Title: Filament Structure, Organization, and Dynamics in MreB Sheets
Abstract: In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several μm long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions. In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several μm long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions. IntroductionDespite usually being constrained by a cell wall, bacterial shapes are highly diverse, reflecting the large phylogenetic range. For example Escherichia coli, Bacillus subtilis, and Thermatoga maritime are straight rods, Vibrio cholera is a curved rod, Borrelia burgdorferi forms flat waves, whereas Spiroplasma species are helical. One of the main cytoskeletal proteins involved in determining the shapes of bacteria is thought to be MreB an actin homolog whose atomic structure is very similar to G-actin, despite the low sequence homology (1van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (605) Google Scholar). It can assemble into polymers both in vitro (1van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (605) Google Scholar) and in vivo (2Jones L.J. Carballido-López R. Errington J. Cell. 2001; 104: 913-922Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). Several studies suggest that MreB plays roles in chromosome segregation (3Kruse T. Møller-Jensen J. Løbner-Olesen A. Gerdes K. EMBO J. 2003; 22: 5283-5292Crossref PubMed Scopus (237) Google Scholar), polar localization of proteins (4Gitai Z. Dye N. Shapiro L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 8643-8648Crossref PubMed Scopus (256) Google Scholar), and maintenance of cell shape and resistance to external mechanical stresses (2Jones L.J. Carballido-López R. Errington J. Cell. 2001; 104: 913-922Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). Peptidoglycan cell wall synthesis has been linked to the role of the MreB homolog MbI in B. subtilis (5Daniel R.A. Errington J. Cell. 2003; 113: 767-776Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar); however, mechanisms by which MreB may provide mechanical support either directly to the cell or indirectly by affecting peptidoglycan wall integrity remain unclear.In vivo studies of MreB have mainly been limited to visualization under the fluorescence microscope (2Jones L.J. Carballido-López R. Errington J. Cell. 2001; 104: 913-922Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar, 5Daniel R.A. Errington J. Cell. 2003; 113: 767-776Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). At the low resolution of this technique (∼0.2 μm), MreB was seen to form cable-like structures, which spiral around the periphery of the cell in B. subtilis, presumably just underneath the cytoplasmic membrane. By electron microscopy (EM), 2The abbreviations used are: EMelectron microscopyHP-SAXShigh pressure small angle x-ray scatteringTIRFtotal internal reflection fluorescence microscopyAMPPNP5′-adenylyl imidodiphosphateGMPPNPguanyl-5′-yl imidodiphosphate. MreB has been observed in vitro to form straight or curved sheets and bundles (1van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (605) Google Scholar, 6Esue O. Cordero D. Wirtz D. Tseng Y. J. Biol. Chem. 2005; 280: 2628-2635Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar).Using cryoelectron tomography of helical-shaped bacteria Spiroplasma melliferum at an intermediate resolution of ∼40 Å, three parallel sheet-like structures underneath the cell membrane were identified. At this low resolution, the molecular structures and organization remained elusive, and it was unclear which of the ribbons may be associated with MreB filaments (7Kürner J. Frangakis A.S. Baumeister W. Science. 2005; 307: 436-438Crossref PubMed Scopus (182) Google Scholar).Because of these shortcomings, we found it necessary to clarify the molecular structure of MreB in vitro by high resolution electron microscopy. We show that MreB from T. maritime forms novel sheets consisting of interwoven helical-like filaments, which may be related to the cables observed by light microscopy and the ribbons observed by cryoelectron tomography in vivo. This design has appeal as it will be mechanically stronger than a simple sheet constructed of linear filaments and may be of importance for maintaining bacterial cell shape. We have characterized various MreB supramolecular structures under a wide variety of conditions by electron microscopy and studied their polymerization properties, steady state dynamics, and stability of MreB filaments by light scattering, TIRF microscopy, and high pressure small angle x-ray scattering (HP-SAXS).DISCUSSIONMutations in MreB resulted in a shape conversion from rod to sphere morphology (25Wachi M. Doi M. Tamaki S. Park W. Nakajima-Iijima S. Matsuhashi M. J. Bacteriol. 1987; 169: 4935-4940Crossref PubMed Scopus (192) Google Scholar), implying a role for MreB in cell shape determination. Disruption of pole-to-pole MreB helically arranged cables by mutational inactivation resulted in failure of lateral murein synthesis and formation of spherical cells (26Dye N.A. Pincus Z. Theriot J.A. Shapiro L. Gitai Z. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 18608-18613Crossref PubMed Scopus (115) Google Scholar). Disruption in the two genes immediately downstream of MreB, MreC, and MreD also yielded spherical phenotypes in E. coli and B.subtilis (27Kruse T. Bork-Jensen J. Gerdes K. Mol. Microbiol. 2005; 55: 78-89Crossref PubMed Scopus (298) Google Scholar, 28Leaver M. Errington J. Mol. Microbiol. 2005; 57: 1196-1209Crossref PubMed Scopus (127) Google Scholar). In B. subtilis the synthesis of lateral murein is governed by MreB homologue MbI (5Daniel R.A. Errington J. Cell. 2003; 113: 767-776Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar) and the localizations of GFP-MreC and GFP-MreD were observed to be similar to that of MbI (28Leaver M. Errington J. Mol. Microbiol. 2005; 57: 1196-1209Crossref PubMed Scopus (127) Google Scholar). Under the light microscope, all of these proteins followed helical patterns underneath the cell membrane.In a bacterial two-hybrid system, E. coli MreC directly interacted with MreB and MreD (27Kruse T. Bork-Jensen J. Gerdes K. Mol. Microbiol. 2005; 55: 78-89Crossref PubMed Scopus (298) Google Scholar). In addition, the bitopic membrane protein MreC interacts with several members of the PBP family of bifunctional transglycosylase-transpeptidases (29Divakaruni A.V. Loo R.R. Xie Y. Loo J.A. Gober J.W. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 18602-18607Crossref PubMed Scopus (113) Google Scholar), which are thought to participate in both lateral and septal murein synthesis. These results suggest that murein synthesis in E. coli is carried out by a helical array of interacting proteins that includes MreB, MreC, MreD, and members of the PBP family. The model predicts that new peptidoglycan is inserted into the lateral cell wall in a helical pattern that reflects the helical pattern of the biosynthesis complex.Despite this knowledge, the actual role of the MreB cytoskeleton remains unclear, as helical distributions of MreC and MreD in E. coli observed by light microscopy were found to be independent of MreB (26Dye N.A. Pincus Z. Theriot J.A. Shapiro L. Gitai Z. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 18608-18613Crossref PubMed Scopus (115) Google Scholar) and in Caulobacter, GFP-PBP2 adopted a helical distribution, which partially overlapped with MreC but not with MreB. In the absence of electron tomography data of T. maritime, the in vivo molecular structure of MreB within the cable-like spirals observed by light microscopy remains elusive. Only one electron tomography study of the helical bacteria Spiroplasma showed ribbon-like structures underneath the cell membrane (7Kürner J. Frangakis A.S. Baumeister W. Science. 2005; 307: 436-438Crossref PubMed Scopus (182) Google Scholar), but the molecular structure and organization within the ribbons could not be unambiguously determined.Here we have shown that in the presence of NTP Thermatoga MreB formed multilayered sheets of apparently interwoven filaments. The mechanical rigidity of these architectures can be expected to be substantially higher than that of a simple set of parallel linear protofilaments. Indeed, the HP-SAX experiments indicate that MreB filaments have a mechanical resilience similar to F-actin, which is the highest of any cytoskeletal protein known.Recent rheology experiments have characterized MreB from T. maritime to have an elastic module that is more than 3-fold higher than F-actin (30Esue O. Wirtz D. Tseng Y. J. Bacteriol. 2006; 188: 968-976Crossref PubMed Scopus (54) Google Scholar). Because the elasticity of a polymer network is predicted to depend weakly on the intrinsic rigidity of the filaments, this high elastic modulus can be mostly accounted for by strong interfilament interactions. The elasticity and concentration of MreB are related by a power law relationship between network elasticity (G′) and protein concentration (C): G′ ∼ C0.44 and G' ∼ C0.29 for MreB-ATP and MreB-GTP, respectively (30Esue O. Wirtz D. Tseng Y. J. Bacteriol. 2006; 188: 968-976Crossref PubMed Scopus (54) Google Scholar), which is consistent with that predicted for cross-linked filaments. In contrast, non-cross-linked and entangled semiflexible polymers, such as F-actin, have a predicted power-law relationship G′ ∼ C1.4 (31Palmer A. Xu J. Kuo S.C. Wirtz D. Biophys J. 1999; 76: 1063-1071Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The lower concentration dependence index of GTP-MreB indicates that GTP-MreB filaments are more strongly cross-linked than ATP-MreB filaments (30Esue O. Wirtz D. Tseng Y. J. Bacteriol. 2006; 188: 968-976Crossref PubMed Scopus (54) Google Scholar). This is consistent with our electron microscopy observations that Thermatoga NTP-MreB forms sheets consisting of interwoven filaments and that GTP-MreB sheets show a higher degree of order than ATP-MreB sheets. The strong interaction of MreB filaments within the sheets observed here should be able to resist large bending and compression forces and help in imposing a rigid cylindrical architecture to rod-shaped bacterial cells.Individual MreB filaments within the sheets formed in the presence of GTP or ATP were not linear protofilaments as described earlier (1van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (605) Google Scholar), which may have to do with the fact that the previous study used a C-terminally His-tagged MreB. Rather untagged MreB assembled into single-stranded sinusoidally varying or helical 3/1 filaments with a repeat of about 200 Å (Fig. 3), whereas in the presence of GDP or ADP linear protofilaments were observed (Fig. 1, B and C). This may indicate that the binding of GTP or ATP to MreB monomers stabilizes a different conformation than ADP or GDP, which in turn leads to the preferential formation of either helical filaments or linear protofilaments. It is unknown whether bacterial cells utilize ATP, GTP, or both nucleotides to assemble MreB in vivo and whether the cell exploits different nucleotides for specific MreB-based functions. In nitrogen-fixing bacteria like T. maritime the rate of ATP regeneration is considered to be growth rate-limiting because of the high ATP requirement for N2 fixation (32Upchurch R.G. Mortenson L.E. J. Bacteriol. 1980; 143: 274-284Crossref PubMed Google Scholar), and the ATP/ADP ratio was estimated to be between 1.4 and 2.0. In E. coli, during exponential growth, the ATP and GTP concentrations are reported to be relatively constant at about 3 mm and 900 μm, respectively (33Jewett M.C. Miller M.L. Chen Y. Swartz J.R. J. Bacteriol. 2009; 191: 1083-1091Crossref PubMed Scopus (45) Google Scholar). Assuming similar concentrations in T. maritime, the nucleotide concentrations can be estimated to be about 2 mm ATP, 1 mm ADP, and 1 mm GTP. Should such concentrations lead to interwoven or linear sheet structures at the molecular level or coexistence of both forms in T. maritime is an open question.It should be noted that opposed to most rod-shaped bacteria described above where MreB is thought to shape the cell wall via MreC, MreC is not present in T. maritime (34Margolin W. Curr. Biol. 2009; 19: R812-R822Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). It may be that different bacteria which evolved during evolution and adopted to specific environments have found different ways to elongate their cell wall while maintaining a constant cell width between each division. Other protein factors like Rod Z regulate MreB assembly in rod-shaped bacteria like E. coli, while cell division protein FtsZ is required for the collapse of MreB cables from extended helices into rings near the future division site (34Margolin W. Curr. Biol. 2009; 19: R812-R822Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Whether any of the MreB-associated proteins preferentially binds to MreB-NTP or MreB-NDP cables is unknown.MreB has been shown to be a determinant of polar protein localization and the translocation of chromosomal origins toward cell poles in both Caulabacter and E. coli (3Kruse T. Møller-Jensen J. Løbner-Olesen A. Gerdes K. EMBO J. 2003; 22: 5283-5292Crossref PubMed Scopus (237) Google Scholar, 35Gitai Z. Dye N.A. Reisenauer A. Wachi M. Shapiro L. Cell. 2005; 120: 329-341Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) leading to the hypothesis that MreB structures possess a uniform polarity that can be interpreted by trafficking factors. The organization of sheets indicates the possibility that both Thermatoga MreB-NTP and MreB-NDP may form polar suprastructures.The kinetic dynamics of Thermatoga MreB-NTP filament formation was faster than F-actin but significantly slower than ParM (8Popp D. Narita A. Oda T. Fujisawa T. Matsuo H. Nitanai Y. Iwasa M. Maeda K. Onishi H. Maéda Y. EMBO J. 2008; 27: 570-579Crossref PubMed Scopus (68) Google Scholar, 21Iwasa M. Maeda K. Narita A. Maeda Y. Oda T. J. Biol. Chem. 2008; 283: 21045-21053Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The critical concentration of MreB-NTP filament formation was similar to ParM, yet assembly under conditions closer to physiological occurred without a nucleation step, making Thermatoga MreB a very efficient polymerizing machine. Polymerization kinetics and critical concentration differed substantially from a previous study (6Esue O. Cordero D. Wirtz D. Tseng Y. J. Biol. Chem. 2005; 280: 2628-2635Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) which had used C-terminal His-tagged Thermatoga MreB, but was more similar to the work of Bean and Amann (18Bean G.J. Amann K.J. Biochemistry. 2008; 47: 826-835Crossref PubMed Scopus (41) Google Scholar), which also employed native MreB, although prepared differently than in our study. The variations in the biochemistry may reflect the differences in molecular structure and organization of Thermatoga MreB suprastructures we have observed here compared with the only previous structural study which used a His-tagged construct (1van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (605) Google Scholar).Time-lapse TIRF microscopy of Thermatoga MreB-NTP sheets at steady state support a treadmilling process consistent with in vivo observations of MreB in Caulobacter (23Kim S.Y. Gitai Z. Kinkhabwala A. Shapiro L. Moerner W.E. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10929-10934Crossref PubMed Scopus (183) Google Scholar). Yet interpreting of treadmilling in terms of monomer motions is complicated by the fact that we are dealing with complex interwoven sheets rather than single filaments. Second, the site of GTP hydrolysis and what triggers hydrolysis is one of the major unknowns. Nevertheless, our findings define clear predictions on the structures, dynamics, and functions of T. maritime MreB, which can be tested by future in vivo light microscopy and electron tomography studies. IntroductionDespite usually being constrained by a cell wall, bacterial shapes are highly diverse, reflecting the large phylogenetic range. For example Escherichia coli, Bacillus subtilis, and Thermatoga maritime are straight rods, Vibrio cholera is a curved rod, Borrelia burgdorferi forms flat waves, whereas Spiroplasma species are helical. One of the main cytoskeletal proteins involved in determining the shapes of bacteria is thought to be MreB an actin homolog whose atomic structure is very similar to G-actin, despite the low sequence homology (1van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (605) Google Scholar). It can assemble into polymers both in vitro (1van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (605) Google Scholar) and in vivo (2Jones L.J. Carballido-López R. Errington J. Cell. 2001; 104: 913-922Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). Several studies suggest that MreB plays roles in chromosome segregation (3Kruse T. Møller-Jensen J. Løbner-Olesen A. Gerdes K. EMBO J. 2003; 22: 5283-5292Crossref PubMed Scopus (237) Google Scholar), polar localization of proteins (4Gitai Z. Dye N. Shapiro L. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 8643-8648Crossref PubMed Scopus (256) Google Scholar), and maintenance of cell shape and resistance to external mechanical stresses (2Jones L.J. Carballido-López R. Errington J. Cell. 2001; 104: 913-922Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). Peptidoglycan cell wall synthesis has been linked to the role of the MreB homolog MbI in B. subtilis (5Daniel R.A. Errington J. Cell. 2003; 113: 767-776Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar); however, mechanisms by which MreB may provide mechanical support either directly to the cell or indirectly by affecting peptidoglycan wall integrity remain unclear.In vivo studies of MreB have mainly been limited to visualization under the fluorescence microscope (2Jones L.J. Carballido-López R. Errington J. Cell. 2001; 104: 913-922Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar, 5Daniel R.A. Errington J. Cell. 2003; 113: 767-776Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). At the low resolution of this technique (∼0.2 μm), MreB was seen to form cable-like structures, which spiral around the periphery of the cell in B. subtilis, presumably just underneath the cytoplasmic membrane. By electron microscopy (EM), 2The abbreviations used are: EMelectron microscopyHP-SAXShigh pressure small angle x-ray scatteringTIRFtotal internal reflection fluorescence microscopyAMPPNP5′-adenylyl imidodiphosphateGMPPNPguanyl-5′-yl imidodiphosphate. MreB has been observed in vitro to form straight or curved sheets and bundles (1van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (605) Google Scholar, 6Esue O. Cordero D. Wirtz D. Tseng Y. J. Biol. Chem. 2005; 280: 2628-2635Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar).Using cryoelectron tomography of helical-shaped bacteria Spiroplasma melliferum at an intermediate resolution of ∼40 Å, three parallel sheet-like structures underneath the cell membrane were identified. At this low resolution, the molecular structures and organization remained elusive, and it was unclear which of the ribbons may be associated with MreB filaments (7Kürner J. Frangakis A.S. Baumeister W. Science. 2005; 307: 436-438Crossref PubMed Scopus (182) Google Scholar).Because of these shortcomings, we found it necessary to clarify the molecular structure of MreB in vitro by high resolution electron microscopy. We show that MreB from T. maritime forms novel sheets consisting of interwoven helical-like filaments, which may be related to the cables observed by light microscopy and the ribbons observed by cryoelectron tomography in vivo. This design has appeal as it will be mechanically stronger than a simple sheet constructed of linear filaments and may be of importance for maintaining bacterial cell shape. We have characterized various MreB supramolecular structures under a wide variety of conditions by electron microscopy and studied their polymerization properties, steady state dynamics, and stability of MreB filaments by light scattering, TIRF microscopy, and high pressure small angle x-ray scattering (HP-SAXS).