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

Abstract

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.

Keywords: assembly; bacterial flagellar motor; disassembly; electron cryo-tomography; in situ imaging.

Figure 1. Stable PL sub‐complexes in three bacterial species imaged by ECT

1. A–O

   (A, F, K) slices through electron cryo‐tomograms of L. pneumophila, P. aeruginosa, and S. oneidensis cells, respectively, highlighting a PL sub‐complex in the outer membrane (dashed yellow circle). (B, G, L) Enlarged views of the sub‐complexes. (C, H, M) Sub‐tomogram averages of PL sub‐complexes from each species. (D, I, N) Schematic representations of the sub‐tomogram averages with different rings colored and labeled. (E, J, O) Sub‐tomogram averages of fully assembled flagella from each species for comparison. Scale bars: (black) 50 nm, (orange) 25 nm, and (red) 20 nm.

Figure 2. Flagellar sub‐complexes in L. pneumophila

1. A

   Sub‐tomogram average of the IM sub‐complex constituting the C‐ ring, MS‐ring, and export apparatus.

2. B

   Schematic representation of the sub‐tomogram average shown in (A) highlighting the different parts of the complex.

3. C

   Sub‐tomogram average of the motors of fully assembled flagella.

4. D–J

   Slices through electron cryo‐tomograms showing neighboring PL and IM sub‐complexes. Dashed yellow circles highlight the IM sub‐complex, while dashed blue arrows highlight the PL sub‐complex. Dashed red lines mark the border between two images used to make a composite image when the PL and IM sub‐complexes were found at different levels in the tomogram.

5. K

   Schematic representation of an IM sub‐complex in the vicinity of a PL sub‐complex.

6. L, M

   Slices through electron cryo‐tomograms of L. pneumophila highlighting the presence of a fully assembled basal body lacking the hook and the filament.

7. N

   Schematic representation of the basal bodies in (L and M).

8. O

   Central slice through an electron cryo‐tomogram of a lysed cell. The dashed yellow circle highlights the flagellar motor.

9. P

   Enlarged view of the same slice shown in (O). The absence of the C‐ring and the export apparatus is highlighted by the dashed orange arrow.

10. Q

    Schematic representation of the complex found in (P).

Data information: Scale bars: (A, C) 20 nm, (D‐J, L, M, and P) 25 nm, and (O) 100 nm.

Figure 3. Flagellar sub‐complexes in P. aeruginosa

1. A–C

   Slices through electron cryo‐tomograms showing fully assembled motors without the hook and filament. The dashed yellow circles indicate the IM sub‐complex.

2. D

   Schematic representation of the P. aeruginosa motors lacking the hook and filament shown in (A–C).

3. E–G

   Slices through electron cryo‐tomograms showing fully assembled motors with the hook and lacking the filament.

4. H

   Schematic representation of the motors with the hook shown in (E–G).

5. I–K

   Slices through electron cryo‐tomograms of intact P. aeruginosa ΔflgI cells showing the presence of the inner‐membrane sub‐complex with the rod.

6. L

   Schematic representation of the inner‐membrane sub‐complex with the rod shown in (I‐K).

7. M–O

   Slices through electron cryo‐tomograms of lysed P. aeruginosa ΔflgI cells showing the presence of the sub‐complex constituting the MS‐ring and the rod.

8. P

   Schematic representation of the sub‐complex described in (M–O).

9. Q

   A slice through an electron cryo‐tomogram of a P. aeruginosa ΔflgG cell highlighting the presence of the inner‐membrane sub‐complex.

10. R

    Schematic representation of the inner‐membrane sub‐complex shown in (Q).

Data information: All scale bars 25 nm.

Figure 4. Flagellar sub‐complexes in S. oneidensis wild‐type and mutant cells

1. A–C

   Slices through electron cryo‐tomograms of wild‐type cells showing fully assembled motors without the hook and filament.

2. D

   Schematic representation of the motors lacking the hook and filament shown in (A–C).

3. E

   Sub‐tomogram average of the motors of fully assembled flagella.

4. F

   Slice through an electron cryo‐tomogram of a ΔflgH cell showing an IM sub‐complex, indicated by the dashed yellow circle.

5. G

   Schematic representation of the IM sub‐complex shown in (F).

6. H, I

   Slices through electron cryo‐tomograms of ΔflgH cells showing the IM sub‐complex with the rod and the P‐ring, indicated by the dashed yellow circles.

7. J

   Schematic representation of the structures shown in (H and I).

8. K–M

   Slices through electron cryo‐tomograms of ΔflaA/B cells highlighting the flagellar motor and the hook (without the filament).

9. N

   Sub‐tomogram average of the flagellar motor and the hook structure found in ΔflaA/B cells.

10. O

    Schematic representation of the sub‐tomogram average shown in (N).

11. P–R

    Slices through electron cryo‐tomograms of ΔflaA/B cells illustrating a disassembly product constituting the PL sub‐complex, the rod, and the hook.

12. S

    Sub‐tomogram average of the disassembly complex shown in (P–R). The dashed orange arrow indicates the absence of the IM sub‐complex in this structure.

13. T

    Schematic representation of the disassembly product shown in (P–S).

14. U–W

    Slices through electron cryo‐tomograms of ΔflaA/B cells highlighting PL sub‐complexes (dashed yellow circles).

15. X

    Sub‐tomogram average of the PL sub‐complexes in ΔflaA/B cells.

16. Y

    Schematic representation of the sub‐tomogram average shown in (X).

Data information: Scale bars: (red) 25 nm, (orange and white) 20 nm.

Figure 5. Summary of observations in the three bacterial species investigated in this study

1. A–C

   Schematic representations of the various assembly and disassembly sub‐complexes observed in this study in L. pneumophila, P. aeruginosa, and S. oneidensis (wild‐type and mutant strains), respectively. The blue shaded area in (A) represents an intact IM sub‐complex in the vicinity of a PL sub‐complex. Note that the boundaries between the different parts in these schematics are tentative.