Characterization of ori and parS-like functions in secondary genome replicons in Deinococcus radiodurans

(A, C) The cisII and cisMP elements were cloned in E. coli cloning vector pNOKOUT, and the resultant plasmids pNOKcisII and pNOKcisMP were transformed into wild-type (A) and recA mutant (C). These transformants were grown in the presence of kanamycin (6 μg/ml) and growth was monitored on TGY agar plate with rqkA deletion mutant (a deletion mutant made by inserting kanamycin cassettes) as described in Rajpurohit & Misra (2010) as positive control and vector as a negative control. (B) The growth characteristic of these transformants grown in TGY broth supplemented with antibiotic was compared with wild-type cells grown in TGY broth without antibiotics (B). (D) The recombinant plasmids isolated from D. radiodurans cells growing in the presence of antibiotic were digested with restriction enzymes, and the release of cisII and cisMP fragments was analysed on an agarose gel and compared with empty vector (D).

(A, B, C, D, E, F, G, H) The radiolabelled full-length cisII containing 11 repeats (A) and cisMP containing eight repeats (B) as well as eight repeats (C), three repeats (E) and one repeat (G) of cisII (Fig S4A), and five repeats (D), three repeats (F), and single repeat (H) of cisMP (Fig S4B) were incubated with the increasing concentration of DnaA. A saturating concentration of the DnaA-DNA ratio was chased with increasing molar ratio of non-specific DNA (NS-DNA) and products were analysed on native PAGE. The fractions of DNA bound with proteins were quantified densitometrically and plotted as a function of protein concentration using GraphPad Prism 6. The Kd for the curve fitting of individual plots was determined and given in Table 1.

(A, B, C, D) The radiolabelled full-length cisII containing 11 repeats (A), 8 repeats (B), 3 repeats (C), and 2 repeats (D) (Table S2) were incubated with the increasing concentration of ParB2. (E, F, G, H) Similarly, the radiolabelled full-length cisMP containing eight repeats (E), five repeats (F), three repeats (G), and two repeats (H) were incubated with the increasing concentration of ParB3. A saturating concentration of DNA–protein ratio was chased with increasing molar ratio of non-specific DNA (NS-DNA) and products were analysed on native PAGE. The fractions of DNA bound with proteins were quantified densitometrically and plotted as a function of protein concentration using GraphPad Prism 6. The Kd for the curve fitting of individual plots was determined and given in Table 2.

(A) The exponentially growing D. radiodurans R1 (WT) and its cisII (ΔcisII) and cisMP (ΔcisMP) deletion mutants were used for the determination of copy number of each replicon (A). (B) Both wild type and cis mutants of D. radiodurans were checked for growth in the presence and absence of kanamycin under normal conditions (B). (C) These mutants were maintained in the presence (+Kan) and absence (−Kan) of antibiotics as indicated on x-axis, and the CFUs in the absence and presence of kanamycin were monitored and plotted on y-axis (C).

Chromosome I was tagged with tetO/TetR-GFP–based fluorescent reporter–operator system in both wild type (WT) and cis mutants (ΔcisII and ΔcisMP) as described in the Materials and Methods section and grown exponentially. (A) These cells were stained with Nile Red and DAPI and visualized microscopically in differential interference contrast, TRITC (Nile Red), DAPI (DAPI), and FITC (GFP) channels and images were merged (Merge) (A). The scale bar of 1 µm is used in each figure. (B) The schematic diagrams showing the foci position with respect to nucleoid and septum are presented for better clarity (B). (C) Data shown are from a single tetrad where most of these cells show a similar pattern as quantified from ∼200 cells (C).

Chromosome II was tagged with a tetO-TetR-GFP–based fluorescent reporter–operator system in both wild type (WT) and cis mutants (ΔcisII and ΔcisMP) as described in the Materials and Methods section and grown exponentially. (A) These cells were stained with Nile Red and DAPI and visualized microscopically in differential interference contrast, TRITC (Nile Red), DAPI (DAPI), and FITC (GFP) channels and images were merged (Merge) (A). The scale bar of 1 µm is used in each figure. (B) The schematic diagrams showing the foci position with respect to nucleoid and septum are presented for better clarity (B). (C) Data shown are from a single tetrad where most of these cells show a similar pattern as quantified from ∼200 cells (C).

The megaplasmid was tagged with a tetO-TetR-GFP–based fluorescent reporter–operator system in both wild type (WT) and cis mutants (ΔcisII and ΔcisMP) as described in the Materials and Methods section and grown exponentially. (A) These cells were stained with Nile Red and DAPI and visualized microscopically in differential interference contrast, TRITC (Nile Red), DAPI (DAPI), and FITC (GFP) channels and images were merged (Merge) (A). The scale bar of 1 µm is used in each figure. (B) The schematic diagrams showing the foci position with respect to nucleoid and septum are presented for better clarity (B). (C) Data shown are from a single tetrad where most of these cells show a similar pattern as quantified from ∼200 cells (C).

(A, B, C) The chromosome I (ChrI), chromosome II (ChrII and megaplasmid [MP]) of wild type (A), cisII (B), and cisMP (C) mutant of D. radiodurans were tagged with GFP-based fluorescent reporter–operator system and cells were imaged under fluorescence microscopy as described in Figs 6–8. Nearly 200 cells of each type were analysed for the absence (0) and the presence of varying numbers (1, 2, ≥ 3) of foci corresponding to all three replicons. Data presented here are mean ± SD (n = 200).

The γ radiation response of wild type (WT) and its cisII (ΔCII) and cisMP (ΔMP) mutants grown in the presence (+Kan) and absence (−Kan) of kanamycin was monitored. Data presented here are mean ± SD (n = 6) from the reproducible experiments.