Abstract

Deinococcus radiodurans is a polyextremophilic bacterium well known for its extreme resistance to irradiation, oxidative stress, and other damaging conditions. Many small noncoding RNAs (ncRNAs) in D. radiodurans have been identified by deep sequencing analysis and computational predictions. However, the precise roles of ncRNAs and their target genes in the oxidative stress response have not been investigated. Here, we report the identification and characterization of a novel ncRNA named OsiR (for oxidative stress-induced ncRNA). Oxidative stress tolerance analysis showed that deleting osiR significantly decreased viability, total antioxidant capacity, and catalase activity in D. radiodurans under oxidative stress conditions. Comparative phenotypic and qRT-PCR analyses of an osiR mutant identify a role of OsiR in regulating the expression of the catalase gene katE2. Microscale thermophoresis and genetic complementation showed that a 21-nt sequence in the stem–loop structure of OsiR (204–244 nt) directly base pairs with its counterpart in the coding region of katE2 mRNA (843–866 nt) via a 19 nt region. In addition, deletion of katE2 caused a significant reduction of catalase activity and oxidative stress tolerance similar to that observed in an osiR mutant. Our results show that OsiR positively regulates oxidative stress tolerance in D. radiodurans by increasing the mRNA stability and translation efficiency of katE2. This work provides a new regulatory pathway mediated by ncRNA for the oxidative stress response that most likely contributes to the extreme tolerances of D. radiodurans.

Highlights

  • The polyextremophilic bacterium Deinococcus radiodurans can survive under high-intensity ionizing radiation, ultraviolet radiation, desiccation, and oxidative stresses [1,2,3,4]

  • Our results indicate that OsiR act as a crucial regulator to positively regulate the oxidative stress resistance of D. radiodurans by directly base pairing with katE2 mRNA, a catalase gene

  • More than a hundred genes have been identified to contribute to oxidative stress tolerance in D. radiodurans; to date, the regulatory mechanism underlying the extreme oxidative stress tolerance in D. radiodurans has not been fully elucidated [5,11]. ncRNA-mediated regulation plays an important role in stress adaptation and gene regulation in almost all bacteria [20,42,43]

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Summary

Introduction

The polyextremophilic bacterium Deinococcus radiodurans can survive under high-intensity ionizing radiation, ultraviolet radiation, desiccation, and oxidative stresses [1,2,3,4]. The oxidative stress-induced sRNA OxyS from E.coli was the first characterized ncRNA involved in oxidative stress response [30], and has a negative regulatory effect on the mRNAs of transcription factors such as FhlA, RpoS, and FlhDC, as well as some other proteins [31,32]. 41 potential ncRNAs have been identified in D. radiodurans by combining deep sequencing analysis and computational predictions [37], but their biological functions and precise molecular mechanisms have not been characterized in detail except for Dsr (DnrH), which positively influences heat tolerance by increasing the transcription of hsp mRNA [38]. Our results indicate that OsiR act as a crucial regulator to positively regulate the oxidative stress resistance of D. radiodurans by directly base pairing with katE2 mRNA, a catalase gene. The significant roles of OsiR in the oxidative stress response highlight the importance of ncRNA-mediated regulation under stress conditions in the Deinococcus species

Results
Discussion
Construction of the Deletion Mutants and Complementation Strains
Northern Blot Analysis
Western Blot Analysis
Oxidative Stress Survival Assays
Half-Life Experiment
Determination of Total Antioxidant Capacity and Catalase Activity
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