In Situ Structures of Polar and Lateral Flagella Revealed by Cryo-Electron Tomography

Abstract

The bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, including Pseudomonas aeruginosa, Vibrio spp., and Salmonella enterica. The bacterial flagellum has been studied extensively in the model systems Escherichia coli and Salmonella enterica serovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determine in situ structures of polar flagella in P. aeruginosa and peritrichous flagella in S. Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in both P. aeruginosa and S. Typhimurium. IMPORTANCE The bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolution in situ structures of polar flagella in Pseudomonas aeruginosa and peritrichous flagella in Salmonella enterica serovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in both P. aeruginosa and S. Typhimurium.

Dynamic production and loss of flagellar filaments during the bacterial life cycle

“…As for the flagellar basal structure, it is composed of the ring structures, which are C-ring, MS-ring, and LP-ring, and the rod which penetrates and connects the rings [32-34]. MS-ring, which is composed of ca.…”

“…C-ring, which is composed of three kind proteins, FliG, FliM and FliN, is assembled beneath MS-ring to interact N-terminal domain of FliG and the C-terminal domain of FliF [36, 37]. The flagellar outer membrane complexes, which seems to contain LP-ring, and many incomplete flagellar subassemblies have been observed by cryo-electron tomography in various bacteria [32, 38, 39]. The polar flagellum of Vibrio alginolyticus has extra ring structures around LP-ring, called T-ring and H-ring [33, 40].…”

Taming the flagellar motor of pseudomonads with a nucleotide messenger

“…Historically, pseudomonads were not used as model organisms for studying the structure and function of flagellar motors. The overall architecture of the flagellar motor complex for pseudomonads was unknown till very recently (Kaplan et al, 2019;Zhu et al, 2019;Fig. 1), with the high-resolution structure of many individual protein components and sub-complexes still missing today.…”

“…For example, one ancillary proteins (MotY) forms the additional 'T-ring'-like structure and another one forms the 'H-ring' structure ( Fig. 1; Kaplan et al, 2019;Zhu et al, 2019). The stator complex of P. aeruginosa contains additional starter units and is wider than the stator complexes of E. coli and S. Typhimurium, which presumably enables P. aeruginosa to generate greater torque for traversing through more viscous media (Kuchma et al, 2010).…”

“…Because P. aeruginosa relies on the MotC/D stator units for generating high torque to swarm on hydrated and viscous semisolid surfaces (Doyle et al, 2004;Toutain et al, 2005;Hook et al, 2019), disengagement of the MotC/D units would lead to fewer engaged stator units, and thus, loss of motor torque and speed. Hence, the FlgZmediated mechanism provides a molecular explanation for the repressing effect of c-di-GMP on swarming motility The drawing is based on the data obtained from the Cryo-electron tomographic studies (Kaplan et al, 2019;Zhu et al, 2019). [Color figure can be viewed at wileyonlinelibrary.com] and the role of c-di-GMP in promoting the motile-tosessile transition.…”