Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane

Abstract

The bacterial flagellum exemplifies a system where even small deviations from the highly regulated flagellar assembly process can abolish motility and cause negative physiological outcomes. Consequently, bacteria have evolved elegant and robust regulatory mechanisms to ensure that flagellar morphogenesis follows a defined path, with each component self-assembling to predetermined dimensions. The flagellar rod acts as a driveshaft to transmit torque from the cytoplasmic rotor to the external filament. The rod self-assembles to a defined length of ~25 nanometers. Here, we provide evidence that rod length is limited by the width of the periplasmic space between the inner and outer membranes. The length of Braun's lipoprotein determines periplasmic width by tethering the outer membrane to the peptidoglycan layer.

Fig. 1. The hook basal body of Salmonella enterica

Construction of the flagellum begins with the assembly of the transmembrane membrane supramembrane ring (MS-ring), the flagellar type 3 secretion system (fT3SS), and the cytoplasmic ring (C-ring) rotor complex. The proximal rod polymerizes on top of the MS-ring followed by the FlgG distal rod to span the distance between the peptidoglycan layer and the outer membrane. FlgJ, the rod cap, allows the growing structure to pass through the peptidoglycan layer through localized hydrolysis of the peptidoglycan (28). Once the distal rod has reached the outer membrane, formation of the periplasmic ring–lipopolysaccharide ring (P-ring–L-ring) complex simultaneously forms a hole in the outer membrane and dislodges the rod cap. Rod cap removal allows the hook cap (FlgD) to form on the tip of the nascent structure and to promote assembly of the hook by FlgE subunits.

Fig. 2. Overexpression of flgG resulted in longer distal rods

To test the FlgG intrinsic model for length control of the distal rod, flgG was overexpressed from a plasmid vector to supplement flgG expression from its native chromosomal locus in a strain lacking the genes required for termination of distal rod assembly (flgH) and subsequent hook assembly (flgD and flgE). (A) Flagellar basal bodies were purified from the flgG-overexpression background and compared with those from an empty-vector control strain by transmission electron microscopy (TEM). (B) Measurements were taken from the top of the MS-ring [dashed line in (A)] to the tip of the rod (P, proximal rod; D, distal rod). Overexpression of flgG was found to significantly increase the length of the rod (P < 0.0001, Student’s two-tailed t test, N = 2).

Fig. 3. The inner-to outer-membrane distance and flagellar rod length varied with LppA lengths

Resin-embedded LppA-length mutants, as well as WT control (TH22579 and TH22634 to TH22637) were thin-sectioned and observed by electron tomography. (A and B) The peptidoglycan-layer (PG)–to–outer-membrane (OM) distances for each strain were measured. As the length of LppA increased, we observed a corresponding increase in PG-to-OM distance [P < 0.0001, one-way analysis of variance (ANOVA)]. (C) To verify these results, cells from −21, +21, and WT LppA strains (TH22579, TH22634, and TH22635) were imaged via cryo-EM, and the distances between the inner membrane (IM) and OM were measured for each. The distances varied with LppA lengths (P < 0.0001, Student’s two-tailed t test). (D and E) Flagellar rods from lengthened LppA variants (TH22574 to TH22577) were purified, imaged via TEM and measured. The average length of the rod increased ~1.5 to 2 nm for every three heptad repeats (21 residues) added [P < 0.0001, one-way ANOVA, N = ≥ 3, data in (D) are means ± SEM].

Fig. 4. LppA functions as an outer-membrane tether

Deletion of lppAB (strain TH22543) resulted in the formation of taut outer-membrane blebs that pulled away from the cell body. The severity of blebbing was increased in the ΔlppAB Δpal double mutant (strain TH22569).