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The dynamic nature and territory of transcriptional machinery in the bacterial chromosome.

Jin DJ, Cagliero C, Martin CM, Izard J, Zhou YN - Front Microbiol (2015)

Bottom Line: In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells.Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized.Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

View Article: PubMed Central - PubMed

Affiliation: Transcription Control Section, Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, National Institutes of Health Frederick, MD, USA.

ABSTRACT
Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

No MeSH data available.


Related in: MedlinePlus

Inhibition ofrrnexpression leads to homogeneous distribution of RNAP across the nucleoid in both living and fixed cells. Fast-growing cells (LB, 37°C) were treated with either rifampicin (100 μg/ml) for 15 min (A) or with SHX (500 μg/ml) for 30 min (B). Cells were sampled and agarose embedded on coverslips. Live-cell imaging (live cell); Fixed-cell imaging (fixed cell), cells were fixed by formaldehyde. The scale bar represents 2 μm.
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Figure 3: Inhibition ofrrnexpression leads to homogeneous distribution of RNAP across the nucleoid in both living and fixed cells. Fast-growing cells (LB, 37°C) were treated with either rifampicin (100 μg/ml) for 15 min (A) or with SHX (500 μg/ml) for 30 min (B). Cells were sampled and agarose embedded on coverslips. Live-cell imaging (live cell); Fixed-cell imaging (fixed cell), cells were fixed by formaldehyde. The scale bar represents 2 μm.

Mentions: It soon became evident that the distribution of RNAP is extremely sensitive to physiological perturbations, including those caused by the sampling and imaging processes; such a property has not been reported for other GFP reporters, such as the parS-ParB-GFP system (Nielsen et al., 2007) and the SSB-GFP fusion used in tracking the replisome (Reyes-Lamothe et al., 2008). Initially, no difference was observed in the RNAP distribution in living cells under different growth conditions. Figure 2A shows a set of images of living cells growing in a rapidly shaking flask (LB, 37°C), a condition in which most RNAP molecules are engaged in active rRNA synthesis. These cells were imaged after sampling from the flask and then agarose-embedded on coverslips at room temperature (25°C). The pattern of RNAP distribution changes depending on the length of time between sampling preparation and imaging. For example, RNAP foci could be observed in some cells after a short time (3 min); however, as the length of time increases (6 min), only few cells have RNAP foci, and RNAP is homogeneously distributed in the nucleoid in the cell population after 12 min. Note that nucleoid structure also changes in parallel and becomes expanded as time increases during the imaging process, indicating also the dynamic nature of the nucleoid. Experimentally, the time elapsed between sampling and image acquisition is usually longer than 10 min. The same distribution pattern was also observed in fast-growing cells treated either with rifampicin (Figure 3A, living cell), or with SHX (Figure 3B, living cell), which inhibit rrn transcription. RNAP is distributed homogeneously in the nucleoid regardless of growth conditions and status of rrn synthesis in living cells under the conditions used.


The dynamic nature and territory of transcriptional machinery in the bacterial chromosome.

Jin DJ, Cagliero C, Martin CM, Izard J, Zhou YN - Front Microbiol (2015)

Inhibition ofrrnexpression leads to homogeneous distribution of RNAP across the nucleoid in both living and fixed cells. Fast-growing cells (LB, 37°C) were treated with either rifampicin (100 μg/ml) for 15 min (A) or with SHX (500 μg/ml) for 30 min (B). Cells were sampled and agarose embedded on coverslips. Live-cell imaging (live cell); Fixed-cell imaging (fixed cell), cells were fixed by formaldehyde. The scale bar represents 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440401&req=5

Figure 3: Inhibition ofrrnexpression leads to homogeneous distribution of RNAP across the nucleoid in both living and fixed cells. Fast-growing cells (LB, 37°C) were treated with either rifampicin (100 μg/ml) for 15 min (A) or with SHX (500 μg/ml) for 30 min (B). Cells were sampled and agarose embedded on coverslips. Live-cell imaging (live cell); Fixed-cell imaging (fixed cell), cells were fixed by formaldehyde. The scale bar represents 2 μm.
Mentions: It soon became evident that the distribution of RNAP is extremely sensitive to physiological perturbations, including those caused by the sampling and imaging processes; such a property has not been reported for other GFP reporters, such as the parS-ParB-GFP system (Nielsen et al., 2007) and the SSB-GFP fusion used in tracking the replisome (Reyes-Lamothe et al., 2008). Initially, no difference was observed in the RNAP distribution in living cells under different growth conditions. Figure 2A shows a set of images of living cells growing in a rapidly shaking flask (LB, 37°C), a condition in which most RNAP molecules are engaged in active rRNA synthesis. These cells were imaged after sampling from the flask and then agarose-embedded on coverslips at room temperature (25°C). The pattern of RNAP distribution changes depending on the length of time between sampling preparation and imaging. For example, RNAP foci could be observed in some cells after a short time (3 min); however, as the length of time increases (6 min), only few cells have RNAP foci, and RNAP is homogeneously distributed in the nucleoid in the cell population after 12 min. Note that nucleoid structure also changes in parallel and becomes expanded as time increases during the imaging process, indicating also the dynamic nature of the nucleoid. Experimentally, the time elapsed between sampling and image acquisition is usually longer than 10 min. The same distribution pattern was also observed in fast-growing cells treated either with rifampicin (Figure 3A, living cell), or with SHX (Figure 3B, living cell), which inhibit rrn transcription. RNAP is distributed homogeneously in the nucleoid regardless of growth conditions and status of rrn synthesis in living cells under the conditions used.

Bottom Line: In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells.Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized.Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

View Article: PubMed Central - PubMed

Affiliation: Transcription Control Section, Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, National Institutes of Health Frederick, MD, USA.

ABSTRACT
Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment and highlights the challenges in the study. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

No MeSH data available.


Related in: MedlinePlus