Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli

Ho, Thanh Quoc

doi:10.1093/emboj/21.7.1864

Cited by 71 publications

(64 citation statements)

References 66 publications

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“…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)(7)(8).…”

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confidence: 99%

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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confidence: 99%

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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“…Contrary to the generally accepted view that plasmids randomly diffuse throughout the cell, it has now been shown that the low-copy-number plasmids F (16, 18, 49), P1 (16, 45), and R1 (31, 63) and the multicopy plasmids RK2 and pUC (4,25,53,54) are not randomly distributed throughout out an Escherichia coli cell but are present as clusters at preferred locations. Using differentially labeled probes and fluorescence in situ hybridization analysis, it has been shown that except for plasmid R1, which is located at mid-cell or at the cell poles (31), clusters of plasmids F, P1, RK2, and pUC generally are located at the mid-cell position in newborn E. coli cells and at the 1/4 and 3/4 positions in larger cells (16,25,44).…”

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confidence: 76%

“…Using differentially labeled probes and fluorescence in situ hybridization analysis, it has been shown that except for plasmid R1, which is located at mid-cell or at the cell poles (31), clusters of plasmids F, P1, RK2, and pUC generally are located at the mid-cell position in newborn E. coli cells and at the 1/4 and 3/4 positions in larger cells (16,25,44). For these plasmids it has been shown further that the movement of newly duplicated plasmid clusters from the mid-cell to the quarter-cell positions occurs with relatively rapid kinetics (16,18,25,44,49).Much has yet to be learned about cell-or plasmid-specified factors that are responsible for the localization and movement of plasmid clusters. It has been shown for plasmids F (18, 49), P1 (6, 44), R1 (63), R27 (39), and pB171 (5) that the mutation of plasmid-encoded partition loci perturbs the regular pattern of plasmid localization or, in the case of plasmid R1, results in an interference with the separation of plasmid pairs and the movement of plasmids to opposite poles in the cell (31).…”

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confidence: 99%

Gyrase Inhibitors and Thymine Starvation Disrupt the Normal Pattern of Plasmid RK2 Localization in Escherichia coli

2005

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Multicopy plasmids in Escherichia coli are not randomly distributed throughout the cell but exist as defined clusters that are localized at the mid-cell, or at the 1/4 and 3/4 cell length positions. To explore the factors that contribute to plasmid clustering and localization, E. coli cells carrying a plasmid RK2 derivative that can be tagged with a green fluorescent protein-LacI fusion protein were subjected to various conditions that interfere with plasmid superhelicity and/or DNA replication. The various treatments included thymine starvation and the addition of the gyrase inhibitors nalidixic acid and novobiocin. In each case, localization of plasmid clusters at the preferred positions was disrupted but the plasmids remained in clusters, suggesting that normal plasmid superhelicity and DNA synthesis in elongating cells are not required for the clustering of individual plasmid molecules. It was also observed that the inhibition of DNA replication by these treatments produced filaments in which the plasmid clusters were confined to one or two nucleoid bodies, which were located near the midline of the filament and were not evenly spaced throughout the filament, as is found in cells treated with cephalexin. Finally, the enhanced yellow fluorescent protein-RarA fusion protein was used to localize the replication complex in individual E. coli cells. Novobiocin and nalidixic acid treatment both resulted in rapid loss of RarA foci. Under these conditions the RK2 plasmid clusters were not disassembled, suggesting that a completely intact replication complex is not required for plasmid clustering.The development of methods for tagging bacterial plasmids with specific fluorescent fusion proteins or by fluorescence in situ hybridization has greatly facilitated the microscopic analysis of the location and movement of plasmids in a bacterial cell during growth and division. Contrary to the generally accepted view that plasmids randomly diffuse throughout the cell, it has now been shown that the low-copy-number plasmids F (16, 18, 49), P1 (16, 45), and R1 (31, 63) and the multicopy plasmids RK2 and pUC (4,25,53,54) are not randomly distributed throughout out an Escherichia coli cell but are present as clusters at preferred locations. Using differentially labeled probes and fluorescence in situ hybridization analysis, it has been shown that except for plasmid R1, which is located at mid-cell or at the cell poles (31), clusters of plasmids F, P1, RK2, and pUC generally are located at the mid-cell position in newborn E. coli cells and at the 1/4 and 3/4 positions in larger cells (16,25,44). For these plasmids it has been shown further that the movement of newly duplicated plasmid clusters from the mid-cell to the quarter-cell positions occurs with relatively rapid kinetics (16,18,25,44,49).Much has yet to be learned about cell-or plasmid-specified factors that are responsible for the localization and movement of plasmid clusters. It has been shown for plasmids F (18, 49), P1 (6, 44), R1 (63), R27 (39), and pB171 (5) that the...

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“…In addition, the selection of a particular expression system requires a cost down in terms of process, design, and other economic consideration. The combination of recombinant DNA technology and large-scale culture process has enabled recombinant proteins to be produced in massive quantities [26]. High cell density culture techniques for culturing E. coli have been developed to improved productivity and also to provide advantages such as reduced culture volume, enhanced downstream process, reduced wastewater, lower production costs and reduced investment in equipment [27,28].…”

Section: Invitro Protein Expression Strategymentioning

confidence: 99%

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

Verma¹

2012

IOSJPBS

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Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli

Cited by 71 publications

References 66 publications

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

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

Gyrase Inhibitors and Thymine Starvation Disrupt the Normal Pattern of Plasmid RK2 Localization in Escherichia coli

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

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