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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

The ΔDR2462 bacteria show an increased delay in cell division and in reconstitution of genomic DNA after γ-irradiation.A. Growth delay after irradiation. Wild type (blue squares) and ΔDR2462 (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 NotI prior to analyses by PFGE. B: wild type; C: ΔDR2462.
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pone.0124358.g006: The ΔDR2462 bacteria show an increased delay in cell division and in reconstitution of genomic DNA after γ-irradiation.A. Growth delay after irradiation. Wild type (blue squares) and ΔDR2462 (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 NotI prior to analyses by PFGE. B: wild type; C: ΔDR2462.

Mentions: The kinetics of DNA DSB repair in the ΔDR2462 mutant exposed to 3.8 kGy γ-irradiation was measured by pulse-field gel electrophoresis. Cells devoid of DR2462 protein showed a delay shorter than one hour in the restoration of an intact genome after irradiation compared to the wild type bacteria (Fig 6B) but it seems that replication did not restart immediately after reconstitution of the genome (Fig 6B), a delay that was also observed for cell division restart (Fig 6A).


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)

The ΔDR2462 bacteria show an increased delay in cell division and in reconstitution of genomic DNA after γ-irradiation.A. Growth delay after irradiation. Wild type (blue squares) and ΔDR2462 (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 NotI prior to analyses by PFGE. B: wild type; C: ΔDR2462.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124358.g006: The ΔDR2462 bacteria show an increased delay in cell division and in reconstitution of genomic DNA after γ-irradiation.A. Growth delay after irradiation. Wild type (blue squares) and ΔDR2462 (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 NotI prior to analyses by PFGE. B: wild type; C: ΔDR2462.
Mentions: The kinetics of DNA DSB repair in the ΔDR2462 mutant exposed to 3.8 kGy γ-irradiation was measured by pulse-field gel electrophoresis. Cells devoid of DR2462 protein showed a delay shorter than one hour in the restoration of an intact genome after irradiation compared to the wild type bacteria (Fig 6B) but it seems that replication did not restart immediately after reconstitution of the genome (Fig 6B), a delay that was also observed for cell division restart (Fig 6A).

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