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

Bacterial flagella are large extracellular protein organelles that drive bacteria motility and taxis in response to environmental changes. Previous research has focused mostly on describing the flagellar assembly, its rotation speed and power output. However, whether flagella are permanent cell structures and, if not, the circumstances and timing of their production and loss during the bacterial life cycle remain poorly understood. Here we used the single polar flagellum of Vibrio alginolyticus as our model and, using in vivo fluorescence imaging, revealed that the percentage of flagellated bacteria (PFB) in a population varies substantially across different bacterial growth phases. In the early-exponential phase, the PFB increases rapidly in respect to incubation time, mostly through widespread flagella production. In the mid-exponential phase, the PFB peaks at around 76% and the partitioning of flagella between the daughter cells is 1:1 and strictly at the old poles. After entering the stationary phase, the PFB starts to decline, mainly because daughter cells stop making new flagella after cell division. Interestingly, we discovered that bacteria can actively abandon flagella after prolonged stationary culturing, though cell division has long been suspended. Lack of glucose was found to be a major factor promoting flagellar disassembly. We also revealed that the active loss of flagella was initiated by breakage in the rod connecting the extracellular filament to the basal body formed by MS- and C-rings. Our results highlight the dynamic production and loss of flagellar filaments during the bacterial life cycle.


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Bacterial flagella are large extracellular protein organelles that drive bacteria motility 24 and taxis in response to environmental changes. Previous research has focused mostly 25 on describing the flagellar assembly, its rotation speed and power output. However, 26 whether flagella are permanent cell structures and, if not, the circumstances and timing 27 of their production and loss during the bacterial life cycle remain poorly understood. 28 Here we used the single polar flagellum of Vibrio alginolyticus as our model and, using 29 in vivo fluorescence imaging, revealed that the percentage of flagellated bacteria (PFB) 30 in a population varies substantially across different bacterial growth phases. In the 31 early-exponential phase, the PFB increases rapidly in respect to incubation time, mostly 32 through widespread flagella production. In the mid-exponential phase, the PFB peaks 33 at around 76% and the partitioning of flagella between the daughter cells is 1:1 and 34 strictly at the old poles. After entering the stationary phase, the PFB starts to decline, 35 mainly because daughter cells stop making new flagella after cell division. Interestingly, 36 we discovered that bacteria can actively abandon flagella after prolonged stationary 37 culturing, though cell division has long been suspended. Lack of glucose was found to 38 be a major factor promoting flagellar disassembly. We also revealed that the active loss    the subsequent bacterial growth was in its early-exponential phase (0-3 hrs of 113 incubation), the PFB increased dramatically ( Figure 1B), peaking at 76% in the mid-114 exponential phase (3-3.67 hrs of incubation). As we continued to culture the bacteria, 115 the PFB started to decline after reaching the late-exponential phase (5 hrs of incubation).

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Once the cells began the stationary phase, the PFB dropped sharply and finally returned 117 to about 10% ( Figure 1B). We confirmed the flagella lost from cell in the late-118 exponential phase to detect the filament proteins (Fig. S1).

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To further understand the dynamics of flagellar production and loss, we plotted the 120 PFB versus cell concentration ( Figure 1C) and calculated the flagellated cell 121 concentration (cell OD × PFB) with respect to the total cell concentration (cell OD), as 122 shown in Figure 1D. Our findings indicated that in the early-exponential phase, many  In the early-exponential phase flagella are widely produced 144 Following our finding that the PFB increased rapidly in the early-exponential 145 phase of bacterial growth, we examined the mechanism underlying these dynamic PFB          Figure 4A, the PFB in a medium with no glucose decreased rapidly to 5% in 5 hrs.

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However, the addition of 5 mM glucose slowed down the PFB drop to 30% in 5 hrs and  To differentiate these 2 possible mechanisms, we traced the movement of EGFP-306 FliG clusters with time-lapse imaging at 10 secs intervals during flagellar loss.
307 Surprisingly, we saw that many polar, localized FliG clusters suddenly moved ( Figure   308 6A). We described this movement using a custom computer program developed to track 309 FliG cluster trajectory (see Material and Methods). Interestingly, moving FliG clusters 310 were found to travel along the periphery, not the center, of bacterial cells ( Figure 6B).

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Next, we measured the distances between mobile FliG clusters and the edges of 312 bacterial cells. The distance distribution was found to be similar to that of localized 313 FliG clusters to the edges of bacterial cells, suggesting that these mobile FliG clusters 314 were moving on the cells' inner membranes ( Figure 6C). This finding also implied that 315 flagellar loss starts with detachment, rather than destruction, of the C-ring complex.       Fig. S1. Detection of the filament protein in the culture supernatant. VIO5 cells were grown in VC medium and 1 ml culture was aliquoted at indicated time. Cells were precipitated by centrifugation, and then culture supernatant were ultracentrifuged. Both pellets (low speed spin for "whole cell lysate" and ultracentrifugation for "culture supernatant") were suspended to the normalized volume (equivalent to OD660 of 10) and analyzed by SDS-PAGE followed by immunoblotting.