Structural and Functional Analysis of Flagellar Filaments of Caulobacter crescentus

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The bacterial flagellum is a complex, multi-protein structure consisting of a basal body, motor, hook, and filament [1]. The filament acts as an Archimedes’ screw propelling the cell body forward and is comprised of thousands of monomers called flagellins [2]. Flagellum biogenesis is a tightly regulated process involving over 60 genes and comes with considerable energetic costs [3]. Genetic sequencing of the oligotrophic bacterium Caulobacter crescentus highlights that six different flagellin genes are present on two separate loci and are readily synthesized in the wild-type strain [4]. The genes denoted fljJ, fljK, and fljL are located on the α-locus while fljM, fljN, and fljO are located on the β-locus [4]. Although nearly 45% of bacterial species synthesize multiple flagellins, little is known about the advantages of filaments comprised of multiple flagellins [5]. Here we begin to assess the role of flagellin packing in C. crescentus by analyzing the energetically favorable states of purified single flagellin filaments and comparing key molecular contacts between non-straightened filaments. To resolve structures of helical assemblies, suspensions of isolated flagellar filaments were applied to EM grids and vitrified using a Vitrobot Mark IV. Imaging was performed on a ThermoFisher Titan Krios G3i FEG-TEM operated at 300 kV equipped with a Gatan Bioquantum energy filter and K3 direct electron detector. Dose-fractionated micrographs were collected in counting mode over a defocus range of -0.5 to -2.5 μm with increment steps of 0.25 μm at a pixel size of 0.834 Å with a total dose of 45 e-/Å2 (1 e-/Å2/frame). Movies were acquired with 3 shots per hole acquiring multiple holes per stage position by using EPU/AFIS, on average ∼250 movies were collected per hour. Helical reconstruction was performed in RELION 4.0, Model Angelo was used for initial model building followed by model improvement in ChimeraX and COOT, and real-space refinement was performed in PHENIX [6-10]. Motility assays were conducted by stab inoculating 0.3% (wt/vol) PYE agar plates with isolated colonies and imaging every 24 hours for 72 hours. To study the effects of flagellin composition on filament supercoiling, molecular packing, and motility, we resolved the structures of native, non-straightened single flagellin filaments of C. crescentus. The helical reconstructions resulted in a ΔFljJLMNO (FljK only) flagellum structure with an 11-start angle of -2.14°, a ΔFljJKMNO (FljL only) flagellum structure with an 11-start angle of +1.82°, and a ΔFljJKLNO (FljM only) flagellum structure with an 11-start angle of +5.12°. The three reconstructions resulted in maps with FSC0.143 resolutions at or below 3.4 Å. Motility assays resulted in reduced motility for all strains when compared to the wild-type filament. We explored how the filament composition and, specifically, the molecular packing of flagellin subunits alters the filament architecture and its impact on motility by studying single flagellin filaments of C. crescentus. We confirm that no single flagellin strain is able of recover wild-type levels of motility, suggesting that the molecular composition of the filament is critical for “normal” swimming. Analysis of isolated flagellar filaments via cryo-EM and helical reconstruction provides insight into the energetically stable states of the flagellum and experimental evidence for comparison to the bi-stable state polymorphism model. We note that of the single flagellin filaments of C. crescentus, ΔFljJLMNO (FljK only) is the only structure with a negative 11-start angle and is closest to a “normal” based on Calladine’s polymorphism model, additionally this strain has the highest motility of the single flagellin strains. We propose that a filament that has an energetically stable state closest to a “normal” swimming filament may be better suited for swimming [11]. Helical reconstructions of single flagellin filaments. 1A) FljK reconstruction, 1B) FljL reconstruction, and 1C) FljM reconstruction of flagellar filaments. Red arrows denote glycosylation sites on surface exposed threonine residues. Scale bar of 50 Å. Flagellar filament models. 2A) FljK model, 2B) FljL model, and 2C) FljM model with individual flagellin monomers denoted in different colors. Scale bar of 50 Å.

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