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ABSTRACT: The Escherichia coli RNA degradosome proteins are organized into a helical cytoskeletal-like structure within the cell. Here we describe the ATP-dependent assembly of the RhlB component of the degradosome into polymeric filamentous structures in vitro, which suggests that extended polymers of RhlB are likely to comprise a basic core element of the degradosome cytoskeletal structures.
Journal of bacteriology 04/2010; 192(12):3222-6. · 3.94 Impact Factor
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ABSTRACT: Ribonuclease E (RNase E) is a component of the Escherichia coli RNA degradosome, a multiprotein complex that also includes RNA helicase B (RhlB), polynucleotide phosphorylase (PNPase) and enolase. The degradosome plays a key role in RNA processing and degradation. The degradosomal proteins are organized as a cytoskeletal-like structure within the cell that has been thought to be associated with the cytoplasmic membrane. The article by Khemici et al. in the current issue of Molecular Microbiology reports that RNase E can directly interact with membrane phospholipids in vitro. The RNase E-membrane interaction is likely to play an important role in the membrane association of the degradosome system. These findings shed light on important but largely unexplored aspects of cellular structure and function, including the organization of the RNA processing machinery of the cell and of bacterial cytoskeletal elements in general.
Molecular Microbiology 12/2008; 70(4):780-2. · 5.01 Impact Factor
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ABSTRACT: The RNA degradosome of Escherichia coli is a multiprotein complex that plays an essential role in normal RNA processing and decay. It was recently shown that the major degradosome constituents are organized in a coiled cytoskeletal-like structure that extends along the length of the cell. Here we show that the endoribonuclease E (RNaseE) and RNA helicase B (RhlB) components of the degradosome can each independently form coiled structures in the absence of the other degradosome proteins. In contrast, the cytoskeletal organization of the other degradosome proteins required the presence of the RNaseE or RhlB coiled elements. Although the RNaseE and RhlB structures were equally competent to support the helical organization of polynucleotide phosphorylase, the cytoskeletal-like organization of enolase occurred only in the presence of the RNaseE coiled structure. The results indicate that the RNA degradosome proteins are components of the bacterial cytoskeleton rather than existing as randomly distributed multiprotein complexes within the cell and suggest a model for the cellular organization of the components within the helical degradosomal structure.
Journal of Biological Chemistry 06/2008; 283(20):13850-5. · 4.77 Impact Factor
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ABSTRACT: RNaseE is the main component of the RNA degradosome of Escherichia coli, which plays an essential role in RNA processing and decay. Localization studies showed that RNaseE and the other known degradosome components (RNA helicase B, polynucleotide phosphorylase, and enolase) are organized as helical filamentous structures that coil around the length of the cell. These resemble the helical structures formed by the MreB and MinD cytoskeletal proteins. Formation of the RNaseE cytoskeletal-like structure requires an internal domain of the protein that does not include the domains required for any of its known interactions or the minimal domain required for endonuclease activity. We conclude that the constituents of the RNA degradosome are components of the E. coli cytoskeleton, either assembled as a primary cytoskeletal structure or secondarily associated with another underlying cytoskeletal element. This suggests a previously unrecognized role for the bacterial cytoskeleton, providing a mechanism to compartmentalize proteins that act on cytoplasmic components, as exemplified by the RNA processing and degradative activities of the degradosome, to regulate their access to important cellular substrates.
Proceedings of the National Academy of Sciences 02/2007; 104(5):1667-72. · 9.68 Impact Factor
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ABSTRACT: Division site placement in Escherichia coli involves interactions of the MinD protein with MinC and MinE and with other MinD molecules to form membrane-associated polymeric structures. In this work, as part of a study of these interactions, we established that heterologous membrane-associated proteins such as MinD can be targeted to the yeast nuclear membrane, dependent only on the presence of a membrane-binding domain and a nuclear targeting sequence. Targeting to the nuclear membrane was equally effective using the intrinsic MinD membrane-targeting domain or the completely unrelated membrane-targeting domain of cytochrome b(5). The chimeric proteins differing in their membrane-targeting sequences were then used to establish the roles of membrane association and specificity of the membrane anchor in MinD interactions, using the yeast two-hybrid system. The chimeric proteins were also used to show that the membrane association of MinD and MinE in E. coli cells had no specificity for the membrane anchor, whereas formation of MinDE polar zones and MinE rings required the presence of the native MinD membrane-targeting sequence.
Journal of Bacteriology 05/2006; 188(8):2993-3001. · 3.83 Impact Factor
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ABSTRACT: The site of cell division in bacterial cells is placed with high fidelity at a designated position, usually the midpoint of the cell. In normal cell division in Escherichia coli this is accomplished by the action of the Min proteins, which maintain a high concentration of a septation inhibitor near the ends of the cell, and a low concentration at midcell. This leaves the midcell site as the only available location for formation of the division septum. In other species, such as Bacillus subtilis, this general paradigm is maintained, although some of the proteins differ and the mechanisms used to localize the proteins vary. A second mechanism of negative regulation, the nucleoid-occlusion system, prevents septa forming over nucleoids. This system functions in Gram-negative and Gram-positive bacteria, and is especially important in cells that lack the Min system or in cells in which nucleoid replication or segregation are defective. Here, we review the latest findings on these two systems.
Nature Reviews Microbiology 01/2006; 3(12):959-68. · 21.18 Impact Factor
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ABSTRACT: A lthough eukaryotic cells contain a complex internal cytoskeleton, until recently microbiologists believed that bacteria contained no compara-ble elements except for the murein exoskeleton located outside the cytoplasmic membrane. Indeed, the absence of a cytoskele-ton was one of the hallmarks used to distinguish bacteria from eukaryotic cells. That view changed dramatically in 2001 when Laura Jones, Rut Carballido-Ló pez, and Jeff Errington of Oxford University, Oxford, United Kingdom, described the cytoskeletal nature of the actin-like MreB and Mbl proteins of Bacillus subtilis. Within these rod-shaped gram-positive cells, the two proteins form structures that extend be-tween the two poles and help to regulate cell shape. This finding prompted a period of rapid progress that changed our view of bacterial cel-lular organization. The eukaryotic cytoplasm has long been known to contain several types of extended cy-toskeletal networks, composed of microtubules, intermediate filaments, and actin filaments, that communicate with other intracellular and mem-brane-associated components. It is now clear that bacteria contain proteins resem-bling both the actin and nonactin cytoskel-etal proteins of eukaryotic cells as well as proteins unrelated to the eukaryotic cy-toskeletal proteins, organized into extended membrane-associated structures. These structures provide long-range order to the cell—far more than microbiologists once recognized. Homologs of Actin Help To Shape Bacterial Cells Cells of B. subtilis contain three actin ho-mologs, designated MreB, Mbl (Fig. 1B), and MreBH. Further, most other types of rod-shaped bacteria, including E. coli, contain actin-like proteins homologous to MreB (Fig. 1C). In contrast, MreB-related proteins are absent from those coccal species whose genomic sequences are known, supporting the idea that a major role of MreB is to support establishment of a rod shape. The three-dimensional structures of MreB and actin are very similar, as shown by F. van den Ent, Linda Amos, and Jan Lö we of the MRC Laboratory of Molecular Biology, Cam-bridge, United Kingdom. Within the cell, MreB is organized into two helical strands composed of actin-like protofila-ments, running along the inner surface of the cytoplasmic membrane and usually coiling around the rod-shaped cell along its long axis (Fig. 1C). The coiled structure changes its ap-pearance and position during the cell cycle, ac-cording to work from the laboratories of James Gober of the University of California, Los An-geles (UCLA) and Lucy Shapiro of Stanford University, Stanford, Calif. At a specific stage of the C. crescentus cell cycle, MreB rearranges to form a ring-like structure near the division site. • Bacteria, long thought to lack a cytoskeleton,
ASM news 01/2005; 71(12):582-586. · 0.95 Impact Factor
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ABSTRACT: In this report, we show that yccV, a gene of unknown function, encodes a protein having an affinity for a hemimethylated oriC DNA and that the protein negatively controls dnaA gene expression in vivo.
Journal of Bacteriology 06/2003; 185(9):2967-71. · 3.83 Impact Factor
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ABSTRACT: Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo.We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region.SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.
Genes to Cells 12/2001; 5(11):873 - 884. · 2.68 Impact Factor
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ABSTRACT: The lacZ–hobH fusion clone, containing an Escherichia coli DNA segment located at 92 min on the chromosomal map, was screened as a producer of E. coli oriC hemi-methylated binding activity. We have purified the protein encoded by this locus to near homogeneity. The protein corresponds to the monomeric form of a non-specific acid phosphatase (NAP) whose gene has been designated aphA. oriC DNA footprinting experiments showed protection of hemi-methylated probe by partially purified NAP, but not by purified preparations. Yet, gel retardation experiments with an oriC oligonucleotide demonstrated DNA binding activity of purified NAP in the presence of Mg2+. This experiment also showed an increased affinity of the protein for the hemi-methylated probe compared with the fully or unmethylated form. Indirect immunofluorescence microscopy revealed the existence of discrete NAP foci at mid-cell in cells with two nucleoids, but at cell poles in those with one nucleoid.
Molecular Microbiology 12/1998; 31(1):167 - 175. · 5.01 Impact Factor
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ABSTRACT: The hemimethylated oriC binding activity of the E. coli heavy density membrane fraction (outer membrane) was investigated by DNase I footprinting experiments using membranes obtained from different replication stages of PC-2 (dnaCts) cells. The maximal binding activity was found at the beginning of replication cycle and then decreased gradually. The same pattern of variation was observed with SeqA protein detected in the membranes by immunoblotting. Both binding activity and the presence of SeqA were conserved in the outer membrane even after floating centrifugation of the heavy density membrane fraction in a sucrose gradient, indicating that SeqA in fact can associate with the membrane and that this association varies according to replication cycle. Site specific binding to hemimethylated oriC, of the heavy density membrane obtained from seqA mutant, could be restored by addition of a low amount of His-tagged SeqA protein.
Biochimie.
