Flotillin-mediated membrane fluidity controls peptidoglycan synthesis and MreB movement.

*For correspondence: [email protected] (MB); [email protected] (D-JS) † These authors contributed equally to this work Competing interests: The authors declare that no competing interests exist. Funding: See page 18 Received: 24 March 2020 Accepted: 12 June 2020 Published: 14 July 2020 Reviewing editor: Taˆm Mignot, CNRS-Aix Marseille University, France Copyright Zielin´ ska et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Flotillin-mediated membrane fluidity controls peptidoglycan synthesis and MreB movement Aleksandra Zielin ´ska 1† , Abigail Savietto 2,3† , Anabela de Sousa Borges 1 , Denis Martinez 4 , Melanie Berbon 4 , Joe ¨l R Roelofsen 1 , Alwin M Hartman 5,6,7 , Rinse de Boer 8 , Ida J Van der Klei 8 , Anna KH Hirsch 5,6,7 , Birgit Habenstein 4 , Marc Bramkamp 2,3 *, Dirk-Jan Scheffers 1 * 1 Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands; 2 Biozentrum, LudwigMaximilians-Universita ¨t Mu ¨nchen, Mu ¨nchen, Germany; 3 Institute for General Microbiology, Christian-Albrechts-University, Kiel, Germany; 4 Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), IECB, CNRS, Universite´ Bordeaux, Institut Polytechnique Bordeaux, Pessac, France; 5 Department of Drug Design and Optimization (DDOP), Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI), Saarbru ¨cken, Germany; 6 Department of Pharmacy, Saarland University, Saarbru ¨cken, Germany; 7 Stratingh Institute for Chemistry, University of Groningen, Groningen, Netherlands; 8 Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands Abstract The bacterial plasma membrane is an important cellular compartment. In recent years it has become obvious that protein complexes and lipids are not uniformly distributed within membranes. Current hypotheses suggest that flotillin proteins are required for the formation of complexes of membrane proteins including cell-wall synthetic proteins. We show here that bacterial flotillins are important factors for membrane fluidity homeostasis. Loss of flotillins leads to a decrease in membrane fluidity that in turn leads to alterations in MreB dynamics and, as a consequence, in peptidoglycan synthesis. These alterations are reverted when membrane fluidity is restored by a chemical fluidizer. In vitro, the addition of a flotillin increases membrane fluidity of liposomes. Our data support a model in which flotillins are required for direct control of membrane fluidity rather than for the formation of protein complexes via direct protein-protein interactions. Introduction The shape of a bacterium is predominantly defined by the structure of its peptidoglycan. Although there is a great variety in bacterial shapes, the overall chemistry of peptidoglycan is very similar between bacteria and thus the shape of peptidoglycan is primarily determined by the temporal and spatial regulation of peptidoglycan synthesis. In rod-shaped bacteria, peptidoglycan synthesis is thought to be mediated by two protein assemblies, the elongasome and the divisome, that synthesise peptidoglycan along the long axis and across the division plane of the cell, respectively (Typas et al., 2012;Zhao et al., 2017). These complexes contain a set of proteins required for the final steps of synthesis and translocation of the peptidoglycan precursor, LipidII, from the inner to the outer leaflet of the cytoplasmic membrane, and proteins that incorporate LipidII into peptidoglycan. These include SEDS (Shape, Elongation, Division and Sporulation) proteins that can perform Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 1 of 21 RESEARCH ARTICLE glycosyl transferase reactions (Cho et al., 2016;Meeske et al., 2016;Taguchi et al., 2019), and Penicillin Binding Proteins (PBPs) that are divided in class A PBPs (aPBPs) that catalyse both glycosyl transferase and transpeptidase reactions, class B PBPs (bPBPs) that only catalyse transpeptidase reactions and low molecular weight PBPs that modify peptidoglycan, as well as hydrolases (Zhao et al., 2017;Morales Angeles and Scheffers, 2017). Coordination of these complexes is linked to cytoskeletal elements, MreB (-like proteins) for the elongasome and FtsZ for the divisome. In models, the cytoplasmic membrane is often depicted as a passive environment in which these machineries are embedded. However, it is becoming clear that the structure of the membrane plays a critical role in the coordination of peptidoglycan synthesis (Strahl and Errington, 2017). Inward membrane curvature serves as a localisation trigger for MreB and the elongasome, and enhanced local synthesis at bulges straightens out the membrane sufficient to convert spherical cells to a rod shape (Hussain et al., 2018;Ursell et al., 2014). In Bacillus subtilis, the motion of MreB along the membrane is associated with elongasome activity (Domı´- nguez-Escobar et al., 2011;Garner et al., 2011), and the velocity of MreB patches is related to growth rate (Billaudeau et al., 2017), indicating that MreB motion can be used as a marker for elongasome activity. Interestingly, MreB localises to and organises regions of increased membrane fluidity (RIF) (Strahl et al., 2014), which in turn is linked to the presence of LipidII, which favours a more fluid membrane and promotes local membrane disorder (Ganchev et al., 2006;Witzke et al., 2016). Inhibition of LipidII synthesis by genetic or chemical means results in a dissolution of membrane structures observed with the dye FM 4–64 and release of MreB from the membrane (Domı´- nguez-Escobar et al., 2011;Garner et al., 2011;Muchova ´et al., 2011;Schirner et al., 2015). Next to RIFs, membrane regions of decreased fluidity have been identified in bacteria (Strahl and Errington, 2017;Bramkamp and Lopez, 2015;Lopez and Koch, 2017). These so-called functional membrane microdomains (FMMs) are thought to be organised by the bacterial flotillin proteins, are enriched in isoprenoid lipids (Garcı´a-Ferna ´ndez et al., 2017;Lo ´pez and Kolter, 2010), and can be found in so-called Detergent Resistant Membrane (DRM) fractions of the membrane. Since the formulation of the FMM hypothesis, FMMs have been linked to many processes, such as protein secretion, biofilm formation, competence and cell morphology (Mielich-Su ¨ss and Lopez, 2015;MielicheLife digest Every living cell is enclosed by a flexible membrane made of molecules known as phospholipids, which protects the cell from harmful chemicals and other threats. In bacteria and some other organisms, a rigid structure known as the cell wall sits just outside of the membrane and determines the cell’s shape. There are several proteins in the membrane of bacteria that allow the cell to grow by assembling new pieces of the cell wall. To ensure these proteins expand the cell wall at the right locations, another protein known as MreB moves and organizes them to the appropriate place in the membrane and controls their activity. Previous studies have found that another class of proteins called flotillins are involved in arranging proteins and phospholipid molecules within membranes. Bacteria lacking these proteins do not grow properly and are unable to maintain their normal shape. However, the precise role of the flotillins remained unclear. Here, Zielin´ ska, Savietto et al. used microscopy approaches to study flotillins in a bacterium known as Bacillus subtilis. The experiments found that, in the presence of flotillins, MreB moved around the membrane more quickly (suggesting it was more active) than when no flotillins were present. Similar results were observed when bacterial cells lacking flotillins were treated with a chemical that made membranes more ‘fluid’ – that is, made it easier for the molecules within the membrane to travel around. Further experiments found that flotillins allowed the phospholipid molecules within an artificial membrane to move around more freely, which increases the fluidity of the membrane. These findings suggest that flotillins make the membranes of bacterial cells more fluid to help cells expand their walls and perform several other processes. Understanding how bacteria control the components of their membranes will further our understanding of how many currently available antibiotics work and may potentially lead to the design of new antibiotics in the future. Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 2 of 21 Research article Cell Biology Microbiology and Infectious Disease Su ¨ss et al., 2013;Bach and Bramkamp, 2013;Dempwolff et al., 2012). Cell morphology defects are linked to cell wall synthesis, and analysis of the protein content of Bacillus subtilis DRMs identified several PBPs, MreC and other proteins involved in cell wall metabolism as well as the two flotillins, FloA and FloT (Lo ´pez and Kolter, 2010;Bach and Bramkamp, 2013;Yepes et al., 2012). FloA is constitutively expressed, whereas FloT is expressed primarily during stationary growth, cell wall stress and sporulation (Schneider et al., 2015a;Huang et al., 1999;Nicolas et al., 2012). Super resolution microscopy showed that the flotillins and other proteins found in DRMs do not colocalise and have different dynamics (Dempwolff et al., 2016), so it is unlikely that FMMs are regions in the membrane that offer a favourable environment in which these membrane proteins are continuously present and active. Recently, the hypothesis has been put forward that FMMs/flotillins form a platform for the formation of functional protein oligomers, as work in Staphylococcus aureus showed that multimerisation of Type 7 secretion systems and PBP2a depends on FMMs (Lopez and Koch, 2017;Garcı´a-Ferna ´ndez et al., 2017;Mielich-Su ¨ss et al., 2017). Here, we have analysed the role of flotillins in peptidoglycan synthesis in B. subtilis. Our results show that, at high growth rates, flotillins control membrane fluidity in a manner that is critical for peptidoglycan synthesis and MreB dynamics, but have no effect on PBP oligomerisation. This results in a new model for flotillin function in the physical organisation of membranes during fast growth. Results Absence of flotillins shifts peptidoglycan synthesis to division Septa In previous studies, a double deletion of floA/floT was either reported to suffer severe shape defects and perturbed membrane structure (Dempwolff et al., 2012), or to not have strong shape defects but with a change in the overall lipid ordering of the membrane (Bach and Bramkamp, 2013). We grew wild type and DfloAT strains and analysed exponentially growing cells. We did not observe striking shape defects but did see an increase in median cell length and distribution of cell lengths in the absence of flotillins (Figure 1A,G). To look at effects on peptidoglycan synthesis, we labelled cells with HADA, a fluorescent D-Alanine analogue that reports on sites of active peptidoglycan synthesis (Kuru et al., 2012), and with fluorescent vancomycin (Van-FL), which labels LipidII and peptidoglycan containing pentapeptide side chains (Daniel and Errington, 2003;Morales Angeles et al., 2017). This revealed a significant accumulation of peptidoglycan synthesis stains at division septa in the DfloAT strain (Figure 1A–C). To look at membrane structure, cells were labelled with FM4-64, Nile-Red and DiI-C12, which are lipid dyes that accumulate in zones enriched in fluid lipids (Strahl et al., 2014). Again, the stains accumulated at the septa in the DfloAT strain, which also showed some accumulation of FM4-64 and DiI-C12 in patches, suggesting that the more fluid regions of the membrane are coalescing into larger regions (Figure 1A,D–F). The HADA, FM4-64 and Nile-Red measurements were repeated using a wild type strain expressing endogenous GFP, allowing simultaneous imaging of both strains on the same slide, and gave similar results, confirming that the observed signal increase is not due to variation between microcopy experiments (Figure 1— figure supplement 1A,B). In this mixed-strain experiment, Nile-Red labelling at the lateral membrane was the same between wild type and DfloAT strains, indicating that there is no difference in dye diffusion between the strains (Figure 1—figure supplement 1D). Inspection of the septa by electron microscopy revealed that there was no difference between the thicknesses of the septa between the wild type and DfloAT strain, ruling out that the increase in signal was due to formation of thicker septa (Figure 1—figure supplement 1C). The shift of peptidoglycan synthesis to the division site could hint at stress in the overall peptidoglycan synthesis route. This was confirmed by growing cells at a sublethal concentration of fosfomycin, which limits synthesis of LipidII (Kahan et al., 1974), but that does not impact growth rate at the concentration used. This resulted in bulging cells and some lysis, which was exacerbated in the DfloAT strain (Figure 1—figure supplement 1F,G). It should be noted that the peptidoglycan synthesis stress caused by fosfomycin is not the same as the stress caused by the absence of flotillins, as the phenotypes of wild type cells with sublethal fosfomycin are quite distinct from DfloAT cells without fosfomycin. We ruled out that the peptidoglycan synthesis stress was caused by a change in the folding or complex formation by PBPs in the absence of flotillins, as there were no differences in the overall PBP-profiles of Bocillin-FL labelled wild type or flotillin deletion strains (Figure 1—figure Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 3 of 21 Research article Cell Biology Microbiology and Infectious Disease Figure 1. Accumulation of peptidoglycan synthesis and membrane material at division sites in a flotillin mutant. (A) Morphology of the exponentially growing wild type (WT) and DfloAT strains labelled with HADA, fluorescent Vancomycin (Van-FL), FM 4–64, Nile Red, and DiI-C12. Scale bar: 5 mm. (B–F) Peak intensity of HADA (B), Van-FL (C), Nile Red (D), FM4-64 (E) and DiI-C12 (F) labelled division sites of the cells shown in (A). Cells from each strain (n 100, except E, n = 60) were analysed using the ObjectJ macro tool PeakFinder followed by statistical analysis with Prism. Significant differences are based on the two-tailed Mann-Whitney test (*p<0.05; **p<0.01). (G) Distribution of the cell length of the strains analysed in (A). Statistical analysis of the data (n = 100, two tailed Mann-Whitney test, *p<0.05) was performed with Prism, resulting in box plot graphs. The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Fluorescence intensity and cell length measurements. Figure supplement 1. Control experiments showing that differences in septal labeling intensity are not due to microscopy settings, septum thickness, or dye diffusion. Figure supplement 1—source data 1. Growth data plotted in FS1F. Figure supplement 1—source data 2. Data plotted in F1F1Sb, F1FS1C, F1FS1. Figure 1 continued on next page Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 4 of 21 Research article Cell Biology Microbiology and Infectious Disease supplement 2A). PBP complex formation was analysed using a combination of Native-PAGE and SDS-PAGE with Bocillin-labelled membrane fractions (Trip and Scheffers, 2016) and showed that various PBPs can be found in a high-MW complex (notably PBPs 1, 2, 3 and 4), but that complex formation is similar in the DfloAT strain (Figure 1—figure supplement 2B). Also, none of the five functional GFP-PBPs examined changed their localisation in the DfloAT strain (Figure 1—figure supplement 2C). Overall, the data suggest that in the absence of flotillins, peptidoglycan synthesis is affected and relatively increased at division septa, with a concomitant accumulation of membrane dyes that are indicative of higher membrane fluidity. The absence of both flotillins and PBP1 causes a severe phenotype, linked to a loss of membrane fluidity We reasoned that a non-lethal defect in septal peptidoglycan synthesis could reveal more about the role of flotillins and constructed a flotillin mutant that lacks PBP1, a bifunctional glycosyl transferase/ transpeptidase that is required for efficient cell division (Scheffers and Errington, 2004). Simultaneous deletion of pbp1,floA, and floT resulted in strong filamentation and delocalisation of peptidoglycan synthesis as well as membrane dyes to patches (Figure 2A,Figure 2—figure supplement 1A). Deletion of single flotillin genes and PBP1 had similar, albeit less severe effects (Figure 2—figure supplement 1B,C). To exclude the possibility that an alteration of peptidoglycan modification resulted in the delocalisation of HADA and Van-FL, we used D-Alanine-D-Propargylglycine (D-Ala-DPra), a clickable dipeptide analogue which is exclusively incorporated into peptidoglycan via LipidII (Sarkar et al., 2016). D-Ala-D-Pra incorporation was delocalised in the Dpbp1DfloAT strain, indicating that peptidoglycan synthesis itself is delocalised (Figure 2—figure supplement 1D). So far, our experiments were done with fast growing cells and Lysogeny Broth (LB) as the growth medium. Strikingly, none of the mutant strains had an apparent phenotype when cultivated in Spizizen’s minimal medium (SMM, Figure 2B), and peptidoglycan synthesis and lipid dyes were no longer accumulating at division sites in the DfloAT strain (Figure 2—figure supplement 2). SMM has a higher Mg 2+ concentration, which is known to rescue various cell shape mutations by inhibition of cell wall hydrolysis (Dajkovic et al., 2017). However, the increase in Mg 2+ was not sufficient to explain the reversal of phenotype as cells grown on LB supplemented with Mg 2+ (6 mM, concentration in SMM, or 20 mM) still displayed the elongated phenotype with delocalised peptidoglycan synthesis (Figure 2— figure supplement 3). This indicated that the phenotypes associated with the absence of flotillins are growth-rate and/or nutrient related. Next, we determined lipid packing order in the different strains using the fluorescent dye Laurdan, a reporter for flotillin-mediated lipid ordering (Bach and Bramkamp, 2013). LB-grown cells lacking flotillins displayed an increased generalised polarisation (GP) (Bach and Bramkamp, 2013), indicative of an overall increase in ordered lipid packing in the membrane, but the effect of flotillins on membrane ordering completely disappeared when cells were grown on SMM (Figure 3). The resolution obtained with Laurdan does not allow the detection of local differences in fluidity between the lateral membrane and the septa, but does report on overall lipid ordering. Overall, lipid order was increased in cells grown on SMM compared to LB (Figure 3), whereas the absence of PBP1 had no significant effect on membrane fluidity, also not when combined with flotillin deletions (Figure 3). The changes in lipid ordering were not due to changes in the overall fatty acid composition of the membranes - the ratios of C17/C15 side chains and iso/anteiso fatty acids, which are indicative of fluidity (Strahl et al., 2014), were identical for wild type and DfloAT strains grown on LB, and very similar for cells grown on SMM (Figure 3—figure supplement 1). Restoring membrane fluidity rescues normal peptidoglycan synthesis The GP values indicated that membranes are more ordered when cells are grown on minimal medium, and this suggests that the flotillin-associated increase in overall membrane fluidity is important for cell shape control at high growth rates. This was tested by growing the strains lacking flotillins and PBP1 on LB in the presence of benzyl alcohol, an extensively used membrane fluidiser that Figure 1 continued Figure supplement 2. Absence of flotillins does not affect expression, oligomerisation or localisation of PBPs. Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 5 of 21 Research article Cell Biology Microbiology and Infectious Disease Figure 2. Cell morphology and cell wall synthesis localisation is dependent on growth conditions. Morphology of the WT,DfloAT,Dpbp1, and Dpbp1DfloAT strains grown in (A) rich (LB), (B) minimal (SMM) medium, and in (C) rich medium with membrane fluidising conditions (0.1% benzyl alcohol, LB+BnOH). Cells were labelled with HADA, and aberrant cell shape and peptidoglycan synthesis are indicated with arrowheads. Panels on the right indicate corresponding cell length distributions (n 100). Distributions were analysed using Dunn’s multiple comparison tests after Kruskal–Wallis. Statistically significant cell length distribution classes (p<0.001) are represented as letters above each graph – in B and C there were no significant differences. Scale bar: 4 mm. The online version of this article includes the following source data and figure supplement(s) for figure 2: Source data 1. Cell length measurements. Figure supplement 1. Deletion of both flotillins and PBP1 induces filamentation and delocalisation of peptidoglycan synthesis. Figure supplement 1—source data 1. Cell length measurements plotted in F2F1C. Figure supplement 2. Septum labelling of wild type and flotillin mutant cells grown on minimal medium. Figure supplement 2—source data 1. Fluorescence intensity measurements plotted in F2FS2. Figure supplement 3. Filamentation and delocalisation of peptidoglycan synthesis in the absence of flotillins and PBP1 is not rescued by the addition of magnesium. Figure supplement 4. Growth curves and growth rates show similar growth for wt, DfloAT,Dpbp1, and Dpbp1DfloAT (as well as DfloA, DfloT, Dpbp1DfloA and Dpbp1DfloT) strains grown on LB or on LB supplemented with BnOH (0.1% (w/v)). Figure supplement 4—source data 1. Growth curve data. Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 6 of 21 Research article Cell Biology Microbiology and Infectious Disease increases membrane hydration due to disordering of membrane structure (Konopa ´sek et al., 2000). Notably, the addition of benzyl alcohol increased membrane fluidity to similar extents in the wildtype and the mutant strains (see Figure 3C), but did not affect the growth rates of the strains (Figure 2—figure supplement 4). The increase in membrane fluidity restored normal cell length and normal peptidoglycan synthesis patterns to the pbp1/floA/floT strain (Figure 2C). In B. subtilis, the rate of growth and of peptidoglycan synthesis is linked to the speed of MreB movement – in minimal media, the speed of MreB patches is reduced compared to the speed in rich media (Billaudeau et al., 2017). Analysis of the movement of a fully functional mRFPruby-MreB fusion (Domı´nguez-Escobar et al., 2011) by time lapse TIRF (Total Internal Reflection Fluorescence) microscopy, confirmed that MreB patch mobility is higher in cells grown on LB than in cells grown on SMM, with MreB speeds similar to those reported previously (Billaudeau et al., 2017;Figure 4,Figure 4—videos 1 and 2). Strikingly, in the absence of flotillins, MreB patch mobility was notably decreased in cells grown on LB, while in SMM grown cells MreB patch mobility was independent of the presence of flotillins (Figure 4,Figure 4—videos 3 and 4). Fluidising the membrane with benzyl alcohol, which does not alter the growth rate, almost completely restored MreB mobility in LB grown cells (Figure 4,Figure 4—videos 5 and 6). These results indicate that the MreB patch mobility is not only controlled by growth rate, but also by membrane fluidity. Thus, in fast growing cells with decreased membrane fluidity there is a decrease in elongasome mediated peptidoglycan synthesis, Figure 3. 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 red indicates regions of decreased fluidity (scale bar: 4 mm). Correspondent theoretical GP measurements in the graphs vary from 1 (more fluid) to 1 (less fluid). Significant statistical differences according to Dunn’s multiple comparison tests after Kruskal–Wallis are represented as letters above each graph in panel (A). Data labelled ‘A’ are significantly different from data labelled ‘B’; data with the same letter are not significantly different. No statistically significant difference was observed for the data in panels (B) and (C) (p<0.001; n 150, two biological replicates). The online version of this article includes the following source data and figure supplement(s) for figure 3: Source data 1. GP measurement. Figure supplement 1. Fatty acid composition analysis. Figure supplement 1—source data 1. Fatty acid composition data. Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 7 of 21 Research article Cell Biology Microbiology and Infectious Disease reflected by the reduction of MreB mobility. This fits with an observed increase in peptidoglycan synthesis at the division site which may act as a compensatory mechanism. Flotillin increases fluidity of model membranes in vitro To assess whether the influence of flotillins on membrane fluidity is direct, we determined the membrane fluidity of model membranes with purified flotillin using solid-state NMR (ssNMR). 2 H ssNMR is a biophysical tool that assesses lipid mobility in native-like model membranes on the atomic level, by monitoring the carbon-deuterium order parameter of a deuterated lipid along the acyl chain (here POPC-d31) (Molugu et al., 2017;Legrand et al., 2019). We purified B. subtilis FloT and tested the impact of FloT on the membrane, when reconstituted in POPC-d31 liposomes (Schematically depicted in Figure 5A). FloT decreases the spectral width of the 2 H quadrupolar splitting, reflecting an increase in motion on the atomic scale (Figure 5B). The 2 H spectrum encodes the local order parameter S CD of the carbon-deuterium in absence and in presence of FloT. Strikingly, FloT Figure 4. MreB speed is linked to membrane fluidity. (A) The MreB speed in different strain backgrounds and growth conditions was analysed by timelapse TIRF microscopy. Scatter plot of the speed of patches obtained from individual tracks in 5 different cells are represented per fusion and condition. Average speeds are shown; error bars indicate the standard deviation. Significant statistical differences according to Dunn’s multiple comparison tests after Kruskal–Wallis are represented (p<0.001). (B) Representative kymographs showing fast and slow moving patches of mRFPrubyMreB in B. subtilis cells lacking endogenous mreB (WT) or mreB and floAT (DfloAT). See Figure 4—videos 1–6for corresponding raw image series. The online version of this article includes the following video and source data for figure 4: Source data 1. MreB patch mobility measurements determined by TIRFM. 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. https://elifesciences.org/articles/57179#fig4video1 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. https://elifesciences.org/articles/57179#fig4video2 Figure 4—video 3. Visualisation of xylose inducible mrfpRuby-MreB in DfloAT patches dynamics (strain 4076) during exponential growth in LB medium at 37˚C by TIRF microscopy. https://elifesciences.org/articles/57179#fig4video3 Figure 4—video 4. Visualisation of xylose inducible mrfpRuby-MreB in DfloAT patches dynamics (strain 4076) during exponential growth in SMM medium at 37˚C by TIRF microscopy. https://elifesciences.org/articles/57179#fig4video4 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. https://elifesciences.org/articles/57179#fig4video5 Figure 4—video 6. Visualisation of xylose inducible mrfpRuby-MreB in DfloAT patches dynamics (strain 4076) during exponential growth in LB medium supplemented with BnOH (0.1%) at 37˚C by TIRF microscopy. https://elifesciences.org/articles/57179#fig4video6 Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 8 of 21 Research article Cell Biology Microbiology and Infectious Disease has an important impact on the order parameter along the entire acyl chain. It is remarkable that the protein significantly decreases the order parameter S CD , reaching even the inner carbon atoms of the acyl chain, indicating a different packing behaviour and increased membrane fluidity upon interaction with FloT (Figure 5B). The strong fluidising effect described for FloT is notably different from the effects other proteins have when reconstituted into liposomes, such as plant remorins (Legrand et al., 2019) or the membrane binding peptide of the nonreceptor tyrosine kinase Src (Scheidt and Huster, 2009). The anisotropic lineshape of the 31 P spectra indicates that the membrane is in the lamellar phase as expected for POPC at the chosen temperature (298K) (Huster, 2014). Upon interactions with FloT the lamellar phase remains intact with formation of a few smaller objects, indicating that the overall liposome structure is not affected and that its phase is maintained (Figure 5—figure supplement 1). Discussion Our data provide evidence that flotillins play a direct role in controlling membrane fluidity and that membrane fluidity is critical for peptidoglycan synthesis at certain growth conditions. In vitro, flotillins enhance the fluidity of a model membrane, and in vivo, the membranes of fast growing flotillinmutant cells are less fluid even though the fatty acid composition in these cells is identical. Therefore, we propose that the effect of flotillins on membrane fluidity is direct, through a change in the packing behaviour of the lipids resulting in an efficient separation of states of liquid ordered and disordered lipid domains in the membrane bilayer (Bach and Bramkamp, 2013). We found that Figure 5. Lipid ordering of FloT probed by 2 H solid-state NMR. (A) Wide-line 2 H spectra of POPC-d31 liposomes with or without FloT at a lipid-toprotein molar ratio of 25:1 acquired at 298 K. (B) Effect of FloT on the C2 H order parameters of the PC acyl chain. De-Pake-ing and simulations were applied on the 2 H solid-state NMR spectra to determine accurately individual quadrupolar splittings. Order parameters of POPC-d31 acyl chain were derived from experimental quadrupolar splittings and plotted as a function of the labelled carbon position. Insert: schematic depiction of a liposome with added FloT which attaches to the membrane via a hairpin loop (Bach and Bramkamp, 2015). The online version of this article includes the following figure supplement(s) for figure 5: Figure supplement 1. 31 P solid-state NMR experiments of POPC liposomes with or without FloT at a lipid-to-protein molar ratio of 25:1. Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 9 of 21 Research article Cell Biology Microbiology and Infectious Disease Bocillin labelling Cells were grown until an OD600 of 0.4–0.5, and washed twice with PBS. Next, samples were resuspended in 50 ml PBS containing Bocillin-FL (5 mg/ml) and incubated at room temperature for 10 min. Subsequently cells were harvested, lysed by sonication and cell-free extracts were prepared. Samples, equalised for culture OD, were prepared with SDS-PAGE sample buffer and run on a 12% SDSPAGE gel. Fluorescent bands were visualised using a Typhoon Trio (GE Healthcare) scanner. Isolation of membranes Membrane isolation was adapted from Schneider et al., 2015b. Briefly, cells were grown until an OD600 of 0.4–0.5, cell fractions were collected and resuspended in PBS with Lysozyme (1 mg/ml), EDTA (5 mM), 1/10 tablet cOmplete protease inhibitor (Roche), and DNAse (5 mg/ml) and incubated for 30 min on ice. Samples were sonicated, cell which did not lyze were spun down (8000 rpm, 2 min, 4˚C), and the supernatant fraction was centrifuged at 4˚C and 40000 rpm for 1 hr. The membrane pellet was dissolved in ACA750 buffer (750 mM aminocaproic acid, 50 mM Bis-Tris, pH 7.0) to a final protein concentration of 1 mg/ml. Membranes were solubilised overnight at 4˚C in 1% (w/v) dodecylmaltoside (DDM) and either used directly or stored at 20˚C. Blue native PAGE (BN-PAGE) The experiment was performed as described (Trip and Scheffers, 2016). Samples were prepared by mixing sample buffer (0.1% Ponceau S, 42.5% Glycerol) with solubilised membranes in a 1:3 ratio. Samples were resolved on a mini-PROTEAN TGX Stain-Free gradient gel (4–15%, BioRad) using cathode (50 mM Tricine and 15 mM BisTris), and anode (50 mM BisTris pH 7.0) buffers. The Novex NativeMark Unstained Protein Standard marker was used as a Mw marker. Second dimension SDS PAGE (2D SDS-PAGE) A lane of interest was excised from the Native-PAGE gel and immobilised horizontally on top of a SDS-PAGE gel (5% stacking, 12% resolving). The excised fragment was flanked with a piece of Whatman paper soaked with PageRuler Prestained Protein Ladder. The gel fragment to be resolved in the second dimension was topped with a mix of 1% (w/v) LowTemperature agarose, 0.5% (w/v) SDS and bromophenol blue. After the agarose had solidified, standard SDS-PAGE electrophoresis was performed. TEM Cultures were harvested by centrifugation and a small amount of pellet was placed on a copper dish. A 400 copper mesh grid and a 75 mm aperture grid was placed on top of the cells to create a thin layer. The sandwiched cells were plunged rapidly into liquid propane. Sandwiches were then disassembled and placed on frozen freeze-substitution medium containing 1% osmium tetroxide, 0.5% uranyl acetate and 5% water in acetone. Cells were dehydrated and fixed using the rapid freeze substitution method (McDonald, 2014). Samples were embedded in epon and ultrathin sections were collected on formvar coated and carbon evaporated copper grids and inspected using a CM12 (Philips) transmission electron microscope. For each strain 70 random septa were imaged with pixel resolution of 1.2 nm. Using ImageJ the cell wall thickness for each septum was measured at 4 places from which the average was taken. Statistical analysis Each set of micrographs to be analysed was imaged with the same exposure time. For the septum intensity analysis of HADA, FM4-64 and Nile Red, the wildtype strain (expressing GFP) and the DfloAT strains were mixed, labelled and imaged on the same agarose pad. Intensity of the fluorescently labelled septa was measured using the ObjectJ macro tool PeakFinder (https://sils.fnwi.uva. nl/bcb/objectj/examples/PeakFinder/peakfinder.html) (Vischer et al., 2015). A perpendicular line was drawn across the septal plane, the background intensity was removed resulting in a maximum peak intensity. The number of septa compared was indicated for every individual experiment. Populations were compared using the non-parametric Mann-Whitney test. The null hypothesis was tested with the pvalue of 0.05. The statistical analyses and their graphical representation (box plots) were generated with GraphPad Prism 8.1 (San Diego, California, USA). Box plots show the median and Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 16 of 21 Research article Cell Biology Microbiology and Infectious Disease the interquartile range (box), the 5th and 95th percentile (whiskers). Laurdan fluorescence generalised polarisation, cell length and MreB speed statistical analyses were performed using KruskalWallis with Dunn’s multiple comparison post-hoc test. Fatty acid composition analysis The fatty acid composition of B. subtilis wild-type cells and the flotillin/PBP mutants was analysed with gas chromatography as fatty acid methyl esters. Cells for the analyses were grown at 37˚C in LB or SMM until mid-exponential (OD 600 ~0.5), harvested (6000 rpm, 10 min, 4˚C) and washed with 100 mM NaCl. Next, the cells were freeze dried at 50˚C, 0.012 mbar for a minimum of 18 hr. All analyses were carried out on biological duplicates by the Identification Service of the DSMZ, Braunschweig, Germany. Sample preparation for solid-state NMR FloT was essentialy purified as described (Bach and Bramkamp, 2013), in solubilised form, and stored in buffer A (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM MgCl 2 ) supplemented with 0.05% Triton X-100. Liposomes containing POPC-d31 were prepared by mixing appropriate lipid powders in organic solvents (chloroform/methanol, 2:1 ratio). Solvents were evaporated under a flow of N 2 to obtain a thin lipid film. Lipids were rehydrated with ultrapure water before lyophilisation over night. The lipid powder was hydrated with an appropriate amount of buffer A with 10% glycerol and homogenised by three cycles of vortexing, freezing (liquid nitrogen, 196˚C, 1 min) and thawing (40˚C in a water bath, 10 min). This protocol generated a milky suspension of micrometer-sized multilamellar vesicles. FloT was solubilised in Buffer A supplemented with 0.05% Triton X-100 and added to preformed liposomes and incubated for 1 hr at room temperature. A dialysis step was then performed against Buffer A at 4˚C under agitation to remove the detergent. Samples were centrifuged at 100,000 g at 4˚C for 1 hr to pellet the proteoliposomes. 2 H solid-state NMR spectra were recorded of liposomes in the presence or absence of FloT at a lipid/protein ratio of 25:1 at 298 K. Solid-state NMR 2 H NMR spectroscopy experiments were performed using a Bruker Avance III 500 MHz WB (11.75 T) spectrometer. They were recorded on 2 H-labelled POPC at 76.77 MHz with a phase-cycled quadrupolar echo pulse sequence (90˚x-t-90˚y-t-acq). Acquisition parameters were as follows: spectral window of 500 kHz for 2H NMR spectroscopy, p/2 pulse width of 3.90 ms for 2 H, interpulse delays (t) were of 40 ms, recycled delays of 1.3 s for 2H; 3000 and 8000 scans were used for 2 H NMR spectroscopy on liposomes and liposomes with FloT, respectively. Spectra were processed using a Lorentzian line broadening of 300 Hz for 2 H NMR spectra before Fourier transformation from the top of the echo. Samples were equilibrated for 30 min at a given temperature before data acquisition. All spectra were processed and analysed using Bruker Topspin 3.2 software. Spectral moments were calculated for each temperature using the NMR Depaker 1.0rc1 software [Copyright (C) 2009 Se´ bastien Buchoux]. Orientational order parameters (SCD) were calculated from experimental quadrupolar splittings (DnQ) as described in Huster, 2014. For 31 P ssNMR, we applied a static Hahn spin echo sequence at the 31 P frequency of 162 MHz on a 400 MHz (9.4T) Bruker Avance III HD spectrometer, with a 90pulse of 8 ms, a delay of 40 ms, a recycle delay of 5 s, a spectral window of 400 ppm and a number of scans of 4000 and 3400 was used on liposomes and liposomes with FloT, respectively. Spectra were processed using a Lorentzian line broadening of 100 Hz. Acknowledgements We thank Henrik Strahl for discussions and sharing of unpublished data, Rut Carballido-Lopez for strain RWBS5 and Luiza Morawska and Oscar Kuipers for the PrrnB-gfp plasmid. This work was funded by NWO grant 864.09.010 (DJS), DFG grants BR 2915/4–1; INST 86/1452– 1 (MB), ERC starting grant 757913; NWO grant 721.014.008 (AKHH); PhD fellowships DAAD-GSSP to AS; and SFRH/BD/78061/2011POPH/FSE/FCT to ASB. Zielin´ska et al. eLife 2020;9:e57179. DOI: https://doi.org/10.7554/eLife.57179 17 of 21 Research article Cell Biology Microbiology and Infectious Disease Additional information Funding Funder Grant reference number Author Nederlandse Organisatie voor Wetenschappelijk Onderzoek Vidi 864.09.010 Dirk-Jan Scheffers Deutsche Forschungsgemeinschaft BR 2915/7-1 Marc Bramkamp Deutsche Forschungsgemeinschaft INST 86/1452-1 Marc Bramkamp Nederlandse Organisatie voor Wetenschappelijk Onderzoek 721.014.008 Anna KH Hirsch European Research Council starting grant 757913 Anna KH Hirsch Deutscher Akademischer Austauschdienst PhD fellowship DAAD-GSSP Abigail Savietto Fundac¸a ˜o para a Cie ˆncia e a Tecnologia PhD fellowship SFRH/BD/ 78061/2011POPH/FSE/ FCT Anabela de Sousa Borges The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions Aleksandra Zielin´ ska, Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing; Abigail Savietto, Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing; Anabela de Sousa Borges, Conceptualization, Funding acquisition, Investigation, Visualization, Methodology; Denis Martinez, Melanie Berbon, Formal analysis, Investigation, Methodology; Joe ¨l R Roelofsen, Investigation; Alwin M Hartman, Anna KH Hirsch, Resources; Rinse de Boer, Formal analysis, Investigation; Ida J Van der Klei, Supervision, Investigation; Birgit Habenstein, Conceptualization, Supervision, Investigation, Methodology, Writing - original draft; Marc Bramkamp, Dirk-Jan Scheffers, Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing Author ORCIDs Ida J Van der Klei https://orcid.org/0000-0001-7165-9679 Marc Bramkamp http://orcid.org/0000-0002-7704-3266 Dirk-Jan Scheffers https://orcid.org/0000-0002-9439-9168 Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.57179.sa1 Author response https://doi.org/10.7554/eLife.57179.sa2 Additional files Supplementary files .Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files. 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