Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.
The function of the essential inner membrane protein (IMP) YidC in Escherichia coli has been studied for a limited number of model IMPs and primarily using targeted approaches. These studies suggested that YidC acts at the level of insertion, folding, and quality control of IMPs, both in the context of the Sec translocon and as a separate entity. To further our understanding of YidC's role in IMP biogenesis, we screened a random overexpression library for factors that rescued the growth of cells upon YidC depletion. We found that the overexpression of the GadX and GadY regulators of the glutamate-dependent acid resistance system complemented the growth defect of YidC-depleted cells. Evidence is presented that GadXY overexpression counteracts the deleterious effects of YidC depletion on at least two fronts. First, GadXY prepares the cells for the decrease in respiratory capacity upon the depletion of YidC. Most likely, GadXY-regulated processes reduce the drop in proton-motive force that impairs the fitness of YidC-depleted cells. Second, in GadXY-overproducing cells increased levels of the general chaperone GroEL cofractionate with the inner membranes, which may help to keep newly synthesized inner membrane proteins in an insertion-competent state when YidC levels are limiting.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. eLife 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, Zielińska, 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. 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 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 (Domínguez-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 (Domínguez-Escobar et al., 2011; Garner et al., 2011; Muchová 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 (García-Fernández et al., 2017; López 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-Süss and Lopez, 2015; Mielich-Süss 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 (López 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; García-Fernández et al., 2017; Mielich-Süss 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 ΔfloAT 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 ΔfloAT 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 ΔfloAT 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 ΔfloAT 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 ΔfloAT 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 ΔfloAT 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 ΔfloAT cells without fosfomycin. Figure 1 with 2 supplements see all Download asset Open asset 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 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. Figure 1—source data 1 Fluorescence intensity and cell length measurements. https://cdn.elifesciences.org/articles/57179/elife-57179-fig1-data1-v1.xlsx Download elife-57179-fig1-data1-v1.xlsx 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 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 ΔfloAT strain (Figure 1—figure supplement 2B). Also, none of the five functional GFP-PBPs examined changed their localisation in the ΔfloAT 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-D-Pra), 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 ∆pbp1ΔfloAT 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 ΔfloAT strain (Figure 2—figure supplement 2). SMM has a higher Mg2+ concentration, which is known to rescue various cell shape mutations by inhibition of cell wall hydrolysis (Dajkovic et al., 2017). However, the increase in Mg2+ was not sufficient to explain the reversal of phenotype as cells grown on LB supplemented with Mg2+ (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. Figure 2 with 4 supplements see all Download asset Open asset 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, 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 µm. Figure 2—source data 1 Cell length measurements. https://cdn.elifesciences.org/articles/57179/elife-57179-fig2-data1-v1.xlsx Download elife-57179-fig2-data1-v1.xlsx 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 ΔfloAT strains grown on LB, and very similar for cells grown on SMM (Figure 3—figure supplement 1). Figure 3 with 1 supplement see all Download asset Open asset 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 µm). 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). Figure 3—source data 1 GP measurement. https://cdn.elifesciences.org/articles/57179/elife-57179-fig3-data1-v1.xlsx Download elife-57179-fig3-data1-v1.xlsx 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 increases membrane hydration due to disordering of membrane structure (Konopásek 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 (Domínguez-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, 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. Figure 4 with 6 supplements see all Download asset Open asset 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 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 mRFPruby-MreB in B. subtilis cells lacking endogenous mreB (WT) or mreB and floAT (ΔfloAT). See Figure 4—videos 1–6 for corresponding raw image series. Figure 4—source data 1 MreB patch mobility measurements determined by TIRFM. https://cdn.elifesciences.org/articles/57179/elife-57179-fig4-data1-v1.xlsx Download elife-57179-fig4-data1-v1.xlsx 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). 2H 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 2H quadrupolar splitting, reflecting an increase in motion on the atomic scale (Figure 5B). The 2H spectrum encodes the local order parameter SCD of the carbon-deuterium in absence and in presence of FloT. Strikingly, FloT has an important impact on the order parameter along the entire acyl chain. It is remarkable that the protein significantly decreases the order parameter SCD, 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 31P 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). Figure 5 with 1 supplement see all Download asset Open asset 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. De-Pake-ing and simulations were applied on the 2H 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). 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 flotillin-mutant 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 membrane fluidity is not solely a function of temperature, but also of growth conditions. In vivo, flotillins may also recruit specific, more rigid lipids, such as hopanoids and carotenoids (Bramkamp and Lopez, 2015; García-Fernández et al., 2017; López and Kolter, 2010) which have been found in association with FMMs, and whose synthesis could be growth condition dependent. The predominantly physical role in membrane organisation for flotillins fits with our observation that adding a chemical fluidiser is sufficient to restore MreB dynamics and cell shape to fast growing cells that lack flotillins. We propose that in fast-growing cells on rich medium, flotillin-mediated control of membrane fluidity is critical and sufficient to allow essential membrane bound processes, such as peptidoglycan synthesis, to proceed normally. A sufficiently fluid membrane is necessary for the efficient recruitment and movement of MreB, and provides a more favourable environment for the peptidoglycan precursor LipidII (Hussain et al., 2018; Ursell et al., 2014; Schirner et al., 2015). It has recently been shown that modulation of either MreBCD or PBP1 levels is sufficient to alter the shape of B. subtilis cells (Dion et al., 2019), underscoring the importance of both systems. In the absence of flotillins, the activity of the MreBCD component is strongly reduced – as evidenced by the reduction of MreB speed – and the overall rigidity of the membrane is increased. This results in a less favourable environment for the peptidoglycan precursor LipidII, which prefers more liquid, disordered membrane phases (Ganchev et al., 2006; Witzke et al., 2016; Calvez et al., 2019). Our data indicate that the reduction in elongasome activity, which does not impact the growth rate itself, is compensated by increased peptidoglycan synthesis activity around division sites in flotillin mutants, which is sufficient to keep the overall cell shape intact, although cells are elongated. The accumulation of lipid dyes indicative of increased fluidity at division sites is in line with a recent study that showed phases of different fluidity in Streptococcus pneumoniae membranes, with more fluid membranes and LipidII localising at midcell (Calvez et al., 2019) where the membrane is most bent. Our findings are also in agreement with the recent observation that B. subtilis cells elongate and lose organisation of MreB when membrane fluidity is decreased by altering the membrane fatty acid composition (Gohrbandt, 2019) (H. Strahl, personal communication). It could very well be that the shift of fluidity towards the septum is only relative as the overall fluidity of the membrane is decreased in the absence of flotillins. This is yet to be determined, as the resolution of Laurdan imaging does not allow conclusive statements about local fluidity changes at the septum. The observation that reduced MreB mobility and therefore altered lateral cell wall synthesis lead to accumulation of Van-FL and HADA staining at the septum is not immediately conclusive. Since septal PG synthesis is MreB independent in B. subtilis, a direct effect of MreB seems unlikely. Rather, a reduction of overall membrane fluidity in a flotillin knock-out might impair LipidII dynamics within the membrane. MurG is the enzyme that catalyses the final step of LipidII synthesis. There are several reports that MurG localises to the septum in different organisms (Aaron et al., 2007; Mohammadi et al., 2007). Thus, it seems likely that the septum is a place of increased LipidII synthesis and a change in membrane fluidity would create problems for LipidII molecules to diffuse away from their insertion site, resulting in reduced lateral PG synthesis and MreB mobility. Alternatively, the reduced MreB mobility and reduced lateral PG synthesis lead to a reduced LipidII consumption at the lateral wall, and the excess LipidII
Xanthomonas citri subsp. citri (Xcc) causes citrus canker, affecting sweet orange-producing areas around the world. The current chemical treatment available for this disease is based on cupric compounds. For this reason, the objective of this study was to design antibacterial agents. In order to do this, we analyzed the anti-Xcc activity of 36 alkyl dihydroxybenzoates and we found 14 active compounds. Among them, three esters with the lowest minimum inhibitory concentration values were selected; compounds 4 (52 μM), 16 (80 μM) and 28 (88 μM). Our study demonstrated that alkyl dihydroxybenzoates cause a delay in the exponential phase. The permeability capacity of alkyl dihydroxybenzoates in a quarter of MIC was compared to nisin (positive control). Compound 28 was the most effective (93.8), compared to compound 16 (41.3) and compound 4 (13.9) by percentage values. Finally, all three compounds showed inhibition of FtsZ GTPase activity, and promoted changes in protofilaments, leading to depolymerization, which prevents bacterial cell division. In conclusion, heptyl dihydroxybenzoates (compounds 4, 16 and 28) are promising anti-Xcc agents which may serve as an alternative for the control of citrus canker.
The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.
A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology. More recently, light microscopy in combination with the use of fluorescent dyes has become an attractive technique for protein localization with the natural membrane. However, the resolution problem in light microscopy remains and overinterpretation of observed phenomena is a pitfall. Thus, light microscopy as a stand-alone technique is not sufficient to prove, for instance, the long-range helical distribution of proteins in membrane such as MinD spirals in <i>Bacillus subtilis</i>. Electron tomography is an emerging electron microscopy technique that can provide three-dimensional reconstructions of small, nonchemically fixed bacteria. It will become a useful tool for studying prokaryotic membranes in more detail and is expected to collect information complementary to those of advanced light microscopy. Together, microscopy techniques can meet the challenge of the coming years: to specify membrane structures in more detail and to bring them to the level of specific protein-protein interactions.
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.
In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.
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