Flotillin-mediated membrane fluidity controls peptidoglycan synthesis and MreB movement
- Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Netherlands
- Biozentrum, Ludwig-Maximilians-Universität München, Germany
- Institute for General Microbiology, Christian-Albrechts-University, Germany
- Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), IECB, CNRS, Université Bordeaux, Institut Polytechnique Bordeaux, France
- Department of Drug Design and Optimization (DDOP), Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI), Germany
- Department of Pharmacy, Saarland University, Germany
- Stratingh Institute for Chemistry, University of Groningen, Netherlands
- Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Netherlands
Figures
Figure 1 with 2 supplements
Accumulation of peptidoglycan synthesis and membrane material at division sites in a flotillin mutant.
(A) Morphology of the exponentially growing wild type (WT) and ΔfloAT strains labelled with HADA, fluorescent Vancomycin (Van-FL), FM 4–64, Nile Red, and DiI-C12. Scale bar: 5 μm. (B–F) Peak …
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Figure 1—source data 1
Fluorescence intensity and cell length measurements.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Control experiments showing that differences in septal labeling intensity are not due to microscopy settings, septum thickness, or dye diffusion.
(A) Morphology of the exponentially growing 4259 (WT-GFP) and ΔfloAT strains labelled simultaneously with Nile Red or FM 4–64 and HADA. (B) Septal peak intensity of FM4-64, Nile Red and HADA …
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Figure 1—figure supplement 1—source data 1
Growth data plotted in FS1F.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
Data plotted in F1F1Sb, F1FS1C, F1FS1.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig1-figsupp1-data2-v1.xlsx
Figure 1—figure supplement 2
Absence of flotillins does not affect expression, oligomerisation or localisation of PBPs.
(A) Expression pattern of PBPs in wild type and flotillin deficient strains visualised with Bocillin-FL. Membrane fractions isolated from cells (wt, ΔfloA, ΔfloT and ΔfloAT) in exponential and …
Figure 2 with 4 supplements
Cell morphology and cell wall synthesis localisation is dependent on growth conditions.
Morphology of the WT, ΔfloAT, Δpbp1, and ∆pbp1ΔfloAT strains grown in (A) rich (LB), (B) minimal (SMM) medium, and in (C) rich medium with membrane fluidising conditions (0.1% benzyl alcohol, …
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Figure 2—source data 1
Cell length measurements.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Deletion of both flotillins and PBP1 induces filamentation and delocalisation of peptidoglycan synthesis.
(A) Exponentially growing wt, Δpbp1 and Δpbp1ΔfloAT strains were labelled with Nile Red and Vancomycin-FL (Van-FL). Arrows indicate accumulation of the dye. (B) Exponentially growing ΔfloAT, Δpbp1Δfl…
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Figure 2—figure supplement 1—source data 1
Cell length measurements plotted in F2F1C.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Septum labelling of wild type and flotillin mutant cells grown on minimal medium.
(A, C) Morphology of the exponentially growing wt and ΔfloAT strains in the minimal medium labelled with fluorescent Vancomycin (Van-FL, (A) and Nile Red (C). Scale bar 5 μm. (B, D) Peak intensity …
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Figure 2—figure supplement 2—source data 1
Fluorescence intensity measurements plotted in F2FS2.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
Filamentation and delocalisation of peptidoglycan synthesis in the absence of flotillins and PBP1 is not rescued by the addition of magnesium.
Cell morphology of wt, ΔfloAT, ∆pbp1, and ∆pbp1ΔfloAT strains grown in LB supplemented with (A) 6 mM magnesium (Mg2+) or (B) 20 mM magnesium, labelled with Nile Red and Vancomycin-FL (Van-FL). …
Figure 2—figure supplement 4
Growth curves and growth rates show similar growth for wt, ΔfloAT, ∆pbp1, and ∆pbp1ΔfloAT (as well as ΔfloA, ΔfloT, ∆pbp1ΔfloA and ∆pbp1ΔfloT) strains grown on LB or on LB supplemented with BnOH (0.1% (w/v)).
Each datapoint represents the average from biological triplicates and error bars indicate standard deviation.
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Figure 2—figure supplement 4—source data 1
Growth curve data.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig2-figsupp4-data1-v1.xlsx
Figure 3 with 1 supplement
Flotillins increase overall membrane fluidity at high growth rate.
Changes in overall membrane fluidity were assessed by Laurdan microscopy in cells grown on LB (A), SMM (B) and LB+BnOH (C). Micrographs show colour-coded generalised polarisation (GP) maps in which …
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Figure 3—source data 1
GP measurement.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Fatty acid composition analysis.
(A, C) Ratios between chain lengths of the major fatty acids (C17 and C15) and ratios between the iso and anteiso forms of fatty acids of the exponentially growing wt, ∆floAT and ∆pbp1∆floAT strains …
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Figure 3—figure supplement 1—source data 1
Fatty acid composition data.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 6 supplements
MreB speed is linked to membrane fluidity.
(A) The MreB speed in different strain backgrounds and growth conditions was analysed by time-lapse TIRF microscopy. Scatter plot of the speed of patches obtained from individual tracks in 5 …
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Figure 4—source data 1
MreB patch mobility measurements determined by TIRFM.
- https://cdn.elifesciences.org/articles/57179/elife-57179-fig4-data1-v1.xlsx
Figure 4—video 1
Visualisation of xylose inducible mrfpRuby-MreB patches dynamics (strain 4070) during exponential growth in LB medium at 37°C by TIRF microscopy.
Exposure time was 2 s and frame rate 1 image/sec over 30 s. MreB patches rotate perpendicularly to the longitudinal cell axis with an average speed of 92.98 nm/s (+/- 21.37). This movie refers to …
Figure 4—video 2
Visualisation of xylose inducible mrfpRuby-MreB patches dynamics (strain 4070) during exponential growth in SMM medium at 37°C by TIRF microscopy.
Exposure time was 2 s and frame rate 1 image/sec over 30 s. MreB patches rotate perpendicularly to the longitudinal cell axis with an average speed of 58.09 nm/s (+/- 15.08). This movie refers to …
Figure 4—video 3
Visualisation of xylose inducible mrfpRuby-MreB in ΔfloAT patches dynamics (strain 4076) during exponential growth in LB medium at 37°C by TIRF microscopy.
Exposure time was 2 s and frame rate 1 image/sec over 30 s. MreB patches rotate perpendicularly to the longitudinal cell axis with an average speed of 41.59 nm/s (+/- 10.48). This movie refers to …
Figure 4—video 4
Visualisation of xylose inducible mrfpRuby-MreB in ΔfloAT patches dynamics (strain 4076) during exponential growth in SMM medium at 37°C by TIRF microscopy.
Exposure time was 2 s and frame rate 1 image/sec over 30 s. MreB patches rotate perpendicularly to the longitudinal cell axis with an average speed of 65.88 nm/s (+/- 15.68). This movie refers to …
Figure 4—video 5
Visualisation of xylose inducible mrfpRuby-MreB patches dynamics (strain 4070) during exponential growth in LB medium supplemented with BnOH (0.1%) at 37°C by TIRF microscopy.
Exposure time was 2 s and frame rate 1 image/sec over 30 s. MreB patches rotate perpendicularly to the longitudinal cell axis with an average speed of 79.00 nm/s (+/- 22.25). This movie refers to …
Figure 4—video 6
Visualisation of xylose inducible mrfpRuby-MreB in ΔfloAT patches dynamics (strain 4076) during exponential growth in LB medium supplemented with BnOH (0.1%) at 37°C by TIRF microscopy.
Exposure time was 2 s and frame rate 1 image/sec over 30 s. MreB patches rotate perpendicularly to the longitudinal cell axis with an average speed of 69.22 nm/s (+/- 25.03). This movie refers to …
Figure 5 with 1 supplement
Lipid ordering of FloT probed by 2H solid-state NMR.
(A) Wide-line 2H spectra of POPC-d31 liposomes with or without FloT at a lipid-to-protein molar ratio of 25:1 acquired at 298 K. (B) Effect of FloT on the C-2H order parameters of the PC acyl chain. …
Figure 5—figure supplement 1
31P solid-state NMR experiments of POPC liposomes with or without FloT at a lipid-to-protein molar ratio of 25:1.
All the spectra were acquired at 298 K and a Lorentzian line broadening of 50 Hz was applied before the Fourier transformation.
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3) | Thermo Fisher Scientific | EC0114 | Chemically competent cells |
| Strain, strain background (Bacillus subtilis) | BB001 | Bach and Bramkamp, 2013 | trpC2 yqfA::tet | |
| Strain, strain background (Bacillus subtilis) | BB003 | Bach and Bramkamp, 2013 | trpC2 yuaG::pMUTIN4 yqfA::tet | |
| Strain, strain background (Bacillus subtilis) | DB003 | Donovan and Bramkamp, 2009 | trpC2 yuaG::pMUTIN4 | |
| Strain, strain background (Bacillus subtilis) | RWBS5 | Domínguez-Escobar et al., 2011 | trpC2 amyE::spc Pxyl-mrfpruby-mreB | |
| Strain, strain background (Bacillus subtilis) | PS832 | Popham and Setlow, 1995 | Prototrophic revertant of 168 | |
| Strain, strain background (Bacillus subtilis) | 2082 | Scheffers et al., 2004 | trpC2 pbpD::cat Pxyl–gfp–pbpD 1–510 | |
| Strain, strain background (Bacillus subtilis) | 2083 | Scheffers et al., 2004 | trpC2 ponA::cat Pxyl–gfp–ponA 1–394 | |
| Strain, strain background (Bacillus subtilis) | 2085 | Scheffers et al., 2004 | trpC2 dacA::cat Pxyl–gfp–dacA 1–423 | |
| Strain, strain background (Bacillus subtilis) | 3105 | Scheffers et al., 2004 | trpC2 pbpC::cat Pxyl-gfp–pbpC 1–768 | |
| Strain, strain background (Bacillus subtilis) | 3122 | Scheffers et al., 2004 | trpC2 pbpB::cat Pxyl-gfp-pbpB 1–825 | |
| Strain, strain background (Bacillus subtilis) | 3511 | Scheffers and Errington, 2004 | trpC2 ponA::spc | |
| Strain, strain background (Bacillus subtilis) | 4042 | Lages et al., 2013 | trpC2 pbpA::cat Pxyl-mkate2-pbpA 1−804 | |
| Strain, strain background (Bacillus subtilis) | 4056 | Morales Angeles et al., 2017 | trpC2 dacA::kan | |
| Strain, strain background (Bacillus subtilis) | 4059 | This work | trpC2 dacA::cat Pxyl-gfp–dacA 1–423yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4064 | This work | trpC2 dacA::kan yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4090 | This work | trpC2 ponA::spc yuaG::pMUTIN4 | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4091 | This work | PS832 ponA::spc yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4092 | This work | trpC2 ponA::spc yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4095 | This work | trpC2 ponA::cat Pxyl-gfp–ponA 1–394 yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4099 | This work | trpC2 pbpB::cat Pxyl-gfp-pbpB 1–825yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4102 | This work | trpC2 pbpA::cat Pxyl-mkate2-pbpA 1−804 yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4108 | This work | trpC2 pbpD::cat Pxyl-gfp–pbpD 1–510yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4122 | This work | trpC2 pbpC::cat Pxyl-gfp–pbpC 1–768yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4128 | This work | trpC2 ponA::spc pbpD::cat Pxyl-gfp–pbpD 1–510 | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4129 | This work | trpC2 ponA::spc yuaG::pMUTIN4 pbpD::cat Pxyl-gfp–pbpD 1–510 | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4070 | This work | trpC2 mreB::kan amyE::spc Pxyl-mrfpruby-mreB | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4076 | This work | trpC2 mreB::kan amyE::spc Pxyl-mrfpruby-mreB yuaG::pMUTIN4 yqfA::tet | Scheffers lab |
| Strain, strain background (Bacillus subtilis) | 4259 | This work; Veening et al., 2009 | trpC2 amyE::PrrnB-gfp | Scheffers lab |
| Other | Bocillin | Thermo Fisher Scientific | BOCILLIN FL Penicillin, Sodium Salt | 5 µg/ml |
| Other | HADA | Synthesised as described (Morales Angeles et al., 2017) | 7-hydroxycoumarin 3-carboxylic acid-amino-D-alanine | 50 µM |
| Other | Vancomycin-FL | Sigma-Aldrich and Molecular Probes (Zhao et al., 2017) | Van-FL | 1:1 mixture of Vancomycin and BODIPYFL Vancomycin (Zhao et al., 2017), final concentration 1 µg/ml |
| Other | Nile Red | Thermo Fisher Scientific | 5H-Benzo[α]phenoxazin-5-one, 9-(diethylamino)- 7385-67-3 | 0.5 µg/ml |
| Other | 16:0-d31-18:1 PC | Avanti | 860399 | Phospholipids |
| Other | Laurdan | Sigma-Aldrich | 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine | - |
| Other | Benzyl alcohol | Sigma-Aldrich | Benzyl alcohol | - |
| Other | DiI-C12 | Thermo Fisher Scientific | 1,1'-Didodecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate | 2.5 µg/ml |
| Other | FM4-64 | Thermo Fischer Scientific | (N-(3-Triethylammoniumpropyl)−4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide) | 0.5 µg/ml, Invitrogen FM 4–64 Dye |
| Software, algorithm | Prism 5 | 1992–2010 GraphPad Software | RRID:SCR_002798 | - |
| Software, algortithm | ImageJ 1.52p/FIJI | Wayne Rasband – National Institutes of Health, USA | RRID:SCR_002285 | Free software |
| Software, algorithm | SPSS | SPSS | RRID:SCR_002865 | software |
| Software, algorithm | NMR Depaker 1.0rc1 software | [Copyright (C) 2009 Sébastien Buchoux] | software | |
| Software, algorithm | Bruker Topspin 3.2 software | Bruker | RRID:SCR_014227 | software |
