Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane of<i>Escherichia coli</i>

Ghosh, Anindya S.; Young, Kevin

doi:10.1128/jb.187.6.1913-1922.2005

Cited by 70 publications

(80 citation statements)

References 60 publications

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“…Spiral localizations have also been observed for other proteins that polymerize [such as FtsZ (41,42) and MinD (15)], proteins that cannot polymerize [such as Pbp2 (12) and SetB (43)], and even nonprotein molecules [such as LPS (44) and nascent peptidoglycan (14)]. Here, we show that MreC appears as a spiral in the periplasmic compartment of Caulobacter cells.…”

Section: Discussionmentioning

confidence: 65%

Two independent spiral structures control cell shape inCaulobacter

Dye

Pincus

Theriot

et al. 2005

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

123

158

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The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter.actin ͉ MreB ͉ MreC ͉ Pbp2 P rokaryotes exhibit a wide variety of cell shapes (including rods, spheres, spirals, squares, and stars), but the mechanisms by which these shapes are achieved are poorly understood. The extracellular peptidoglycan layer provides structural rigidity for bacterial cells and is of central importance in the establishment and maintenance of cell shape (1, 2). This layer is a meshwork of disaccharide chains (alternating N-acetylglucosamine and N-acetylmuramic acid sugars) cross-linked by short peptide bridges. Rod-shaped bacteria are believed to possess two peptidoglycan synthesis complexes with distinct activities: one for elongation along the cell length and the other for cell division (1, 3, 4). It is thought that maintenance of a regular rod shape requires the activities of these two complexes to be carefully balanced (3).The bacterial cytoskeleton also plays a role in the establishment and maintenance of cell shape (2). Homologs for the three major types of eukaryotic cytoskeletal elements have been identified in bacteria: the actin homolog is MreB, the tubulin homolog, FtsZ, and the intermediate filament homolog, Crescentin (5). Of these known cytoskeletal elements, MreB is the only one required to establish an underlying rod-like character. Mutations in mreB confer a spherical-like morphology to the normally rod-like cells of Escherichia coli, Bacillus subtilis, and Caulobact...

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Section: Discussionmentioning

confidence: 65%

Two independent spiral structures control cell shape inCaulobacter

Dye

Pincus

Theriot

et al. 2005

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

123

158

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“…3G). This pattern was reminiscent of many helical patterns present in bacterial cells (20,(22)(23)(24)(25)(26)(27)(28). Note, however, that these RNArelated patterns were more diffuse than most helical patterns revealed by protein labeling.…”

Section: Estimation Of Number Of Egfp Molecules Per Cell After Rna Inmentioning

confidence: 84%

Spatiotemporal patterns and transcription kinetics of induced RNA in single bacterial cells

Valencia-Burton

Shah

Sutin

et al. 2009

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

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Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells. Our results reveal that the target RNA is not evenly distributed within the cell and localizes laterally along the long cell axis, in a pattern suggesting the existence of ordered helical RNA structures reminiscent of known bacterial cytoskeletal cellular elements.espite their relatively small dimensions, bacterial cells show a remarkable, rich internal subcellular organization that has captured the interest of researchers over the past decade (1-4). Many cytoplasmic and membrane proteins, particularly those involved in cell division, DNA replication, and chromosome segregation, have specific subcellular localizations that can change quickly over time in response to cell cycle progression, motility, and environmental cues. This dynamic and organized behavior is also true for bacterial chromosomal DNA. The use of GFP fusions and in situ fluorescence hybridization (FISH) have shown that every chromosomal locus has a defined subcellular address and is replicated and segregated into the new cell as part of an active and directed process (4, 5). Bacterial plasmids, both low and high copy, also have specific cellular addresses and segregate in a fashion that is unique for a given plasmid (6-8).Little is known, however, about RNA dynamics in bacteria. With the advent of new methods to label RNA in live cells, the transcription kinetics, localization, and movement of RNA in the bacterium Escherichia coli has begun to be discerned only recently (9-13).To understand RNA dynamics in live cells better, it would be useful to develop RNA labeling methods that would allow direct visualization and real-time quantitation of RNAs with low background levels. We recently reported a system based on protein complementation that uses binding of a split and inactive protein complex to a short interacting sequence on a target RNA. The marker protein re-associates and becomes fluorescent only upon binding to RNA (13), which makes this approach more desirable than alternative techniques relying on expression of full-size fluorescent proteins. Briefly, the method consists of fusing the Nterminal fragment of EGFP to the N-terminal domain of an RNA-binding protein, the eukaryotic initiation factor 4A (eIF4A), via a polypeptide linker. Similarly, the C-terminal fragment of EGFP is fused to the C-terminal domain of eIF4A. The target RNA is tagged at the 3Ј end with an aptamer sequence known to bind eIF4A with high affinity (14) (Fig. 1A). Expression of the labeling components in E. coli cells generates a fluorescent signal only in the presence of the target RNA, caused by the reassociation of the two EGFP fragments and forma...

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“…However, it is now clear that physical linkages between the membranes do occur in flagella basal bodies (53); drug efflux pumps (54); and type I (55), III (56), and IV (57) protein secretion (58) and capsule export machinery (22). More recent studies have supported the notion that the insertion sites are not random but instead follow a helical pattern (59). The discovery of a soluble periplasmic LPS-binding protein (LptA) does not necessarily preclude additional components in a molecular scaffold spanning the periplasm nor does it argue against specific sites of insertion, as these could be determined by the distribution and location of the outer membrane components LptD and LptE.…”

Section: Discussionmentioning

confidence: 95%

The LptA Protein of Escherichia coli Is a Periplasmic Lipid A-binding Protein Involved in the Lipopolysaccharide Export Pathway

Tran

Trent

Whitfield

2008

Journal of Biological Chemistry

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The LptA protein of Escherichia coli has been implicated in the transport of lipopolysaccharide (LPS) from the inner membrane to the outer membrane. Here we provide evidence that LptA binds structurally diverse LPS substrates in vitro and demonstrate that it interacts specifically with the lipid A domain of LPS. These results are consistent with LptA playing a chaperone role in the transport of LPS across the periplasm and have implications for possible assembly models.

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Helical Disposition of Proteins and Lipopolysaccharide in the Outer Membrane ofEscherichia coli

Cited by 70 publications

References 60 publications

Two independent spiral structures control cell shape inCaulobacter

Two independent spiral structures control cell shape inCaulobacter

Spatiotemporal patterns and transcription kinetics of induced RNA in single bacterial cells

The LptA Protein of Escherichia coli Is a Periplasmic Lipid A-binding Protein Involved in the Lipopolysaccharide Export Pathway

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