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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

Description of the transposition procedure used to create the D. radiodurans Tn5-insertion mutant library.(A) The Tn5-based transposon delivery vector, p13554 is a derivative of the temperature sensitive plasmid p13841 [24]. The mini Tn5 (Tn5-hph) consists of a hygromycin resistance cassette (hph) as a selectable marker, flanked by the optimized 19-bp mosaic ends (ME) of Tn5 [28]. The hph gene is expressed under the control of the Pkat promoter. The hyperactive Tn5 transposase [25,26,94] is cloned outside the mobile element to generate stable insertions and is expressed from the Pspac promoter inducible with IPTG [31]. Ter116 is a D. radiodurans transcription terminator. (B) Flow chart of the procedure used to screen the D. radiodurans Tn5-insertion mutant library for sensitivity to DNA damaging agents.
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pone.0124358.g001: Description of the transposition procedure used to create the D. radiodurans Tn5-insertion mutant library.(A) The Tn5-based transposon delivery vector, p13554 is a derivative of the temperature sensitive plasmid p13841 [24]. The mini Tn5 (Tn5-hph) consists of a hygromycin resistance cassette (hph) as a selectable marker, flanked by the optimized 19-bp mosaic ends (ME) of Tn5 [28]. The hph gene is expressed under the control of the Pkat promoter. The hyperactive Tn5 transposase [25,26,94] is cloned outside the mobile element to generate stable insertions and is expressed from the Pspac promoter inducible with IPTG [31]. Ter116 is a D. radiodurans transcription terminator. (B) Flow chart of the procedure used to screen the D. radiodurans Tn5-insertion mutant library for sensitivity to DNA damaging agents.

Mentions: To generate a collection of mutants in D. radiodurans, we have developed an in vivo Tn5-based mutagenesis system. For this purpose, we cloned a mini-Tn5 derived (Tn5-hph) transposable element and a mutant tnp gene encoding a hyperactive Tn5 transposase [26] into a conditionally replicating temperature-sensitive shuttle vector (repUTs), that was shown previously to be stably maintained at 28°C in D. radiodurans and rapidly lost at 37°C [24] (plasmid p13554, Fig 1A). The Tn5-hph mini-transposon was constructed by assembling in vitro a cassette conferring hygromycin resistance and two flanking optimized 19-bp transposase recognition sequences (Mosaic Ends; [28]) (see Material and Methods for details of the construction). The tnp gene encoding the transposase was placed under the control of the Pspac promoter [31] and cloned outside the mobile element to obtain stable insertions upon the loss of the delivery vector.


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)

Description of the transposition procedure used to create the D. radiodurans Tn5-insertion mutant library.(A) The Tn5-based transposon delivery vector, p13554 is a derivative of the temperature sensitive plasmid p13841 [24]. The mini Tn5 (Tn5-hph) consists of a hygromycin resistance cassette (hph) as a selectable marker, flanked by the optimized 19-bp mosaic ends (ME) of Tn5 [28]. The hph gene is expressed under the control of the Pkat promoter. The hyperactive Tn5 transposase [25,26,94] is cloned outside the mobile element to generate stable insertions and is expressed from the Pspac promoter inducible with IPTG [31]. Ter116 is a D. radiodurans transcription terminator. (B) Flow chart of the procedure used to screen the D. radiodurans Tn5-insertion mutant library for sensitivity to DNA damaging agents.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124358.g001: Description of the transposition procedure used to create the D. radiodurans Tn5-insertion mutant library.(A) The Tn5-based transposon delivery vector, p13554 is a derivative of the temperature sensitive plasmid p13841 [24]. The mini Tn5 (Tn5-hph) consists of a hygromycin resistance cassette (hph) as a selectable marker, flanked by the optimized 19-bp mosaic ends (ME) of Tn5 [28]. The hph gene is expressed under the control of the Pkat promoter. The hyperactive Tn5 transposase [25,26,94] is cloned outside the mobile element to generate stable insertions and is expressed from the Pspac promoter inducible with IPTG [31]. Ter116 is a D. radiodurans transcription terminator. (B) Flow chart of the procedure used to screen the D. radiodurans Tn5-insertion mutant library for sensitivity to DNA damaging agents.
Mentions: To generate a collection of mutants in D. radiodurans, we have developed an in vivo Tn5-based mutagenesis system. For this purpose, we cloned a mini-Tn5 derived (Tn5-hph) transposable element and a mutant tnp gene encoding a hyperactive Tn5 transposase [26] into a conditionally replicating temperature-sensitive shuttle vector (repUTs), that was shown previously to be stably maintained at 28°C in D. radiodurans and rapidly lost at 37°C [24] (plasmid p13554, Fig 1A). The Tn5-hph mini-transposon was constructed by assembling in vitro a cassette conferring hygromycin resistance and two flanking optimized 19-bp transposase recognition sequences (Mosaic Ends; [28]) (see Material and Methods for details of the construction). The tnp gene encoding the transposase was placed under the control of the Pspac promoter [31] and cloned outside the mobile element to obtain stable insertions upon the loss of the delivery vector.

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