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The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin.

Dimude JU, Stockum A, Midgley-Smith SL, Upton AL, Foster HA, Khan A, Saunders NJ, Retkute R, Rudolph CJ - MBio (2015)

Bottom Line: We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas.In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises.In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Cell replication and chromosome dynamics in cells lacking RNase HI. (A) Viable cell counts of dnaA derivatives following shift to restrictive temperature. Cells were grown at the permissive temperature to early exponential phase and shifted to 42°C, and samples were taken at the times indicated. Samples were diluted, plated, and incubated at the permissive temperature. The strains used were AU1054 (dnaA46), AU1091 (dnaA recG), and AU1066 (dnaA rnhA). (B) Fluorescence microscopy shows replication of origin (red foci) and terminus (green foci) areas of the chromosome. (Combined phase-contrast and fluorescence images are shown.) The strains used were RCe198 (dnaA recG) and RCe202 (dnaA rnhA). Incubation times following the shift to 42°C are indicated. (C) Imaging flow cytometry analysis of EdU incorporation into newly synthesized DNA, followed by Alexa Fluor 488 click labeling. The strains used were AU1054 (dnaA46), AU1091 (dnaA recG), and AU1066 (dnaA rnhA). The strains were shifted to 42°C for 90 min to inhibit oriC firing without affecting ongoing synthesis. All strains were labeled with EdU for 15 min before fixing and click labeling. The histogram shows the fraction of cells against fluorescence intensity. A minimum of 5,000 in-focus cells were analyzed for each strain. The experiment from which the data are plotted was repeated with a similar number of cells and was reproducible. (D) Images of cells are representative for fluorescence levels, and the distribution in the genetic backgrounds indicated was derived from the imaging flow cytometry data collection. Images of bright-field and Alexa Fluor 488 fluorescence are shown at 60× magnification. The percentages of cells with the shown fluorescence levels are indicated.
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fig4: Cell replication and chromosome dynamics in cells lacking RNase HI. (A) Viable cell counts of dnaA derivatives following shift to restrictive temperature. Cells were grown at the permissive temperature to early exponential phase and shifted to 42°C, and samples were taken at the times indicated. Samples were diluted, plated, and incubated at the permissive temperature. The strains used were AU1054 (dnaA46), AU1091 (dnaA recG), and AU1066 (dnaA rnhA). (B) Fluorescence microscopy shows replication of origin (red foci) and terminus (green foci) areas of the chromosome. (Combined phase-contrast and fluorescence images are shown.) The strains used were RCe198 (dnaA recG) and RCe202 (dnaA rnhA). Incubation times following the shift to 42°C are indicated. (C) Imaging flow cytometry analysis of EdU incorporation into newly synthesized DNA, followed by Alexa Fluor 488 click labeling. The strains used were AU1054 (dnaA46), AU1091 (dnaA recG), and AU1066 (dnaA rnhA). The strains were shifted to 42°C for 90 min to inhibit oriC firing without affecting ongoing synthesis. All strains were labeled with EdU for 15 min before fixing and click labeling. The histogram shows the fraction of cells against fluorescence intensity. A minimum of 5,000 in-focus cells were analyzed for each strain. The experiment from which the data are plotted was repeated with a similar number of cells and was reproducible. (D) Images of cells are representative for fluorescence levels, and the distribution in the genetic backgrounds indicated was derived from the imaging flow cytometry data collection. Images of bright-field and Alexa Fluor 488 fluorescence are shown at 60× magnification. The percentages of cells with the shown fluorescence levels are indicated.

Mentions: To investigate whether synthesis outside the termination area can contribute to cell duplication, we directly followed the viable titer of dnaA, dnaA recG, and dnaA rnhA cells following shift to 42°C. Both dnaA and dnaA recG cells showed approximately 2 cell division events in LB broth before growth arrest (Fig. 4A), with no hint of further divisions. In contrast, dnaA rnhA cells showed continuous growth over several hours (Fig. 4A). Growth is linear rather than exponential, which is likely to be caused by the broth sensitivity of dnaA rnhA cells (Fig. 1B) (8), and dnaA rnhA cells showed robust levels of bromodeoxyuridine (BrdU) incorporation in all chromosomal areas (see Fig. S2A in the supplemental material). Thus, synthesis in dnaA rnhA cells can contribute toward successful cell duplication for significant periods of time, whereas synthesis outside the termination area in recG cells cannot.


The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin.

Dimude JU, Stockum A, Midgley-Smith SL, Upton AL, Foster HA, Khan A, Saunders NJ, Retkute R, Rudolph CJ - MBio (2015)

Cell replication and chromosome dynamics in cells lacking RNase HI. (A) Viable cell counts of dnaA derivatives following shift to restrictive temperature. Cells were grown at the permissive temperature to early exponential phase and shifted to 42°C, and samples were taken at the times indicated. Samples were diluted, plated, and incubated at the permissive temperature. The strains used were AU1054 (dnaA46), AU1091 (dnaA recG), and AU1066 (dnaA rnhA). (B) Fluorescence microscopy shows replication of origin (red foci) and terminus (green foci) areas of the chromosome. (Combined phase-contrast and fluorescence images are shown.) The strains used were RCe198 (dnaA recG) and RCe202 (dnaA rnhA). Incubation times following the shift to 42°C are indicated. (C) Imaging flow cytometry analysis of EdU incorporation into newly synthesized DNA, followed by Alexa Fluor 488 click labeling. The strains used were AU1054 (dnaA46), AU1091 (dnaA recG), and AU1066 (dnaA rnhA). The strains were shifted to 42°C for 90 min to inhibit oriC firing without affecting ongoing synthesis. All strains were labeled with EdU for 15 min before fixing and click labeling. The histogram shows the fraction of cells against fluorescence intensity. A minimum of 5,000 in-focus cells were analyzed for each strain. The experiment from which the data are plotted was repeated with a similar number of cells and was reproducible. (D) Images of cells are representative for fluorescence levels, and the distribution in the genetic backgrounds indicated was derived from the imaging flow cytometry data collection. Images of bright-field and Alexa Fluor 488 fluorescence are shown at 60× magnification. The percentages of cells with the shown fluorescence levels are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Cell replication and chromosome dynamics in cells lacking RNase HI. (A) Viable cell counts of dnaA derivatives following shift to restrictive temperature. Cells were grown at the permissive temperature to early exponential phase and shifted to 42°C, and samples were taken at the times indicated. Samples were diluted, plated, and incubated at the permissive temperature. The strains used were AU1054 (dnaA46), AU1091 (dnaA recG), and AU1066 (dnaA rnhA). (B) Fluorescence microscopy shows replication of origin (red foci) and terminus (green foci) areas of the chromosome. (Combined phase-contrast and fluorescence images are shown.) The strains used were RCe198 (dnaA recG) and RCe202 (dnaA rnhA). Incubation times following the shift to 42°C are indicated. (C) Imaging flow cytometry analysis of EdU incorporation into newly synthesized DNA, followed by Alexa Fluor 488 click labeling. The strains used were AU1054 (dnaA46), AU1091 (dnaA recG), and AU1066 (dnaA rnhA). The strains were shifted to 42°C for 90 min to inhibit oriC firing without affecting ongoing synthesis. All strains were labeled with EdU for 15 min before fixing and click labeling. The histogram shows the fraction of cells against fluorescence intensity. A minimum of 5,000 in-focus cells were analyzed for each strain. The experiment from which the data are plotted was repeated with a similar number of cells and was reproducible. (D) Images of cells are representative for fluorescence levels, and the distribution in the genetic backgrounds indicated was derived from the imaging flow cytometry data collection. Images of bright-field and Alexa Fluor 488 fluorescence are shown at 60× magnification. The percentages of cells with the shown fluorescence levels are indicated.
Mentions: To investigate whether synthesis outside the termination area can contribute to cell duplication, we directly followed the viable titer of dnaA, dnaA recG, and dnaA rnhA cells following shift to 42°C. Both dnaA and dnaA recG cells showed approximately 2 cell division events in LB broth before growth arrest (Fig. 4A), with no hint of further divisions. In contrast, dnaA rnhA cells showed continuous growth over several hours (Fig. 4A). Growth is linear rather than exponential, which is likely to be caused by the broth sensitivity of dnaA rnhA cells (Fig. 1B) (8), and dnaA rnhA cells showed robust levels of bromodeoxyuridine (BrdU) incorporation in all chromosomal areas (see Fig. S2A in the supplemental material). Thus, synthesis in dnaA rnhA cells can contribute toward successful cell duplication for significant periods of time, whereas synthesis outside the termination area in recG cells cannot.

Bottom Line: We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas.In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises.In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom.

No MeSH data available.


Related in: MedlinePlus