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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

RNAP foci are evident in fast-growing cells and dynamic in response to nutrient downshift and upshift using live-cell imaging with continuous-flow microfluidics. Cells (rpoC-venus, hupA-mCherry) were growing in a microfluidic device controlled by the CellASIC ONIX microfluidic perfusion system (EMD Millipore) with continuous flow of LB. After exponential-phase cells in LB were imaged (LB, 0 min), LB was replaced with M63 + glycerol, and time-lapse images were taken, two of which, at 3 min and 20 min, are shown. At 20 min, the minimal medium was replaced with LB, and 30 min later, the image shown was taken. The experiments with microfluidics were performed at 30°C because LB has high background autofluorescence at 37°C. It took about 1 min to complete a medium change in the system. Hu-mCherry was used as a proxy for DNA staining because the microfluidic device used has high autofluorescence in the range of 460–488 nm, which interferes with the detection of DNA-bind dye DAPI or Hoescht 33342. The scale bar represents 2 μm. RNAP foci are indicated by arrows. Note the changes in the cells' position and size during the imaging process.
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Figure 4: RNAP foci are evident in fast-growing cells and dynamic in response to nutrient downshift and upshift using live-cell imaging with continuous-flow microfluidics. Cells (rpoC-venus, hupA-mCherry) were growing in a microfluidic device controlled by the CellASIC ONIX microfluidic perfusion system (EMD Millipore) with continuous flow of LB. After exponential-phase cells in LB were imaged (LB, 0 min), LB was replaced with M63 + glycerol, and time-lapse images were taken, two of which, at 3 min and 20 min, are shown. At 20 min, the minimal medium was replaced with LB, and 30 min later, the image shown was taken. The experiments with microfluidics were performed at 30°C because LB has high background autofluorescence at 37°C. It took about 1 min to complete a medium change in the system. Hu-mCherry was used as a proxy for DNA staining because the microfluidic device used has high autofluorescence in the range of 460–488 nm, which interferes with the detection of DNA-bind dye DAPI or Hoescht 33342. The scale bar represents 2 μm. RNAP foci are indicated by arrows. Note the changes in the cells' position and size during the imaging process.

Mentions: Recently, continuous-flow microfluidics has been introduced for live cell imaging of E. coli (Wang et al., 2010). This technique has the advantage of enabling continuous live-cell imaging of RNAP in fast-growing and/or in changing environments without sampling interruptions. Time-lapse images from a set of such experiments are shown in Figure 4. RNAP foci are evident in fast-growing living cells in LB (RNAP and RNAP/nucleoid overlay), and the foci disappear shortly (~3 min) after the culture is downshifted from LB (at time 0) to nutrient-poor, minimal medium, M9 + glycerol; the process is reversible, as RNAP foci reappear after the culture is upshifted back to LB (RNAP and RNAP/Nucleoid overlay). Treatment of fast-growing cells in LB with SHX for 30 min (Figure 5) causes the RNAP foci to disappear (RNAP and RNAP/Nucleoid overlay) almost completely. In addition, RNAP foci are preserved after formaldehyde treatment of fast-growing cells in microfluidics (Figure 6A), whereas RNAP maintains a homogenous distribution pattern after the formaldehyde treatment of cells that were starved for amino acid by the SHX treatment (Figure 6B). Together, these findings demonstrated that formaldehyde does not cause “artificial” perturbations in the organization of RNAP and DNA in cells, as has been shown in ChIP-chip assays (Davis et al., 2011), and that the distribution of RNAP from fixed-cell images (Cabrera and Jin, 2003b, 2006; Cabrera et al., 2009; Cagliero and Jin, 2013; Endesfelder et al., 2013; Jin et al., 2013), reflects the true dynamic states of RNAP in living cells, thus validating the use of formaldehyde in the study of the cell biology of RNAP.


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)

RNAP foci are evident in fast-growing cells and dynamic in response to nutrient downshift and upshift using live-cell imaging with continuous-flow microfluidics. Cells (rpoC-venus, hupA-mCherry) were growing in a microfluidic device controlled by the CellASIC ONIX microfluidic perfusion system (EMD Millipore) with continuous flow of LB. After exponential-phase cells in LB were imaged (LB, 0 min), LB was replaced with M63 + glycerol, and time-lapse images were taken, two of which, at 3 min and 20 min, are shown. At 20 min, the minimal medium was replaced with LB, and 30 min later, the image shown was taken. The experiments with microfluidics were performed at 30°C because LB has high background autofluorescence at 37°C. It took about 1 min to complete a medium change in the system. Hu-mCherry was used as a proxy for DNA staining because the microfluidic device used has high autofluorescence in the range of 460–488 nm, which interferes with the detection of DNA-bind dye DAPI or Hoescht 33342. The scale bar represents 2 μm. RNAP foci are indicated by arrows. Note the changes in the cells' position and size during the imaging process.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: RNAP foci are evident in fast-growing cells and dynamic in response to nutrient downshift and upshift using live-cell imaging with continuous-flow microfluidics. Cells (rpoC-venus, hupA-mCherry) were growing in a microfluidic device controlled by the CellASIC ONIX microfluidic perfusion system (EMD Millipore) with continuous flow of LB. After exponential-phase cells in LB were imaged (LB, 0 min), LB was replaced with M63 + glycerol, and time-lapse images were taken, two of which, at 3 min and 20 min, are shown. At 20 min, the minimal medium was replaced with LB, and 30 min later, the image shown was taken. The experiments with microfluidics were performed at 30°C because LB has high background autofluorescence at 37°C. It took about 1 min to complete a medium change in the system. Hu-mCherry was used as a proxy for DNA staining because the microfluidic device used has high autofluorescence in the range of 460–488 nm, which interferes with the detection of DNA-bind dye DAPI or Hoescht 33342. The scale bar represents 2 μm. RNAP foci are indicated by arrows. Note the changes in the cells' position and size during the imaging process.
Mentions: Recently, continuous-flow microfluidics has been introduced for live cell imaging of E. coli (Wang et al., 2010). This technique has the advantage of enabling continuous live-cell imaging of RNAP in fast-growing and/or in changing environments without sampling interruptions. Time-lapse images from a set of such experiments are shown in Figure 4. RNAP foci are evident in fast-growing living cells in LB (RNAP and RNAP/nucleoid overlay), and the foci disappear shortly (~3 min) after the culture is downshifted from LB (at time 0) to nutrient-poor, minimal medium, M9 + glycerol; the process is reversible, as RNAP foci reappear after the culture is upshifted back to LB (RNAP and RNAP/Nucleoid overlay). Treatment of fast-growing cells in LB with SHX for 30 min (Figure 5) causes the RNAP foci to disappear (RNAP and RNAP/Nucleoid overlay) almost completely. In addition, RNAP foci are preserved after formaldehyde treatment of fast-growing cells in microfluidics (Figure 6A), whereas RNAP maintains a homogenous distribution pattern after the formaldehyde treatment of cells that were starved for amino acid by the SHX treatment (Figure 6B). Together, these findings demonstrated that formaldehyde does not cause “artificial” perturbations in the organization of RNAP and DNA in cells, as has been shown in ChIP-chip assays (Davis et al., 2011), and that the distribution of RNAP from fixed-cell images (Cabrera and Jin, 2003b, 2006; Cabrera et al., 2009; Cagliero and Jin, 2013; Endesfelder et al., 2013; Jin et al., 2013), reflects the true dynamic states of RNAP in living cells, thus validating the use of formaldehyde in the study of the cell biology of RNAP.

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