Figure 1.
The bacterial flagellar motor (BFM) switch—landmarks. (A) Conceptualization: (i) The switch complex was proposed based on phenotypic characterization of mot, che and fla alleles and their suppressor mutations in swarm plate assays. Its interactions with chemotaxis components and Mot proteins were also identified. {a} Schematic of a swarm plate—the native (WT) strain forms a swarm with chemotactic rings. Strains carrying mot mutations (Mot-) do not swarm while those with che mutations (Che-) have reduced swarms. Suppressor mutations yield pseudo-revertant strain (PR) with partially restored swarming. {b} Color codes are followed in subsequent Figures for the switch complex components (FliG (green), FliM (gold), FliN (cyan)), the CheY protein (salmon) and the MS-ring scaffold (orange) (adapted from [6]). (ii) Gene sequencing identified the mutations. The fliM gene (N–C terminal residue numbers) predominantly contained the che lesions, clustered into distinct CW (green) and CCW (magenta) regions. Arrows mark mot lesions (adapted from [8]). (B) Structural identification: (i) An extended cytoplasmic structure contiguous with the basal body MS-ring (yellow arrow) was isolated using gentler protocols and subsequently established as the switch complex by immuno-EM and biochemistry (from [14]). (ii) Assembly of switch complex by overproduction of plasmid-encoded components allowed biochemical characterization culminating in the determination of the C ring subunit stoichiometry (n = 33–34) (from [23]). (iii) Single-particle analysis resolved FliG domain substructure (yellow arrows) from differences in central sections from wild-type (WT) and ∆FliFFliG (∆) 3D basal-body reconstructions (from [22] with permission). (C) Motor function and mechanism: (i) Temporally resolved measurement of filament rotation, as a sinusoidal variation of laser dark-field spot intensity, characterized aberrant phenotypes in switch complex mutant strains. Panels (top to bottom) show slow rotation (S), pausing (P) and reversal (R) episodes (reproduced from [32] with permission). (ii) The first atomic structure of a switch component (FliGc [33]) followed by the FliGMC structure localized much of the mutant library then available ((mot lesions (black); CW lesions (red); CCW lesions (yellow); CW or CCW, depending on the residue substitution, orange; and motB suppressors (purple)) to generate chemically explicit ideas for motor reversal (PDB: 1lkv (modified from [34])). (D) Switch chemotactic signal transduction: (i) {1}—Determination of switch “ultra-sensitivity” (Hill coefficient, H = 10.3) by simultaneous measurement of the CW bias of beads on flagellar stubs (red) and concentration of a fluorescent GFP-CheY fusion (green) locked in the active state (*) in engineered strains (reproduced from [45] with permission). {2}—Plots show non-cooperative binding of acetate-activated CheY to overproduced C rings [46] compared to the in-vivo change in CW bias. (ii) The atomic structure of beryllium-fluoride (BeF3 (black))-activated CheY (salmon) bound to the FliM N-terminal peptide (yellow) initiated structure guided mutagenesis to explain the switch ultra-sensitivity. Aromatic residue (W58, Y106 (orange)) motions were early diagnostics for activation. Magnesium ion (red) (PDB: 1f4v (modified from [41])).