γ-proteobacteria eject their polar flagella under nutrient depletion, retaining flagellar motor relic structures

Abstract

Bacteria switch only intermittently to motile planktonic lifestyles under favorable conditions. Under chronic nutrient deprivation, however, bacteria orchestrate a switch to stationary phase, conserving energy by altering metabolism and stopping motility. About two-thirds of bacteria use flagella to swim, but how bacteria deactivate this large molecular machine remains unclear. Here, we describe the previously unreported ejection of polar motors by γ-proteobacteria. We show that these bacteria eject their flagella at the base of the flagellar hook when nutrients are depleted, leaving a relic of a former flagellar motor in the outer membrane. Subtomogram averages of the full motor and relic reveal that this is an active process, as a plug protein appears in the relic, likely to prevent leakage across their outer membrane; furthermore, we show that ejection is triggered only under nutritional depletion and is independent of the filament as a possible mechanosensor. We show that filament ejection is a widespread phenomenon demonstrated by the appearance of relic structures in diverse γ-proteobacteria including Plesiomonas shigelloides, Vibrio cholerae, Vibrio fischeri, Shewanella putrefaciens, and Pseudomonas aeruginosa. While the molecular details remain to be determined, our results demonstrate a novel mechanism for bacteria to halt costly motility when nutrients become scarce.

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

“…Although they explained this with a cell membrane breakage event, perhaps these disassembly products resulted from a ‘break in the rod’, as we observed and according to our proposed model. Furthermore, we established that flagellar loss is less affected by the newly produced substances in the stationary phase, but is more dependent on glucose in the medium, thus agreeing with a previous result [39]. In terms of pathogen-host interaction, whether the loss of flagella due to nutrient depletion would intensify or attenuate host immune response warrants continued study.…”

“…Recently, work using electron micrograph revealed that the C. crescentus disassembly product does not include the C-ring, MS-ring, and export apparatus [52]. However, Ferreira et al [39] showed the ejected flagella in 5 γ-proteobacteria would leave relic structures composed of the P-, L-, H-, and T-rings and the basal disk in the outer membrane. All these findings came from biochemical and electron microscopic studies but lacked dynamic imaging of the disassembly process.…”

“…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].…”

In situ imaging of the bacterial flagellar motor disassembly and assembly processes

“…Our experiments (mass spectrometry of isolated membrane fractions and negative-stain electron microscopy) revealed that the flagellar disassembly process happens more frequently at high OD 600 values where the cells enter the stationary phase due to low nutrient levels in the environment. While this manuscript was under review, an independent study appeared describing a similar observation in other Gammaproteobacteria species (Ferreira et al, 2019), suggesting that this phenomenon is ubiquitous in this class of bacteria and raising the question whether a similar behavior is present in other bacterial classes. We also explored the process by which the outer membrane becomes sealed after flagellar disassembly and provided a list of a handful of candidate proteins that might be involved in this process.…”

A guided approach for subtomogram averaging of challenging macromolecular assemblies

“…Large, eukaryotic and bacterial membrane-associated complexes have also benefited greatly from this technique, as alignment of the relatively high-signal membrane bilayer can help drive the initial alignment of the more noisy, low SNR complex of interest. Examples of these complexes include COPI/II vesicle coats 34,84,85 , bacterial secretion systems and flagellar motors [86][87][88][89][90][91][92][93][94][95][96][97][98] .…”