Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria

Swulius, Matthew T.; Chen, Songye; Ding, H.; Li, Zhuo; Briegel, Ariane; Pilhofer, Martin; Tocheva, Elitza I.; Lybarger, Suzanne R.; Johnson, Tanya; Sandkvist, Maria; Jensen, Grant J.

doi:10.1016/j.bbrc.2011.03.062

Cited by 76 publications

(74 citation statements)

References 31 publications

(40 reference statements)

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“…S2). The heterogeneous spotty localization is consistent with the recent report that no long helical filaments are observed in E. coli by electron cryotomography (11) (Fig. 1 and Movies S1 and S2).…”

Section: Resultssupporting

confidence: 80%

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly

Teeffelen

Wang

Furchtgott

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

382

398

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Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yetunidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis.bacterial cytoskeletal dynamics | cell-wall organization | peptidoglycan synthesis | cell growth C ytoskeletal proteins play an important role in bacterial morphogenesis (1). Of the bacterial cytoskeletal proteins, the widely conserved actin homolog MreB is particularly important for bacterial cells to elongate and maintain a rod-like shape. MreB forms polymers that are associated with the cell membrane and distributed along the length of the cell in many rod-shaped bacteria (2). These polymeric MreB structures are essential for the maintenance of rod-like cell shape, as their disruption leads to cell rounding. Although MreB is essential for proper morphogenesis, bacterial cell shape is ultimately determined by the shape of the peptidoglycan cell-wall sacculus, which in turn is controlled by the cell-wall synthesis machinery. The cell wall, which is composed of stiff glycan strands cross-linked by flexible peptide linkers, forms a load-bearing structure that can counteract the intracellular turgor pressure. Cell-wall assembly requires peptidoglycan subunits to be synthesized, polymerized into glycan strands by transglycosylase enzymes, and cross-linked into the existing cell-wall network by transpeptidase enzymes. MreB directly or indirectly associates with a number of proteins that have been implicated in cell-wall assembly, such that MreB is believed to act upstream of the cell-wall assembly machinery to direct the synthesis enzymes to the sites of cell-wall insertion.Previous studies have demonstrated that MreB structures are dynamic in Bacillus subtilis and Caulobacter crescentus (3-7). To date, no motor proteins have been shown to either ...

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Section: Resultssupporting

confidence: 80%

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly

Teeffelen

Wang

Furchtgott

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

382

398

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“…This tightly packed filamentous bundle is different from the cytoplasmic filaments found in other bacteria. For example, MreB is known to form filaments, yet the reported interfilament distance is significantly larger (44). The structural characteristics of the bundle were consistent with the molecular arrangement of DNA observed in Deinococcus radiodurans nucleoids, in which cholesteric liquid crystalline order was observed and the average interfilament distance was 4 nm (10).…”

Section: Cryo-electron Tomography Of Leptospirasupporting

confidence: 66%

Three-Dimensional Structures of Pathogenic and Saprophytic Leptospira Species Revealed by Cryo-Electron Tomography

Raddi¹,

Morado²,

Yan

et al. 2012

J Bacteriol

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e Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. An in-depth structural characterization of L. interrogans is needed to understand its biology and pathogenesis. In this study, cryo-electron tomography (cryo-ET) was used to compare pathogenic and saprophytic species and examine the unique morphological features of this group of bacteria. Specifically, our study revealed a structural difference between the cell envelopes of L. interrogans and Leptospira biflexa involving variations in the lipopolysaccharide (LPS) layer. Through cryo-ET and subvolume averaging, we determined the first three-dimensional (3-D) structure of the flagellar motor of leptospira, with novel features in the flagellar C ring, export apparatus, and stator. Together with direct visualization of chemoreceptor arrays, DNA packing, periplasmic filaments, spherical cytoplasmic bodies, and a unique "cap" at the cell end, this report provides structural insights into these fascinating Leptospira species.

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“…The helices were not simply made detectable to ECT by the additional mass of the YFP, since filaments were not seen in the MreB-RFP SW fusion (where not just many copies but every copy of MreB was fused to a fluorescent protein), and we and others have previously resolved other actin homologs and thin cytoskeletal filaments within intact bacteria by ECT (17,18,20,23,25,29). While it is true that if MreB filaments were embedded within the membrane they would be masked to ECT, MreB is known to bind directly to the membrane and be clearly visible by ECT, at least in vitro (24).…”

Section: Resultsmentioning

confidence: 95%

The Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag

2012

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Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFP SW ) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, "patchy" localization patterns of MreB-RFP SW , even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.

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Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria

Cited by 76 publications

References 31 publications

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly

Three-Dimensional Structures of Pathogenic and Saprophytic Leptospira Species Revealed by Cryo-Electron Tomography

The Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag

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