Deinococcus radiodurans is known for its extreme resistance to ionizing radiation, oxidative stress, and other DNA-damaging agents. The robustness of this bacterium primarily originates from its strong oxidative resistance mechanisms. Hundreds of genes have been demonstrated to contribute to oxidative resistance in D. radiodurans; however, the antioxidant mechanisms have not been fully characterized. In this study, comparative proteomics analysis of D. radiodurans grown under normal and oxidative stress conditions was conducted using label-free quantitative proteomics. The abundances of 852 of 1700 proteins were found to significantly differ between the two groups. These differential proteins are mainly associated with translation, DNA repair and recombination, response to stresses, transcription, and cell wall organization. Highly upregulated expression was observed for ribosomal proteins such as RplB, Rpsl, RpsR, DNA damage response proteins (DdrA, DdrB), DNA repair proteins (RecN, RecA), and transcriptional regulators (members of TetR, AsnC, and GntR families, DdrI). The functional analysis of proteins in response to oxidative stress is discussed in detail. This study reveals the global protein expression profile of D. radiodurans in response to oxidative stress and provides new insights into the regulatory mechanism of oxidative resistance in D. radiodurans.

Deinococcus radiodurans adapts to challenging environments by modulating gene expression in response to oxidative stress. Recently, bacterial small noncoding RNAs (sRNAs) have been presumed to participate in the transcriptional or translational regulation of stress-responsive genes. We found 24 sRNAs that may be involved in the oxidative stress response of D. radiodurans by deep RNA sequencing. Moreover, a typical stress-inducible sRNA, IGR_3053, named OsiA, was predicted to bind to the mRNA of katA, katE, and sodC by the bioinformatics method. An osiA knockout of D. radiodurans displayed increased sensitivity to H₂O₂ and the decreased catalase activity and total antioxidant activity, suggesting that OsiA probably serves as a regulator in the adaptation to oxidative environments. Further microscale thermophoresis results demonstrated that OsiA can directly bind to the mRNA of katA, sodC, and katE. The stability test result of katA mRNA showed that its half-life was 2 min in the osiA mutant compared with 5 min in the wildtype(wt) strain. Our results indicated that OsiA can enhance the stability of katA mRNA and the activity of KatA and consequently the oxidation resistance of D.radiodurans. We are the first one to explore the super-strong oxidative stress resistance of D.radiodurans at the level of post-transcriptional regulation, and found a new pathway that provides a new explanation for the long-term adaptability of D.radiodurans in extreme environments.

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.

Deinococcus radiodurans is best known for its extraordinary resistance to diverse environmental stress factors, such as ionizing radiation, ultraviolet (UV) irradiation, desiccation, oxidation, and high temperatures. The heat response of this bacterium is considered to be due to a classical, stress-induced regulatory system that is characterized by extensive transcriptional reprogramming. In this study, we investigated the key functional genes involved in heat stress that were expressed and accumulated in cells (R48) following heat treatment at 48 °C for 2 h. Considering that protein degradation is a time-consuming bioprocess, we predicted that to maintain cellular homeostasis, the expression of the key functional proteins would be significantly decreased in cells (RH) that had partly recovered from heat stress relative to their expression in cells (R30) grown under control conditions. Comparative transcriptomics identified 15 genes that were significantly downregulated in RH relative to R30, seven of which had previously been characterized to be heat shock proteins. Among these genes, three hypothetical genes (dr_0127, dr_1083, and dr_1325) are highly likely to be involved in response to heat stress. Survival analysis of mutant strains lacking DR_0127 (a DNA-binding protein), DR_1325 (an endopeptidase-like protein), and DR_1083 (a hypothetical protein) showed a reduction in heat tolerance compared to the wild-type strain. These results suggest that DR_0127, DR_1083, and DR_1325 might play roles in the heat stress response. Overall, the results of this study provide deeper insights into the transcriptional regulation of the heat response in D. radiodurans.

Deinococcus radiodurans is an extremely resistant bacteria that has evolved masterful strategies to enable survival under various environmental stress conditions. Heat stress is a major environmental stress factor that can cause denaturation of proteins, membrane disruption, and oxidative stress. Previous studies have examined the mechanisms of the heat stress response by analyzing changes in protein levels; however, little is known about the role of small noncoding RNAs (ncRNAs), which are known to play important regulatory functions in bacteria during various environmental stress response. The ncRNA dsr11 of D. radiodurans was previously identified by RNA-seq and Northern blot. In this study, we showed that the transcription level of dsr11 was up-regulated 4.2-fold under heat stress by qRT-PCR analysis. Heat tolerance assay showed that deleting dsr11 significantly inhibited the viability under high temperature conditions. To assess the influence of dsr11 on the D. radiodurans transcriptome, 157 genes were found differentially expressed in the knock-out mutant by RNA-seq experiment. Combining RNA-seq and in silico analysis, we found that trmE (tRNA modification GTPase) and dr_0651 (arginase) were likely to be the direct targets of dsr11. Further microscale thermophoresis results demonstrated that dsr11 can directly bind to the mRNA of trmE and dr_0651. Our results indicated that dsr11 can enhance the tolerance to heat stress of D. radiodurans by binding to trmE and dr_0651 mRNA. Overall, these results extend our understanding of ncRNA regulation and provide new insights into the heat stress response in D. radiodurans.

Cold-adapted esterases are attracting increasing attention owing to their prospective use in biotechnology. In this study, a novel cold-adapted family Ⅳ esterase EstDR4 was identified and obtained from extremophile Deinococcus radiodurans (D. radiodurans). EstDR4 displayed significant substrate preference towards short and medium chain monoesters (C2–C12). It also showed regioselectivity, enantioselectivity and degradation effects on four insecticides. The optimum temperature and pH for EstDR4 activity were 30 °C and pH 8, respectively. Additionally, EstDR4 exhibited relatively high catalytic activity at 0 °C and high stability from 10–40 °C, with over 80% of its initial activity retained after 1 h of incubation. Moreover, EstDR4 activity was stimulated by Tween 80 and Triton X-100, and inhibited by metal ions such as Co²⁺, Cu²⁺ and Zn²⁺ and several organic solvents. Thus, this enzyme shows development potential for many industrial biotechnological applications, including the manufacture of thermolabile pharmaceutical products, cold-wash detergents and insecticide biodegradation.