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

Dulermo R, Onodera T, Coste G, Passot F, Dutertre M, Porteron M, Confalonieri F, Sommer S, Pasternak C - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Univ. Paris-Sud, Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Orsay, France.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

ΔDR0265 and ΔDR2462 are sensitive to UV but not ΔDR0007 and ΔDR0008.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.
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pone.0124358.g005: ΔDR0265 and ΔDR2462 are sensitive to UV but not ΔDR0007 and ΔDR0008.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.

Mentions: To confirm the importance of these proteins for D. radiodurans recovery after irradiation, several single and double deletion mutants were constructed. Homogenotes of the ΔDR0007 mutant were easily obtained on selective medium and did not display any growth defect under standard cultivation conditions (same doubling time as those of the wild type R1 strain), indicating that DR0007 gene is not essential for cell viability under our culture conditions. The survival of ΔDR0007 was decreased by a factor of 7- and 21-fold after γ-irradiation at 15 and 20 kGy, respectively, when compared to the wild type strain. Ectopic expression of DR0007 gene alone did not fully restore the wild type radiation resistance (Fig 3A). Although we did not find insertions in DR0008 in our initial screening, we constructed a DR0008 deletion mutant to completely inactivate the gene. The ΔDR0008 mutant displayed the same γ-radiation sensitive phenotype as the ΔDR0007 mutant, and again ectopic expression of DR0008 did not restore the wild type phenotype (Fig 3A). The double mutant ΔDR0007 ΔDR0008 showed the same survival rate than those of the single mutants (Fig 3A). In contrast, ectopic expression of both of these genes in the double mutant was sufficient to restore the wild type radiation resistance (Fig 3A). These results suggest that DR0007 and DR0008 are functionally linked and that a coordinated expression of the two genes is required to fulfill their role in radioresistance. To examine whether c-di-AMP could complement the radiosensitive phenotype of ΔDR0007 and ΔDR0008, exogenous c-di-AMP was added immediately after exposure to γ-radiation, in the presence of polyamines to favor c-di-AMP uptake according to the procedure of Oppenheimer-Shaanan et al. [61]. Nevertheless, addition of c-di-AMP did not restore radiation resistance in the single and double mutants. This may be due to the use of polyamines which decrease cell survival even in the wild type (by a factor of 3.5 at 10 kGy and 4 at 15 kGy). Finally, deletion of DR0007, DR0008 or both only slightly sensitizes the cells to MMC (Fig 4A) and to UV light (Fig 5A). Deletion of DR0009 gene did not affect resistance to γ-irradiation, but we cannot exclude that DR0025 that shares 35% identity and 53% similarity with DR0009 may functionally complement the deletion of DR0009. This putative redundancy of function may explain why we did not find insertion mutants inactivated for DR0009 in our initial screening.


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

Dulermo R, Onodera T, Coste G, Passot F, Dutertre M, Porteron M, Confalonieri F, Sommer S, Pasternak C - PLoS ONE (2015)

ΔDR0265 and ΔDR2462 are sensitive to UV but not ΔDR0007 and ΔDR0008.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401554&req=5

pone.0124358.g005: ΔDR0265 and ΔDR2462 are sensitive to UV but not ΔDR0007 and ΔDR0008.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.
Mentions: To confirm the importance of these proteins for D. radiodurans recovery after irradiation, several single and double deletion mutants were constructed. Homogenotes of the ΔDR0007 mutant were easily obtained on selective medium and did not display any growth defect under standard cultivation conditions (same doubling time as those of the wild type R1 strain), indicating that DR0007 gene is not essential for cell viability under our culture conditions. The survival of ΔDR0007 was decreased by a factor of 7- and 21-fold after γ-irradiation at 15 and 20 kGy, respectively, when compared to the wild type strain. Ectopic expression of DR0007 gene alone did not fully restore the wild type radiation resistance (Fig 3A). Although we did not find insertions in DR0008 in our initial screening, we constructed a DR0008 deletion mutant to completely inactivate the gene. The ΔDR0008 mutant displayed the same γ-radiation sensitive phenotype as the ΔDR0007 mutant, and again ectopic expression of DR0008 did not restore the wild type phenotype (Fig 3A). The double mutant ΔDR0007 ΔDR0008 showed the same survival rate than those of the single mutants (Fig 3A). In contrast, ectopic expression of both of these genes in the double mutant was sufficient to restore the wild type radiation resistance (Fig 3A). These results suggest that DR0007 and DR0008 are functionally linked and that a coordinated expression of the two genes is required to fulfill their role in radioresistance. To examine whether c-di-AMP could complement the radiosensitive phenotype of ΔDR0007 and ΔDR0008, exogenous c-di-AMP was added immediately after exposure to γ-radiation, in the presence of polyamines to favor c-di-AMP uptake according to the procedure of Oppenheimer-Shaanan et al. [61]. Nevertheless, addition of c-di-AMP did not restore radiation resistance in the single and double mutants. This may be due to the use of polyamines which decrease cell survival even in the wild type (by a factor of 3.5 at 10 kGy and 4 at 15 kGy). Finally, deletion of DR0007, DR0008 or both only slightly sensitizes the cells to MMC (Fig 4A) and to UV light (Fig 5A). Deletion of DR0009 gene did not affect resistance to γ-irradiation, but we cannot exclude that DR0025 that shares 35% identity and 53% similarity with DR0009 may functionally complement the deletion of DR0009. This putative redundancy of function may explain why we did not find insertion mutants inactivated for DR0009 in our initial screening.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Univ. Paris-Sud, Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Orsay, France.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus