Identification of new genes contributing to the extreme radioresistance of Deinococcus radiodurans using a Tn5-based transposon mutant library.

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network

Synthesis, Photochemical Properties, and Cytotoxicities of 2<i>H</i>‑Naphtho[1,2‑<i>b</i>]pyran and Its Photodimers

A 2<i>H</i>-naphtho­[1,2-<i>b</i>]­pyran, prepared by dimerization of 2-bromo-3-methyl-1,4-naphthoquinone and <i>O</i>-methylation, readily undergoes solid-state [2 + 2] photodimerization to give a photodimer in excellent yield and with excellent selectivity. Retro [2 + 2] cycloaddition can be achieved by irradiation of a solution of the photodimer in chloroform. Interestingly, the 2<i>H</i>-naphtho­[1,2-<i>b</i>]­pyran dimerizes with a skeletal rearrangement to afford 2,5-dihydro-1-benzoxepin dimers upon irradiation in methanol or via irradiation with hexamethylditin. Furthermore, treatment of the resulting dimers with triethylamine regenerates the 2<i>H</i>-naphtho­[1,2-<i>b</i>]­pyran monomer. Significant differences in the color, fluorescence, and cytotoxic properties of the monomer and dimers were observed

Δ<i>DR0265</i> and Δ<i>DR2462</i> are sensitive to UV but not Δ<i>DR0007</i> and Δ<i>DR0008</i>.

<p>Bacteria were grown in TGY2X liquid medium to A₆₅₀ = 1, serially diluted and dilutions were spotted onto TGY agar plates subsequently exposed to UV-irradiation at the indicated doses.</p

Genes detected in our screen for which the corresponding <i>Tn</i>5 insertion mutants exhibit the highest gamma radiation sensitive phenotype.

<p>^{a, b}SS, highly sensitive, survival <10^-6; S, sensitive, survival comprised between 10^-6 and 10^-4; s, slightly sensitive, survival comprised between 10^-4 and 10^-2; R, resistant, survival >10^-2.</p><p>^a onto TGY plates supplemented with 40 ng/mL mitomycin C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124358#sec009" target="_blank">Materials and Methods</a>).</p><p>^b onto TGY plates exposed to 600 J m^-2 UV-rays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124358#sec009" target="_blank">Materials and Methods</a>).</p><p>^c by using the disc inhibition assay as described in Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124358#pone.0124358.g002" target="_blank">Fig 2</a> (for the inhibition diameters corresponding to the different levels of sensitivity).</p><p>^d NA: Not applicable because of growth defect.</p><p>^(o) gene is located in a putative operon (predicted by FGENESB (<a href="http://www.softberry.com" target="_blank">www.softberry.com</a>).</p><p>^(u) transposon inserted in the upstream region of the corresponding CDS.</p><p>Genes detected in our screen for which the corresponding <i>Tn</i>5 insertion mutants exhibit the highest gamma radiation sensitive phenotype.</p

The <i>D</i>. <i>radiodurans</i> mutants deleted for <i>DR0007</i>, <i>DR0008</i> or both (A), <i>DR0265</i> (B) and <i>DR2462</i> (C) show increased sensitivity to γ-irradiation.

<p>Bacteria were exposed to γ-irradiation at doses indicated on the abscissa. Symbols: (A) wild-type (blue squares), Δ<i>DR0007</i> (red closed triangles), Δ<i>DR0008</i> (green closed circles), Δ<i>DR0007</i>Δ<i>DR0008</i> (yellow closed diamonds), Δ<i>DR0007</i>/p11559-<i>DR0007</i>⁺ (red open triangles), Δ<i>DR0008</i>/p11559-<i>DR0008</i>⁺ (green open circles), Δ<i>DR0007</i> Δ<i>DR0008</i>/p11559-<i>DR0007</i>⁺<i>DR0008</i>⁺ (yellow open diamonds). (B) wild-type (blue squares), Δ<i>DR0265</i> (purple closed triangles), Δ<i>DR0265</i>/p11520-<i>DR0265</i>⁺ (pink open triangles). (C) wild-type (blue squares), Δ<i>DR2462</i> (brown closed circles), Δ<i>DR2462</i>/p11520-<i>DR2462</i>⁺ (ochre open circles).</p

Deletion of <i>DR0007</i>, <i>DR0008</i> or both (A), <i>DR0265</i> (B), or <i>DR2462</i> (C) sensitizes <i>D</i>. <i>radiodurans</i> to MMC.

<p>Bacteria were grown in TGY2X liquid medium to A₆₅₀ = 1, serially diluted and dilutions were spotted onto TGY agar plates supplemented or not with MMC at the indicated doses, and supplemented with spectinomycin for strains harboring derivatives of p11559 or p11520 plasmids.</p

The Δ<i>DR2462</i> bacteria show an increased delay in cell division and in reconstitution of genomic DNA after γ-irradiation.

<p>A. Growth delay after irradiation. Wild type (blue squares) and Δ<i>DR2462</i> (brown circles) bacteria were exposed (filled symbols) or not (open symbols) to γ-irradiation at a dose of 3.8 kGy, diluted in TGY2X to an A₆₅₀ of 0.3 and incubated at 30°C. At different times after irradiation, aliquots were taken to measure the number of viable cells per mL. B and C. Kinetics of restoration of genomic DNA. Bacteria were treated as in (A). DNA agarose plugs were prepared at the indicated post-irradiation times and digested with <i>Not</i>I prior to analyses by PFGE. B: wild type; C: Δ<i>DR2462</i>.</p

Measurement of sensitivity to H₂O₂ stress by disc inhibition assay (procedure described in Materials and Methods).

<p>(A) Phenotype of the wild-type (resistant), the <i>katA</i> mutant (highly sensitive), and the <i>DR0265</i> mutant (middle sensitive) are shown. (B) The mutants are classified into four categories depending on the diameter (in mm) of growth inhibition area as indicated.</p