PprI: The Key Protein in Response to DNA Damage in Deinococcus

Lu, Haijun; Hua, Yuejin

doi:10.3389/fcell.2020.609714

Cited by 10 publications

(5 citation statements)

References 65 publications

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“…The dearth of molecular tools in D. radiodurans has limited the progress in studying its radiation resistance mechanisms. For example, in well-worked out system of DdrO-based regulation of radiation response, it is still not known how PprI is activated ( 44 ). Not all the transcriptional regulators have been characterized in this microbe and the role of sRNAs in radiation response requires further investigation.…”

Section: Discussionmentioning

confidence: 99%

Effective gene silencing using type I–E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans

Misra,

Pandey,

Appukuttan

et al. 2023

Microbiol Spectr

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The extremely radiation-resistant bacterium, Deinococcus radiodurans , is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting, with its multiploid genome presenting an additional challenge. In view of this, the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for the expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A dCas9 system in D. radiodurans . An assayable acid phosphatase gene, phoN integrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown of ssb , a gene important for radiation resistance resulted in poor recovery post-irradiation. Targeting the Radiation and Desiccation Response Motif (RDRM), upstream of the ssb , prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down both phoN and ssb expression simultaneously. The programmable CRISPR interference tool developed in this study will facilitate the study of essential genes, hypothetical genes, and cis -elements involved in radiation response as well as enable metabolic engineering in this organism. Further, the tool can be extended for implementing high-throughput approaches in such studies. IMPORTANCE Deinococcus radiodurans is a microbe that exhibits a very high degree of radiation resistance. In addition, it is also identified as an organism of industrial importance. We report the development of a gene-knockdown system in this organism by engineering a type I-E clustered regularly interspaced short palindromic repeat (CRISPR)-Cascade system. We used this system to silence an assayable acid phosphatase gene, phoN to 10% of its activity. The study further shows the application of the Cascade system to target an essential gene ssb , that caused poor recovery from radiation. We demonstrate the utility of CRISPR-Cascade to study the role of a regulatory cis -element in radiation response as well as for multi-gene silencing. This easy-to-implement CRISPR interference system would provide an effective tool for better understanding of complex phenomena such as radiation response in D. radiodurans and may also enhance the potential of this microbe for industrial application.

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Section: Discussionmentioning

confidence: 99%

Effective gene silencing using type I–E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans

Misra,

Pandey,

Appukuttan

et al. 2023

Microbiol Spectr

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show abstract

“…The dearth of molecular tools in D. radiodurans has limited the progress in studying its radiation resistance mechanisms. For example, in well-worked out system of DdrO-based regulation of radiation response, it is still not known how PprI is activated (Lu & Hua, 2021). Not all the transcriptional regulators have been characterized in this microbe and the role of sRNAs in radiation response requires further investigation.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CRISPRi inDeinococcus radiodurans

Misra¹,

Pandey²,

Appukuttan

et al. 2022

Preprint

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The extremely radiation resistant bacterium,Deinococcus radiodurans, is a microbe of importance, both, for studying stress tolerance mechanisms and as a chassis for industrial biotechnology. However, the molecular tools available for use in this organism continue to be limiting. In view of this, the CRISPR-Cas tools provide a large repertoire of applications for gene manipulation. We show the utility of the type I-E Cascade system for knocking down gene expression in this organism. A single-vector system was designed for expression of the Cascade components as well as the crRNA. The type I-E Cascade system was better tolerated than the type II-A Cas9 system inD. radiodurans. An assayable acid phosphatase gene,phoNintegrated into the genome of this organism could be knocked down to 10% of its activity using the Cascade system. Cascade-based knockdown ofssb, a gene important for radiation resistance resulted in poor recovery post irradiation. Targeting the Radiation and Desiccation Resistance Motif (RDRM), upstream of thessb, prevented de-repression of its expression upon radiation exposure. In addition to this, multi-locus targeting was demonstrated on the deinococcal genome, by knocking down bothphoNandssbexpression simultaneously. The programmable CRISPRi tool developed in this study will facilitate study of essential genes, hypothetical genes, cis-elements involved in radiation response as well as enable metabolic engineering in this organism. Further the tool is amenable for implementing high-throughput approaches for such studies.

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“…The IrrE/DdrO system is crucial for radiation resistance in D. radiodurans and D. deserti, and most likely in other Deinococcus bacteria as well. It is also the best studied radiation response pathway in these bacteria (de Groot et al 2019;Lim et al 2019;Lu et al 2019;Lu and Hua 2020;Blanchard and de Groot 2021;Eugénie et al 2021;Magerand et al 2021;Narasimha and Basu 2021). This system controls expression of genes that are characterized by a palindromic motif called radiation-desiccation response motif (RDRM) in the promoter region, which is the binding site for the transcriptional repressor DdrO (Makarova et al 2007;Wang et al 2015;Blanchard et al 2017;Eugénie et al 2021).…”

Section: Radiation and Oxidative Stress Resistance-associated Regulat...mentioning

confidence: 99%

DNA repair and oxidative stress defense systems in radiation-resistant Deinococcus murrayi

Groot

Blanchard

2023

Can. J. Microbiol.

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Deinococcus murrayi is a bacterium isolated from hot springs in Portugal, and named after Dr. Robert G. E. Murray in recognition of his research on the genus Deinococcus. Like other Deinococcus species, D. murrayi is extremely resistant to ionizing radiation. Repair of massive DNA damage and limitation of oxidative protein damage are two important factors contributing to the robustness of Deinococcus bacteria. Here, we identify, amongst others, the DNA repair and oxidative stress defense proteins in D. murrayi, and highlight special features of D. murrayi. For DNA repair, D. murrayi does not contain a standalone uracil-DNA glycosylase (Ung), but it encodes a protein in which Ung is fused to a DNA photolyase domain (PhrB). UvrB and UvrD contain large insertions corresponding to inteins. One of its endonuclease III enzymes lacks a [4Fe-4S] cluster. Deinococcus murrayi possesses a homolog of the error-prone DNA polymerase IV. Concerning oxidative stress defense, D. murrayi encodes a manganese catalase in addition to a heme catalase. Its organic hydroperoxide resistance protein Ohr is atypical because the redox active cysteines are present in a CXXC motif. These and other characteristics of D. murrayi show further diversity among Deinococcus bacteria with respect to resistance-associated mechanisms.

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PprI: The Key Protein in Response to DNA Damage in Deinococcus

Cited by 10 publications

References 65 publications

Effective gene silencing using type I–E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans

Effective gene silencing using type I–E CRISPR system in the multiploid, radiation-resistant bacterium Deinococcus radiodurans

CRISPRi inDeinococcus radiodurans

DNA repair and oxidative stress defense systems in radiation-resistant Deinococcus murrayi

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