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

View ORCID ProfileGanesh K Maurya, View ORCID ProfileHari S Misra  Correspondence email
Ganesh K Maurya
1Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India
2Homi Bhabha National Institute, Mumbai, India
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  • ORCID record for Ganesh K Maurya
Hari S Misra
1Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India
2Homi Bhabha National Institute, Mumbai, India
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  • ORCID record for Hari S Misra
  • For correspondence: hsmisra@barc.gov.in
Published 16 November 2020. DOI: 10.26508/lsa.202000856
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  • Figure 1.
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    Figure 1. Schematic presentation of direct repeats located upstream to parAB operon in chromosome II (cisII) and megaplasmid (cisMP).

    (A, B) The 99–554 bp region of chromosome II (A) and 177,446- to 417-bp region of megaplasmid (B) were analysed in silico for the structure of direct repeats and their sequence compositions of DnaA boxes and iterons.

  • Figure 2.
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    Figure 2. Role of cisII and cisMP elements in the replication of Escherichia coli plasmid into 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).

  • Figure S1.
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    Figure S1. Purification of recombinant proteins used for DNA–protein interaction.

    (A, B, C) The recombinant DnaA (A), ParB2 (B), and ParB3 (C) were purified from E. coli using metal affinity chromatography.

  • Figure 3.
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    Figure 3. DnaA interaction with cisII and cisMP and their repeats variants.

    (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.

  • Figure 4.
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    Figure 4. ParBs interaction with cisII and cisMP and their repeats variants.

    (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.

  • Figure S2.
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    Figure S2. Generation of cisII and cisMP deletion mutants in Deinococcus radiodurans.

    (A) Schematic representation of strategy for replacement of cis-elements with the expressing cassette of kanamycin and position of primers used for diagnostic PCR (A). (B, C) The recombinant cells expected to have cisII (B) and cisMP (C) deletions grown several generations under selection pressure. (A) The genomic DNA was prepared, and PCR amplification was carried out by diagnostic PCR using primer as shown in (A). Products were analysed on 1% agarose gel.

  • Figure 5.
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    Figure 5. Effect of cisII and cisMP deletions on genome copy number and radioresistance in D. radiodurans.

    (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).

  • Figure S3.
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    Figure S3. Schematic representation of tetO-TetR–mediated fluorescent reporter–operator system development for D. radiodurans.

    (A, B, D) The tetO cassette from was inserted at 1.5° in chromosome I (A), at 4° in chromosome II (B) and at 4.4° in megaplasmid (D) through homologous recombination. (C, E) Insertion of tetO cassettes into respective positions was confirmed by diagnostic PCR using plasmid sequence-specific primers (C, E).

  • Figure 6.
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    Figure 6. The localization pattern of chromosome I in the nucleoid of wild type and secondary genome cis mutants of Deinococcus radiodurans.

    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).

  • Figure 7.
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    Figure 7. The localization pattern of chromosome II in the nucleoid of wild type and secondary genome cis mutants of Deinococcus radiodurans.

    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).

  • Figure 8.
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    Figure 8. The localization pattern of megaplasmid in the nucleoid of wild type and secondary genome cis mutants of Deinococcus radiodurans.

    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).

  • Figure 9.
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    Figure 9. Distribution of genomic replicons in wild type and cis mutants of Deinococcus radiodurans observed under fluorescence microscopy.

    (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).

  • Figure 10.
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    Figure 10. Gamma radiation response of cisII and cisMP mutants of Deinococcus radiodurans.

    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.

  • Figure S4.
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    Figure S4. Schematics of cis elements and their repeat varian

    ts. (A, B) Schematic representation of cisII (A) and cisMP (B) elements and their repeat variants PCR amplified and used for DNA–protein interaction studies.

  • Figure S5.
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    Figure S5. Expression pattern of TetR-GFP in wild type and cis mutants without tetO repeats.

    (A, B) The expression of TetR-GFP in both wild type and cis mutants of D. radiodurans was confirmed microscopically (A) and through immunoblotting (B). The TetR-GFP expression in the absence of tetO element was found to be uniformly spread in the cell.

Tables

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    Table 1.

    The dissociation constant (Kd) value of DnaA interaction with different repeats of cisII and cisMP elements.

    Repeat no.Dissociation constants (Kd) (mean ± SD, μM)
    cisII variantscisMP variants
    Full length0.31 ± 0.0040.56 ± 0.21
    8/5 repeats0.70 ± 0.350.89 ± 0.39
    3 repeats0.80 ± 0.111.4 ± 0.7
    2 repeatsPoor affinityPoor affinity
    1 repeatNo affinityNo affinity
    • View popup
    Table 2.

    The dissociation constant (Kd) value of ParB2 and ParB3 interaction with repeats variants of cisII and cisMP elements, respectively.

    No. of repeatsDissociation constants (Kd) (mean ± SD, μM)
    ParB2 with cisII variantsParB3 with cisMP variants
    Full length0.40 ± 0.0060.59 ± 0.06
    8/5 repeats0.68 ± 0.060.81 ± 0.04
    3 repeats1.06 ± 0.261.01 ± 0.15
    2 repeatsPoor affinityPoor affinity
    1 repeatNo affinityPoor affinity

Supplementary Materials

  • Figures
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  • Table S1 List of oligonucleotides used in this study.

  • Table S2 List of bacterial strains and plasmids used in this study.

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Multipartite genome, ploidy, and radioresistance
Ganesh K Maurya, Hari S Misra
Life Science Alliance Nov 2020, 4 (1) e202000856; DOI: 10.26508/lsa.202000856

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Multipartite genome, ploidy, and radioresistance
Ganesh K Maurya, Hari S Misra
Life Science Alliance Nov 2020, 4 (1) e202000856; DOI: 10.26508/lsa.202000856
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