The actin homologue MreB organizes the bacterial cell membrane

Abstract

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes.

ARTICLE

Received 5 Dec 2013 | Accepted 13 Feb 2014 | Published 7 Mar 2014

The eukaryotic cortical actin cytoskeleton creates specic lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated laments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton inuences uorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specic lipid staining techniques and spectroscopic methods, revealed that MreB laments create specic membrane regions with increased uidity (RIFs). Interference with these uid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The inuence of MreB on membrane organization and uidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes.

DOI: 10.1038/ncomms4442 OPEN

The actin homologue MreB organizes the bacterial cell membrane

Henrik Strahl1, Frank Brmann2 & Leendert W. Hamoen1,3

1 Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Richardson Road, Newcastle NE2 4AX, UK. 2 Max Planck Institute of Biochemistry, Chromosome Organization and Dynamics, Am Klopferspitz 18, Martinsried D-82152, Germany. 3 Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Amsterdam 1098 XH, The Netherlands. Correspondence and requests for materials should be addressed to H.S. (email: mailto:[email protected]

Web End [email protected] ) or to L.W.H. (email: mailto:[email protected]

Web End [email protected] ).

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The morphology of many rod-shaped bacteria is established by the coordinated incorporation of new cell wall material perpendicular to the cell axis1,2. An essential component of

this machinery is the bacterial actin homologue MreB, which polymerizes into laments at the cell periphery3. The peripheral association of MreB is facilitated by a conserved hydrophobic membrane-binding loop, which in some organisms is further supported by a membrane-binding N-terminal amphipathic helix4. Upon binding, MreB forms a complex with the conserved membrane proteins MreC and MreD, and with proteins involved in peptidoglycan synthesis such as RodA, MurG, MraY, and several penicillin-binding proteins1,2,5. Interference with the MreB activity renders cells mechanically less rigid6, and, in the absence of this, protein cells lose their rod-shaped morphology7,8. The prevailing model, in which helical MreB polymers spatially direct the synthesis of new peptidoglycan and as a result determine the general shape of the cell, has recently been revised. It turned out that MreB laments, and the associated cell wall synthetic machinery, move around the cell in a process that is driven by peptidoglycan synthesis911. The MreB cytoskeleton has also been implicated in other cellular processes, including the establishment of cell polarity and chromosome segregation12,13.

In a previous study, we have shown that the MreB cytoskeleton of Bacillus subtilis is sensitive to changes in the membrane potential, and incubation of cells with the proton ionophore CCCP results in a rapid delocalization of MreB14. The mechanism for this membrane potential sensitivity is currently

unknown. During this work, we noticed that the uorescence of the cell membrane, when stained with the lipid dye Nile Red, shows a rapid (within 12 min) transformation from a uniform to a clustered signal, which indicates irregularities in the lipid membrane. Interestingly, these Nile Red foci colocalize with GFPMreB and do not emerge in bacteria that lack MreB. MreB is a homologue of eukaryotic actin, and actin forms an intricate membrane-associated network termed the cortical actin cytoskeleton15. The correlation between MreB and the lipid staining effects was intriguing since the cortical actin cytoskeleton is involved in the formation of lipid domains including lipid rafts and sphingolipid-enriched domains15,16. By applying different lipid staining techniques, and using a variety of mutant strains, we were able to show that the MreB cytoskeleton of B. subtilis is associated with uid lipid domains, and, like the eukaryotic cortical actin cytoskeleton, is involved in the distribution of lipids and proteins. Furthermore, the active and directed movement of MreB appeared to stimulate the diffusion of proteins within the cell membrane. The consequences for membrane protein activity and cell wall synthesis are discussed.

ResultsAltered membrane stain upon MreB delocalization. Dissipation of the membrane potential with CCCP results in delocalization of the cytoskeletal protein MreB in B. subtilis14. We noticed that such treatment also affects uorescent membrane staining (Fig. 1a). In untreated cells, Nile Red stains cell membranes

a b

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AtpA-GFP Nile Red Merge

Figure 1 | Disruption of the MreB cytoskeleton is accompanied by an aberrant membrane stain. (a) Phase contrast images of B. subtilis cells (upper panel) expressing GFP-MreB (middle panel), and uorescent Nile Red membrane stains (lower panel), are depicted in the absence or presence of the proton ionophore CCCP. Some of the Nile Red foci appearing with CCCP are highlighted with arrows (see Supplementary Fig. 5a for more examples). The CCCP-triggered local enrichment of MreB is not caused by the weak dimerization property of GFP, which was recently shown to stimulate protein clustering69

(Supplementary Fig. 5b), or by articial overproduction (Supplementary Fig. 6). Strain used: B. subtilis YK405 (gfp-mreB). (b) Incubation of F1Fo ATP synthasedecient cells with CCCP also results in a rapid (within 2 min) emergence of Nile Red foci, ruling out that the appearance of Nile Red foci is due to a reduction of ATP levels (see Supplementary Fig. 1c for more examples). Strain used: B. subtilis HS13 (Datp). (c) Comparison of Nile Red foci, and the localization of the integral membrane protein F1Fo ATP synthase fused to GFP (AtpA-GFP). The lack of overlap in uorescence signals rules out membrane invagination, or aberrantly formed septa (see arrows) as an explanation for the strong local Nile Red uorescence (see Supplementary Fig. 4 for more examples). The reason that in some cases the Nile red stain appears to protrude into the cytoplasm is a consequence of high membrane uorescence originating from slightly above and below the exact focal plane. Strain used: B. subtilis BS23 (atpA-gfp). (d) CCCP does not affect Nile Red uorescent membrane stain in Staphylococcus aureus and Corynebacterium glutamicum which both lack MreB (see Supplementary Fig. 1d for more examples). Strains used:C. glutamicum RES167 and S. aureus RN4220. (e) Colocalization of GFP-MreB (upper panel) with Nile Red (middle panel) in B. subtilis strains encoding wild type MreD (wt), and a 15 amino acid C-terminal truncation of MreD ( 15aa), which results in a mild delocalization of MreB in low Mg2 medium (see

Supplementary Fig. 1e for more examples). Strains used B. subtilis HS35 (gfp-mreB mreD wt) and HS38 (gfp-mreB mreD1-471/ 15aa). Scale bar, 2 mm.

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without a visible preference for certain membrane areas. A slightly stronger uorescence signal is observed for division septa, which is caused by the presence of the two adjacent cell membranes from the newly formed daughter cells. However, in the presence of CCCP, brightly uorescent foci appear. These membrane staining effects are visible within 2 min after CCCP addition. CCCP is a proton carrier that abolishes the proton motive force (pmf)-driven synthesis of ATP, resulting in a rapid reduction of the cellular ATP pool14. An F1Fo ATP synthase deletion mutant is viable and generates ATP by pmf-independent means via glycolysis and is therefore able to maintain stable ATP levels when CCCP is present for a much longer period of time14. Nevertheless, in an F1Fo ATP synthase deletion strain, the addition of CCCP still results in a spotty Nile Red membrane stain, indicating that the effect is not related to changes in ATP levels (Fig. 1b). Addition of the potassium-ionophore valinomycin also gives rise to an irregular uorescence membrane staining, whereas the proton-potassium antiporter nigericin has no effect (Supplementary Fig. 1a). Valinomycin and nigericin dissipate the membrane potential and proton gradient, respectively. Therefore, the membrane staining effects specically occur when the membrane potential is disturbed. The presence of chloramphenicol did not prevent these effects, indicating that de novo protein synthesis is not required (Supplementary Fig. 1b). The fact that the uorescent foci become visible within 1 or 2 min makes it unlikely that they are formed by local accumulation of newly synthetized membrane material. A detailed analysis of the Nile Red uorescence spectra showed that the increased Nile Red uorescence originates from a regular lipid bilayer environment, and is not caused by Nile Red that is bound to abnormal protein or lipid aggregates (Supplementary Fig. 2). In theory, a loss of cell turgor as a result of CCCP treatment could result in the invagination of lipid membranes through plasmolysis. However, a dissipation of the membrane potential with CCCP does not cause a rapid loss of cell turgor (Supplementary Fig. 3). Moreover, when the Nile Red membrane stain was compared with the uorescent membrane signal of a GFP-labelled transmembrane protein (F1Fo ATP

synthase), the GFP signal remained unaffected by CCCP and did not correlate with the uorescent Nile Red foci (Fig. 1c; Supplementary Fig. 4). For these reasons, we conclude that the increase in uorescence signal is not explained by invagination of the cell membrane, or otherwise abnormal membrane shapes.

Several experiments suggest a link between the CCCP-induced lipid staining effect and the MreB cytoskeleton. First, the Nile Red-stained membrane foci colocalize with clusters of MreB (Fig. 1a; Supplementary Fig. 5). Importantly, these clusters are not caused by articial overproduction of a GFP-fusion protein (Supplementary Fig. 6). Second, both the irregular Nile Red stain and the delocalization of MreB are triggered by valinomycin and not by nigericin14 (Supplementary Fig. 1a). Third, when the effect of CCCP on Nile Red staining was tested in Staphylococcus aureus or Corynebacterium glutamicum, which do not contain MreB homologues, no change in the uorescent membrane stain was observed (Fig. 1d). Finally, the localization of B. subtilis MreB depends on MreD17 and an MreD mutant that lacks the last 15 amino acids causes an irregular MreB pattern but also an uneven Nile Red stain (Fig. 1e). The latter experiment indicates that the membrane staining effect occurs upon delocalization of MreB, even when the membrane potential is not affected.

If the delocalization of MreB causes irregular uorescent membrane signals, then the addition of CCCP should have no effect on the Nile Red membrane stain when MreB is absent. However, it appeared that CCCP still causes uorescent Nile Red foci in the membrane of a B. subtilis mreB deletion mutant (Fig. 2a). B. subtilis encodes two other MreB homologues, Mbl

and MreBH, which are known to be able to complement the activity of MreB7. Individual deletions of mbl or mreBH do not prevent the formation of CCCP-induced Nile Red foci either, but in the absence of all three genes (DmreB, Dmbl, DmreBH) the Nile

Red membrane stain showed a smooth uorescence signal in the presence of CCCP (Fig. 2a, see Supplementary Fig. 7 for the analysis of the foci frequency). Importantly, the absence of Nile Red foci in the triple mutant is not a consequence of the deformation of the cell shape by the inhibition of lateral cell wall synthesis, since inactivation of RodA, which is required for peptidoglycan synthesis in the lateral cell wall18, also results in round cells, yet these deformed cells still display Nile Red foci when CCCP is added (Fig. 2b). Taken together, these results support the suggestion that there is a correlation between MreB delocalization and the irregularities in Nile Red lipid stain.

Involvement of specic lipids. Cardiolipin (CL), phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG) are the major phospholipid species in bacteria. Both CL and PE have been reported to form domains in B. subtilis membranes19,20. Nile Red is an uncharged uorescent dye that partitions into lipid membranes based on its intrinsic hydrophobicity21,22. It is not known whether Nile Red prefers certain lipid head groups, but its uorescence is sensitive to physical changes in its lipid environment23. To test whether an aberrant distribution of CL

a

mreB mbl mreBH

mreB mbl mreBH

CCCP

+ CCCP

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GFP-MreB Nile Red Merge

CCCP

+ CCCP

Figure 2 | Distorted lipid staining requires the MreB cytoskeleton.(a) CCCP-dependent uorescent Nile Red foci occur in B. subtilis strains decient for single MreB homologues, but are absent in strains that lack all three MreB homologues (DmreB, Dmbl, DmreBH). Some of the Nile Red foci appearing with CCCP are highlighted with arrows. See also

Supplementary Fig. 7 for the analysis of the foci frequency. Strains used:B. subtilis 3728 (DmreB), 4261 (Dmbl), 4262 (DmreBH) and 4277 (DmreB, Dmbl, DmreBH). (b) Colocalization of GFP-MreB (left panels) with Nile Red (middle panels) in B. subtilis cells depleted for RodA. The MreB cytoskeleton is disrupted with CCCP. Some of the Nile Red foci appearing with CCCP are highlighted with arrows. Strain used: B. subtilis HS36 (gfp-mreB Pspac-rodA). Scale bar, 2 mm.

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or PE is responsible for the irregular Nile Red stain, CL- or PE-decient B. subtilis mutant strains were treated with CCCP. Despite the absence of CL or PE in these strains24, the uorescent Nile Red foci still appear after depolarization of the membrane (Supplementary Fig. 8a).

Another commonly used membrane dye is FM 4-64, a styryldye that also partitions into lipid membrane surface based on intrinsic hydrophobicity25. Unlike Nile Red, it does carry a positive charge25 and a preference to bind negatively charged lipid species can be speculated. It has been shown that this dye exhibits a uorescent stain in B. subtilis cells that under certain conditions weakly resembles a MreB-like helical pattern26,27. This was interpreted as PG-enriched helical domains since PG is negatively charged, and these structures coalesce when PG is depleted26,28. However, it turned out that this change in an FM 4-64 staining pattern is caused by depolarization of the membrane potential upon PG depletion. As in case of uncharged Nile Red, addition of CCCP readily induced FM 4-64 foci that colocalize with MreB (see Supplementary Figs 8be and 9 for details). Thus, we conclude that FM 4-64 is not a specic indicator for PG. The strong formation of Nile Red foci upon depletion of PG makes it unlikely that these are specic clusters of PG. Together, these results suggest that the uorescent membrane foci are not caused by domains enriched in lipids with specic head groups. A more probable explanation for the intense uorescent Nile Red foci is the accumulation of lipids carrying specic fatty acid species.

Localization of uid lipid domains. Bacterial membranes are composed of lipids that can form different conformations such as gel-phase or a more uid liquid-crystalline phase. These distinct membrane congurations are predominantly determined by the fatty acid moiety of the lipids29,30. The presence of unsaturated bonds or branched chains increases the space that fatty acids occupy within a bilayer31. As a result, lipids containing unsaturated or branched fatty acids assemble into a more disordered liquid-crystalline phase, characterized by a less dense packing of lipids. In contrast, lipids with saturated fatty acids favour the more compact and less uid gel-phase. The uidity of the membrane is known to inuence the uorescent staining of many lipid dyes, including that of Nile Red23. To test whether this is also the case in B. subtilis cells, we measured the uorescence intensity of Nile Red using cells that differ in their membrane uidity. In B. subtilis the uid lipid content is greatly altered as a response to growth temperatures. To maintain a constant membrane uidity at different temperatures, the uid lipid content decreases with increasing growth temperature32. As a consequence, when cells grown at 48 C are cooled down to 30 C, their cell membrane will be less uid compared with cells that are grown at 30 C. As shown in Fig. 3a, this results in a lower Nile Red uorescence intensity. In contrast, if cells are grown at room temperature, and subsequently transferred to 30 C, the membrane uidity is increased compared with cells grown at 30 C. Indeed, this leads to an increase in Nile Red uorescence (Fig. 3a). It is therefore possible that the irregular membrane stain caused by MreB delocalization points towards local differences in membrane uidity.

To test whether the Nile Red foci are caused by the enrichment of uid lipids, we adapted a staining technique used to visualize uidity differences in eukaryotic membranes. The uidity sensitive dye Laurdan undergoes an emission wavelength shift upon changes in uidity that can be detected microscopically3336. As shown in Fig. 3b and Supplementary Fig. 10, incubation with CCCP generates membrane areas with a clear and

statistically signicant reduction in Laurdan Generalized Polarization, which indicates an increase in local membrane uidity. Importantly, these membrane domains colocalize with Nile Red foci (Fig. 3c).

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Figure 3 | Analysis of cell membrane uidity. (a) Nile Red uorescence intensity of B. subtilis cells grown in room temperature, 30 C or 48 C followed by shift to 30 C, was measured uorometrically. To prevent rapid adaptation of membrane uidity67, the measurements were carried out with a lipid desaturase (des)-decient strain. The curves represent average values with s.d. of three replicate measurements. Strain used: B. subtilis HB5134 (Ddes). (b) Microscopic analysis of uid membrane domains using

Laurdan GP. The images show a colour-coded Laurdan GP intensity map in which red indicates regions of increased uidity. Membrane uidity is determined for untreated B. subtilis cells (left panel) and cells treated with CCCP (right panel). See also Supplementary Fig. 10 for the measurement of the average uidity change. (c) Colocalization of Nile Red foci and Laurdan GP in CCCP-treated B. subtilis cells. Strain used: B. subtilis 168 (wild type). Scale bar, 2 mm.

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MreB generates membrane regions with increased uidity. These experiments suggest a direct link between the MreB cytoskeleton and membrane regions with increased uidity (RIFs). So far, we have only observed RIFs when MreB coalesces into large clusters, and the question arises whether RIFs are present under conditions in which MreB is normally localized, rather that delocalized by CCCP or other means. Presumably, visualization of RIFs in normal uninhibited cells requires a lipid dye with a high afnity for membrane areas with increased uidity. It has been shown that a lipid-mimicking dye DiI-C12 localizes with high specicity to uid membrane areas as a consequence of its relative short acyl chains37,38. When B. subtilis cells were stained with this dye, a distinct punctuated uorescent pattern became visible that overlapped signicantly with the GFP-MreB signal, with a Pearsons correlation coefcient of 0.9 (Fig. 4ac; Supplementary Fig. 11a,b)37. As a control, we tested DiI-C18, a variant with longer acyl chains. Importantly, in this case, a smooth membrane stain is observed (Fig. 4ac; Supplementary Fig. 11a,b), indicating that the uorescent pattern of DiI-C12 is based on differences in membrane uidity, and not a consequence of favoured interactions between the DiI head group and certain lipids or proteins.

The ability to visualize RIFs with DiI-C12 without dissipation of membrane potential allowed us to test which of the MreBCD proteins are required for RIF formation. In the absence of all three MreB homologues (DmreB, Dmbl, DmreBH), staining with

DiI-C12 produces a smooth uorescent membrane signal, and RIFs are no longer visible (Fig. 5a; Supplementary Fig. 11c). In contrast, mreC or mreD deletion strains that form comparably round cells still form RIFs (Fig. 5a; Supplementary Fig. 11c), indicating that the MreB homologues rather than the integral membrane proteins MreC or MreD are essential for RIF formation. MreB can bind directly to lipid bilayers4, and, when GFP-MreB is expressed in a DmreBCD strain, a strong colocalization of GFP-MreB clusters with DiI-C12 foci is observed (Fig. 5b). These results show that the formation and spatial organization of RIFs is a specic property of MreB.

Absence of MreB changes membrane properties. Since MreB creates local membrane areas with increased uidity, it might be that this activity affects the general uidity of the cell membrane. Indeed, when the overall membrane uidity was measured using Laurdan generalized polarization (GP), it turned out that the general membrane uidity is signicantly elevated when MreB homologues are absent (Fig. 6a). This increase in uidity is comparable to the addition of 30 mM of the membrane uidizer benzyl alcohol39 (Fig. 6a). It should be added that growth in the absence of all three MreB homologues is only possible when the antisigma factor rsgI is deleted40, however, a DrsgI mutation by itself does not result in altered membrane uidity (Fig. 6a).

DmreC or DmreD strains show identical changes in cell shape and inhibition of lateral cell wall synthesis as the triple mreB mbl mreBH deletion mutant but still contain RIF clusters. Importantly, the overall membrane uidity of these strains is comparable to wild-type cells.

The Laurdan experiments suggest that RIFs formed by MreB affect the overall uidity. However, it is possible that the absence of MreB inuences the actual lipid composition. To investigate this, the fatty acid compositions of the different strains were analysed using gas chromatography (Fig. 6b; Supplementary Table 1). Both the DmreC mutant and mreB triple mutant (DmreB/mbl/mreBH) increase their overall fatty acid chain length (less C15 and more C17 species), which normally lowers the membrane uidity (Fig. 6b; Supplementary Table 1). The DmreC, and to a lesser extent the mreB triple mutant, also increases the fraction of anteiso fatty acids compared with iso fatty acid species. Anteiso fatty acid species carry a methyl group attached to the third last carbon of the chain and are signicantly more uid than iso species, which carry a methyl group attached to the second last carbon41. In case of DmreC, this increase in anteiso fatty acids seems to compensate for the effect of increased fatty acid chain length since the overall membrane uidity of this DmreC mutant is the same as for wild type (Fig. 6a). Importantly, this increase in anteiso fatty acids is lower in the mreB triple mutant compared with the DmreC mutant (Fig. 6b;

GFP-MreB (%)

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Figure 4 | Localization of RIFs in untreated B. subtilis cells. (a) RIFs can be visualized in normally growing untreated B. subtilis cells using the uid membrane dye DiI-C12 (left panel). Staining with the long acyl-chain variant, DiI-C18, reveals a regular (smooth) membrane signal (right panel). Strain used: B. subtilis 168 (wild type). (b) Colocalization of DiI-C12 and GFP-MreB (see Supplementary Fig. 11 for more examples and statistical analysis of colocalization). Strain used: B. subtilis YK405 (gfp-mreB). (c) Fluorescence intensity correlation graphs are shown for the cells in panel b. The graphs display a pixel by pixel intensity correlation between DiI and GFP-MreB uorescence, and shows the Pearsons correlation coefcients (Rr) (see Supplementary Fig. 11 for details). It should be mentioned that some uorescence correlation between DiI-C18 and GFP remains since both DiI-C18 and MreB are present in the cell membrane. Strain used: B. subtilis YK405 (gfp-mreB). Scale bar, 2 mm.

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Supplementary Table 1), and yet the overall uidity of the MreB triple mutant is signicantly higher (Fig. 6a). The strong increase of membrane uidity of the mreB triple mutant can therefore not

be explained by an altered fatty acid composition. This suggests that the MreB protein complex concentrates uid lipids into RIFs, which results in lower overall membrane uidity. These experiments provide further evidence for the specic role of MreB homologues in maintaining a proper lipid homeostasis in the cell membrane.

MreB is important for membrane protein distribution. The cortical actin cytoskeleton is involved in segregation of the eukaryotic cell membrane into domains that differ in uidity, including the formation of lipid rafts15,16,4245. This activity of actin inuences the localization and diffusion of many membrane proteins4650. Since MreB is an actin homologue and creates distinct membrane domains, the question arises whether the MreB cytoskeleton also inuences the distribution of membrane proteins. To test this, the MreB cytoskeleton was disrupted with CCCP, and the localization of several integral membrane proteins was analysed. Indeed, incubation with CCCP induces clustering of several membrane proteins including Fructose permease (FruA), P16.7, YhaP and YqfD and RNase Y (Fig. 7a). Other proteins, such as succinate dehydrogenase (SdhA) and F1Fo ATP

synthase (AtpA), showed no apparent difference in localization pattern (Fig. 7a). The proteins FruA, YhaP, P16.7 and YqfD show a clear clustering with Nile Red foci in CCCP-treated cells (Supplementary Fig. 12). To test whether this clustering depends on MreB, and is not caused by CCCP itself, we analysed the localization of these proteins in the mreB triple mutant strain. Indeed, in this strain background, the localization of these proteins was unaffected by CCCP (Fig. 7b). Thus, the MreB-induced uid lipid domains can inuence the distribution of membrane proteins. It should be added that the localization pattern of RNase Y is still inuenced by dissipation of membrane potential in MreB triple mutant (Fig. 7b), suggesting that in this case the effect is MreB independent.

MreB stimulates membrane protein diffusion. The MreB complexes of B. subtilis move rapidly along the cell membrane, driven by the cell wall synthesis machinery. This movement can be slowed down by inhibiting peptidoglycan synthesis with vancomycin9,10. Since MreB affects the localization of lipids and

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mreB,mbl, mreBH mreD mreC

Dil-C12, 3DDil-C12

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Figure 5 | DiI-C12-stained RIFs are MreB dependent. (a) DiI-C12 uorescence of wideeld (upper panel), and maximum intensity projection of a Z-stack (lower panel) of B. subtilis DmreC and DmreD cells, and cells that lack the three MreB homologues (DmreB, Dmbl, DmreBH). See

Supplementary Movie 1 for 3D-reconstruction, Supplementary Movie 2 for corresponding raw and deconvolved images, and Supplementary Fig. 11c for more examples. Strains used: B. subtilis 3481 (DmreC), 4311 (DmreD), 4277 (DmreB, Dmbl, DmreBH, DrsgI). (b) Colocalization of DiI-C12 and GFP-MreB in the absence of MreBCD. The maximum intensity projections of Z-stacks are depicted. See Supplementary Movie 3 for 3D-reconstruction and Supplementary Movie 4 for corresponding raw and deconvolved images. Strain used: B. subtilis HS35 (gfp-mreB DmreBCD). Scale bar, 2 mm.

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Figure 6 | Membrane uidity increases in the absence of MreB. (a) Overall membrane uidity of several cytoskeletal mutant strains was measured as Laurdan GP of logarithmically growing B. subtilis cells. As a positive control, an increase in uidity ( decrease in Laurdan GP) was established by

adding the membrane uidizer benzyl alcohol (BA; 30 mM) to wild type cells. The diagram depicts the average values and s.d. of three independent measurements. (b) Ratios between chain lengths of the major fatty acids (C17 and C15) and ratios between the iso and anteiso forms of fatty acidsare depicted for the different strains (see Supplementary Table 1 for the detailed composition). High C17/C15 or high iso/anteiso ratios indicate a reduced uidity. The diagram depicts the average values and s.d. of two independent analyses. Strains used: B. subtilis 168 (wild type), 4264 (DrsgI), 3481 (DmreC), 4277 (DmreB, Dmbl, DmreBH, DrsgI).

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a

FruA P16.7 YhaP YqfD Rny AtpA SdhA

+ CCCP CCCP

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P16.7 YhaP YqfD Rny

+ CCCP CCCP

Figure 7 | Aberrant localization of different membrane proteins. (a) The localization of several membrane proteins (FruA, P16.7, YhaP, YqfD and Rny) is changed after the MreB cytoskeleton was disturbed by addition of CCCP. Some of the foci appearing with CCCP are highlighted with arrows. The localization of other proteins (AtpA and SdhA) remains unaffected. See Supplementary Fig. 12 for more examples, and colocalization with Nile Red. Strains used: B. subtilis FruA-GFP, 110WA (p16.7-gfp), yhaP-gfp, yqfD-gfp, 3569 (rny-gfp), BS23 (atpA-gfp), BS112 (sdhA-gfp). (b) The CCCP-induced clustering of membrane proteins FruA, P16.7, YhaP and YqfD is absent in strains decient for MreB homologues. Strains used B. subtilis HS43 (rny-gfp, DmreB*),

HS44 (fruA-gfp, DmreB*), HS45 (P16.7-gfp, DmreB*), HS46 (yhaP-gfp, DmreB*), HS47 (yqfD-gfp, DmreB*). mreB* designates (DmreB, Dmbl, DmreBH, DrsgI). Scale bar, 2 mm.

membrane proteins, it is possible that the active movement of MreB also inuences the diffusion dynamics of membrane proteins. To test this, the diffusion of several membrane proteins was followed using high-speed total internal reection (TIRF) microscopy. As shown in the kymographs of Fig. 8a, the diffusion dynamics of FruA and F1Fo ATP synthase changed signicantly when the movement of MreB was stopped with vancomycin. The same behaviour was observed for two other tested transmembrane proteins (Supplementary Fig. 13a, see also Supplementary Fig. 13b for quantitation based on uorescence intensity uctuation).

In TIRF microscopy, a major determinant is the diffusion of GFP in and out of the range of the evanescent light. The reduction in membrane protein diffusion should thus increase photobleaching. Indeed, for all tested proteins, a signicant increase in diffusion-limited GFP bleaching was observed in the presence of vancomycin (Fig. 8b,c; Supplementary Fig. 14). Thus, the active movement of the MreB cytoskeleton stimulates diffusion of membrane proteins.

DiscussionDespite their small size, the cell membrane of bacteria exhibits a remarkably complex organization. There are lipid species that accumulate at cell division sites and cell poles, and most membrane proteins are not uniformly distributed over the bacterial cell membrane30,51,52. In addition, it has been reported that B. subtilis can form low uidity lipid rafts5355. The capacity of the MreB cytoskeleton to induce uid lipid domains (RIFs)

that determine the distribution of membrane proteins adds a new dimension to this complexity.

The mechanism through which MreB organizes uid lipids remains hypothetical. Peripheral membrane proteins have a capacity to induce lipid domains56,57. This activity is related to the binding of lipid bilayers by means of an intercalating amphipathic helix, but these domains are absent in MreB homologues from Gram-positive bacteria4. However, it has also been postulated that peripheral membrane proteins can directly induce uid lipid domains by reducing the ordering effect of electrostatic attraction and repulsion between charged lipid head groups58. Although this mechanism is in good agreement with the electrostatic membrane-binding mode of MreB4, we cannot rule out the involvement of other MreB-interacting proteins in the formation of RIFs.

Because of the various cellular functions of the cytoplasmic membrane, an organization into membrane areas that differ in their physical properties has a clear advantage. The optimal uid state of the membrane is a compromise between different factors. On the one hand, high membrane uidity promotes protein activity by reducing viscosity, and thus stimulating catalytic activity and diffusion of integral membrane proteins59. A higher membrane elasticity of uid membranes also allows the lipid bilayer to optimally embed membrane proteins60,61. On the other hand, high membrane uidity has the disadvantage that it increases proton permeability32,62, resulting in a less efcient energy and ion homeostasis. The MreB-dependent formation of uid lipid domains (RIFs) could therefore enable the cell to maintain an optimal condition for membrane protein activity in

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limited areas of increased uidity without compromising the proton permeability of the whole membrane.

In a recent study, it was shown that the random motion of chromosomes in bacterial and yeast cells is faster than that can be explained by simple Brownian motion. This increased molecular diffusion rate is caused by ATP-dependent processes such as RNA synthesis63. This bears some similarity with our observation that the diffusion of membrane proteins is also stimulated by an active process, in this case peptidoglycan synthesis, and the subsequent movement of RIFs. It is tempting to speculate that the continuous mixing of the cell membrane by active RIF movement promotes diffusion of proteins in a viscous membrane environment, thereby stimulating proteinprotein and proteinsubstrate interactions.

Inactivation of MreB prevents lateral cell wall synthesis resulting in deformed round cells. This effect can be explained by the inability to recruit the necessary proteins to the proper site

of synthesis. However, the uid membrane environment created by MreB might also be required for optimal cell wall synthesis since Lipid II, the membrane-anchored precursor for peptidoglycan synthesis, has a strong preference for uid membranes64. Possibly, RIFs are enriched with this essential precursor. In fact, the delocalization of MreB with CCCP does coincide with a loss of lateral Lipid II localization, based on staining with uorescently-labelled vancomycin, whereas staining of the cell division septum remains unaffected (Supplementary Fig. 15).

With its ability to organize lipids and proteins in the bacterial cell membrane, MreB bears a functional resemblance to the eukaryotic cortical actin cytoskeleton15,42,44,45. There are, however, differences. The actin cytoskeleton forms a relatively static meshwork at the membrane periphery65, yet the bacterial counterpart is more fragmented911. In eukaryotic cells, the membrane-anchored actin creates fences that restrict diffusion of proteins65, whereas the active movement of the MreB cytoskeleton stimulates diffusion. Interestingly, it was recently shown that short and highly dynamic actin polymers, which inuence clustering of membrane proteins, also exist in eukaryotic cells48. Thus, despite some differences, the ability of MreB and actin to induce and organize membrane domains appears comparable, and may explain why actin and MreB are evolutionarily linked.

Methods

Strains and growth conditions. The growth conditions and construction of strains are described in the Supplementary Materials and Methods. The used strains and conditions for gene induction are listed in Supplementary Table 2.

General uorescence microscopy. For uorescence microscopy, cells were grown to exponential growth phase at 30 C if not stated otherwise. The cells were immobilized on microscope slides covered with a thin lm of 1.2% agarose in water. For the dissipation of membrane potential with CCCP, the mounting medium was supplemented with 0.5% dimethylsulphoxide (DMSO) or 100 mM

CCCP dissolved in DMSO (0.5% nal concentration of DMSO). Imaging was carried out within 2 min after addition of ionophores. Membranes were visualized with Nile Red or FM 4-64 (0.5 mg ml 1). Standard uorescence microscopy was carried out using Zeiss Axiovert 200 M (Zeiss Plan-Neouar 100/1.30 Oil Ph3

objective), Nikon Eclipse Ti (Nikon Plan Fluor 100/1.30 Oil Ph3 DLL objective),

a

van + van

AtpA van + van

3 m

FruA

b FruA van FruA +van

AtpA van AtpA +van

Normalized fluorescence (%)

100

Figure 8 | MreB movement inuences membrane protein diffusion.(a) Kymographs visualizing the diffusion of fructose permease (FruA) and F1Fo ATP synthase (AtpA) in the absence and presence of vancomycin (van), which blocks MreB movement. Time lapse series with 20 s length and 100 ms time resolution was acquired using TIRF microscopy. See Supplementary Movie 5 for corresponding raw image series. (b) In TIRF microscopy, only proteins within the vicinity of the evanescent light (o200 nm distance from the coverslip surface) are subject to uorophore (GFP) bleaching. The graphs depict the averaged, normalized and background-subtracted uorescent signals of fructose permease (FruA) and F1Fo ATP synthase (AtpA) in the presence ( van) and absence

( van) of MreB movement. A signicant increase in bleaching is observed

when movement of MreB is inhibited with vancomycin, which indicates a reduced diffusion of proteins in and out of the range of the evanescent light. The curve ts were performed using a two-phase exponential decay model composed of a rapid bleaching of GFP within the range of the evanescent wave, and slower (diffusion limited) bleaching of the whole cell uorescence (see Supplementary Fig. 14 for details). (c) Rate constants and s.e. of slow (diffusion limited) bleaching kinetics for Fructose permease (FruA), RNaseY (Rny), F1Fo ATP synthase (AtpA) and Succinate dehydrogenase (SdhA) in the absence and presence of vancomycin.

Increased rate constant indicates a reduced exchange of protein between the TIRF-illuminated area and rest of the cell surface. The number of analysed cells, goodness-of-t and s.e. are provided in SupplementaryFig. 14. Strains used: B. subtilis BS23 (atpA-gfp), BS112 (sdhA-gfp), FruA-GFP and 3569 (rny-gfp).

80

60

0 10 20

0 10 20 Time (s)

Time (s)

c

van +van

FruA Rny AtpA SdhA

15

Diffusion limited rate constant (1s1 )

10

5

0

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4442 ARTICLE

and Applied Precision DeltaVision RT (Zeiss Plan-Neouar 100x/1.30 Oil Ph3) microscopes. The images were acquired with Metamorph 6 (Molecular Devices), softWoRx Suite (Applied Precision), and further analysed using ImageJ v.1.38 (National Institutes of Health). Deconvolution was carried from optical sections using Huygens Essentials v.3.3 (Scientic Volume Imaging).

Laurdan GP microscopy. For the microscopic analysis of membrane uidity,B. subtilis cells grown in LB medium were incubated for 5 min with 100 mM Laurdan, washed and then resuspended in fresh prewarmed LB. No inhibitory effect on growth was detected with 100 mM Laurdan. Laurdan was exited at 36020 nm, and uorescence emission was captured at 52819 nm (exposure time: 500 ms) followed by a second image at 45725 nm (exposure time: 500 ms). Image analysis and generation of a colour-coded GP map were performed with Wolfram Mathematica 7 (Wolfram Research). In brief, the background uorescence was subtracted, and GP values were calculated for each pixel, based on intensities values of the separate images, as GP (I457 I528)/(I457 I528), whereby

I457 represents the emission intensity at 45725 nm and I528 emission intensity at 52819 nm. To remove signals not associated with cells, pixel pairs were regarded as insignicant when one of the channel intensities was below a threshold of 10% of the maximal values found in the membrane. Insignicant pixel pairs were assigned a GP of zero. Finally, GP pixel values were represented as a linear colour-coded GP map, whereby blue represents high GP values (low uidity) and red represents low GP values (high uidity). Insignicant pixels (GP 0) are shown in black. The

full Wolfram Mathematica-script can be obtained from the authors upon request. The ability of the microscope-based assay to detect changes in membrane uidity was veried by measuring changes in membrane uidity upon cold shock or incubation with 30 mM benzyl alcohol66 (Supplementary Fig. 16a). For the measurement of mean GP values, the membrane uorescence signal intensities at both emission channels were extracted as maximum values from a uorescence intensity line scan perpendicular to the membrane plane. The values were measured for 70 individual cells, background subtracted, and the Laurdan GP values were calculated as described above. To prevent fast adaptation of membrane uidity, a B. subtilis strain decient for lipid desaturase (Ddes) was used in this case67. Des itself has no inuence on the appearance of RIFs upon incubation with CCCP (Supplementary Fig. 16b).

Laurdan GP spectroscopy. For the measurement of membrane uidity in batch cultures, cells were grown in LB supplemented with 0.1% glucose to an OD600

B0.5, followed by 5 min incubation with 10 mM Laurdan. Subsequently, cells were washed three times with prewarmed buffer containing 50 mM Na2HPO4/NaH2PO4 pH 7.4, 0.1% glucose and 150 mM NaCl with and without the membrane uidizer benzyl alcohol (30 mM). In case of the different Mre-mutant strains, the media and buffers were supplemented with 20 mM MgCl2. The Laurdan uorescence intensities were measured at 4355 nm and 4905 nm upon excitation at 35010 nm, using a Tecan Innite 200 M uorometer. The Laurdan GP was calculated using the formula GP (I435 I490)/(I435 I490). In both microscopic and spectroscopic

measurements, the Laurdan GP assay was able to reliably detect general changes in membrane uidity, although, due to different excitation and emission wavelengths used, the absolute values obtained with microscopy slightly differed from the normal batch uidity measurements (Supplementary Fig. 16a). The uorescence emission spectrum of Laurdan was recorded using 10 mM Laurdan embedded in liposomes upon excitation with 350 nm light using a Tecan Innite 200 M uorometer. The liposomes were formed from E. coli polar lipids using detergent dialysis method. In brief, the lipid extract was solved in chloroform followed by evaporation under dry argon stream. The lipids were resolubilized in 25 mM Tris/ HCl pH 7.4 and 1.5% octylglucoside (25 mg ml 1 lipids), followed by dialysis against 25 mM Tris/HCl pH 7.4. At last, the formed liposomes were diluted to 1:5 in H20 and sized by extrusion through a 0.1-mm membrane.

DiI-C12 staining of uid lipid domains. For staining of B. subtilis cells with DiIC12, an overnight culture was diluted 1:100 in medium supplemented with5 mg ml 1 DiI-C12 and 1% DMSO followed by growth for 2.5 h at 30 C. Because of the high uorescence of unbound dye, the stained cells were washed three times in prewarmed medium supplemented with 1% DMSO, followed by microscopy. The staining of B. subtilis with DiI-C18, which is insoluble in water, was carried out by suspending small amounts of solid DiI-C18 crystals in culture medium followed by 2.5 h incubation at 30 C, based on a method described in Kim et al.68 For microscopy, the crystals were removed by a brief low speed centrifugation. No growth inhibitory effect of the Dil-dyes was observed (Supplementary Fig. 17). For colocalization studies, the concentration of DiI-C12 was reduced to 2.5 mg ml 1.

The reason for this was the extensive overlap of the excitation spectra between DiIC12 and GFP, which results in weak GFP uorescence. The emission spectra of GFP and Dil-C12 are well separated, and no signicant bleach-through of the emitted uorescence was detected for DiI-C12 in the GFP channel, and vice versa.

TIRF time lapse microscopy. The analysis of membrane protein diffusion was carried out using a Nikon Eclipse Ti equipped with, Nikon CFI APO TIRF 100/

1.49 Oil objective, TIRF illumination module, Andor Xion X3 EMCCD-camera and 488 nm solid state laser. The cells were imaged for 20 s with 100 ms time resolution

in a continuous illumination mode. The image analysis including the generation of kymographs was carried out using ImageJ v.1.38 (National Institutes of Health) and Multiple Kymograph plugin (J. Rietdorf and A. Seitz).

TIR-CP analysis. For the analysis of GFP-bleaching kinetics upon TIRF illumination, the average uorescence intensity of cells was measured using an automated detection of cell area based on above background uorescence intensity (ImageJv.1.38, National Institutes of Health). In brief, the cell detection was performed using the rst image frame and subsequently used to measure the average uorescence intensity for each image of the time series. The background uorescence signals were extracted from the same images as the average intensity of the remaining image area not associated with cells. The background subtracted and normalized intensity values were used to t the GFP photobleach kinetics as monoor biexponential decays using GraphPad Prism 5 (GraphPad Software). The xation of GFP, applied to inhibit diffusion was performed by incubation of the cell samples with paraformaldehyde for 5 min at RT, followed by saturation of formaldehyde crosslinking by the addition of 100 mM Glycine.

Spectral imaging uorescence microscopy. The analysis of the emission wavelength spectrum for Nile Red-stained B. subtilis cells was carried out with Nikon A1R point scanning confocal microscope using Nikon Plan Apo VC 60 NA 1.4

oil objective and 561 nm excitation laser. The emission spectrum was recorded with a 4.8-nm window size for the total range of 570749 nm. Image analysis was carried out with NIS-Elements 4.0 (Nikon).

Membrane uidity-dependent Nile Red uorescence intensity. Nile Red exhibits low uorescence in a polar environment such as LB, but emits strongly when incorporated into hydrophobic cell membranes. This property was used to measure the intensity of Nile Red membrane uorescence in B. subtilis cells grown in LB at different growth temperatures. For this, growing cultures were diluted to an optical density of OD600 0.5. Subsequently, the temperature of the cultures

was rapidly adjusted to 30 C, and the uorescence increase was measured after addition of 0.5 mg ml 1 Nile Red. The measurements were carried out at 30 C using a BMG Fluostar Optima uorometer, and 584 nm excitation, and 620 nm emission lters. Background uorescence was subtracted using signals measured from Nile Red in LB. To prevent rapid adaptation of membrane uidity67,the measurements were carried out with a lipid desaturase-decient strain(B. subtilis Ddes).

Analysis of fatty acid composition. The fatty acid composition of B. subtilis wild-type cells and the cytoskeletal mutants was determined from cells grown at 30 C in LB supplemented with 20 mM MgCl2 that were collected when the cultures reached an OD600 of approximately 0.5. Fatty acids were analysed as fatty acid methyl esters using gas chromatography. All analyses were carried out in duplicates by the Identication Service of the DSMZ, Braunschweig, Germany.

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Acknowledgements

We thank Richard Daniel for strains and discussion, Alex Laude for help with spectral imaging microscopy, Darren Wilkinson and Daniel Klose for help with TIR-CP analysis, and Kursad Turgay, Stephan Gruber, Jeremy Lakey, and Tanneke den Blaauwen for critical reading of the manuscript. Funding for this research was provided by a Bio-technology and Biological Sciences Research Council (BBSRC) grant BB/I01327X/1 forL.W.H., and by the Wellcome Trust Value in People-programme for H.S.

Author contributions

H.S and F.B. carried out the experiments; H.S. and L.W.H. designed the project, analysed the data and wrote the paper.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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How to cite this article: Strahl, H. et al. The actin homologue MreB organizes the bacterial cell membrane. Nat. Commun. 5:3442 doi: 10.1038/ncomms4442 (2014).

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