB::aphA ΔkpsD background shows that the decreased sedimentation rate of the of EJC103 in (D) is not a result of increased motility in this background (error bars represent mean +/− SEM).