Thioredoxin (Trx), a ubiquitous protein showing disulfide reductase activity, plays critical roles in cellular redox control and oxidative stress response. Trx is a member of the Trx system, comprising Trx, Trx reductase (TrxR), and a cognate reductant (generally reduced nicotinamide adenine dinucleotide phosphate, NADPH). Bacterial Trx1 contains only the Trx-fold domain, in which the active site CXXC motif that is critical for the disulfide reduction activity is located. Bacterial Trx2 contains an N-terminal extension, which forms a zinc-finger domain, including two additional CXXC motifs. The multi-stress resistant bacterium Deinococcus radiodurans encodes both Trx1 (DrTrx1) and Trx2 (DrTrx2), which act as members of the enzymatic antioxidant systems. In this study, we constructed Δdrtrx1 and Δdrtrx2 mutants and examined their survival rates under H₂O₂ treated conditions. Both drtrx1 and drtrx2 genes were induced following H₂O₂ treatment, and the Δdrtrx1 and Δdrtrx2 mutants showed a decrease in resistance toward H₂O₂, compared to the wild-type. Native DrTrx1 and DrTrx2 clearly displayed insulin and DTNB reduction activity, whereas mutant DrTrx1 and DrTrx2, which harbors the substitution of conserved cysteine to serine in its active site CXXC motif, showed almost no reduction activity. Mutations in the zinc binding cysteines did not fully eliminate the reduction activities of DrTrx2. Furthermore, we solved the crystal structure of full-length DrTrx2 at 1.96 Å resolution. The N-terminal zinc-finger domain of Trx2 is thought to be involved in Trx-target interaction and, from our DrTrx2 structure, the orientation of the zinc-finger domain of DrTrx2 and its interdomain interaction, between the Trx-fold domain and the zinc-finger domain, is clearly distinguished from those of the other Trx2 structures.

Deinococcus radiodurans is a robust bacterium with extraordinary resistance to ionizing radiation and reactive oxygen species (ROS). D. radiodurans produces an antioxidant thiol compound called bacillithiol (BSH), but BSH-related enzymes have not been investigated. The D. radiodurans mutant lacking bshA (dr_1555), the first gene of the BSH biosynthetic pathway, was devoid of BSH and sensitive to hydrogen peroxide (H₂O₂) compared to the wild-type D. radiodurans strain. Three bacilliredoxin (Brx) proteins, BrxA, B, and C, have been identified in BSH-producing bacteria, such as Bacillus. D. radiodurans possesses DR_1832, a putative homolog of BrxC. However, because DR_1832 contains a novel signature motif (TCHKT) and a C-terminal region similar to the colicin-like immunity domain, we named it AbxC (atypical BrxC). The deletion of abxC also sensitized cells to H₂O₂. AbxC exhibited peroxidase activity in vitro, which was linked to nicotinamide adenine dinucleotide phosphate (NADPH) oxidation via the BSH disulfide reductase DR_2623 (DrBdr). AbxC proteins were present mainly as dimers after exposure to H₂O₂ in vitro, and the oxidized dimers were resolved to monomers by the reaction coupled with BSH as an electron donor, in which DrBdr transported reducing equivalents from NADPH to AbxC through BSH recycling. We identified 25 D. radiodurans proteins that potentially interact with AbxC using AbxC-affinity chromatography. Most of them are associated with cellular metabolisms, such as glycolysis and amino acid biosynthesis, and stress response. Interestingly, AbxC could bind to the proposed peroxide-sensing transcription regulator, DrOxyR. These results suggest that AbxC may be involved in the H₂O₂ signaling mechanism mediated by DrOxyR.

Cell growth is inhibited by abiotic stresses during industrial processes, which is a limitation of microbial cell factories. Microbes with robust phenotypes are critical for its maximizing the yield of the target products in industrial biotechnology. Currently, there are several reports on the enhanced production of industrial metabolite through the introduction of Deinococcal genes into host cells, which confers cellular robustness. Deinococcus is known for its unique genetic function thriving in extreme environments such as radiation, UV, and oxidants. In this study, we established that Deinococcus proteolyticus showed greater resistance to oxidation and UV-C than commonly used D. radiodurans. By screening the genomic library of D. proteolyticus, we isolated a gene (deipr_0871) encoding a response regulator, which not only enhanced oxidative stress, but also promoted the growth of the recombinant E. coli strain. The transcription analysis indicated that the heterologous expression of deipr_0871 upregulated oxidative-stress-related genes such as ahpC and sodA, and acetyl-CoA-accumulation-associated genes via soxS regulon. Deipr_0871 was applied to improve the production of the valuable metabolite, poly-3-hydroxybutyrate (PHB), in the synthetic E. coli strain, which lead to the remarkably higher PHB than the control strain. Therefore, the stress tolerance gene from D. proteolyticus should be used in the modification of E. coli for the production of PHB and other biomaterials

Regulatory T cells (Tregs) harness a central role in peripheral immune tolerance. Since its discovery, there have been numerous efforts to dissect the role of Tregs in the context of disease condition. Recently, Treg fields are moving from the academia to industry since many evidences have been accumulated for the potential of treatment of inflammatory disease by administering genetically engineered Tregs. However, in the pre-clinical or clinical trials, this rare cell subset needs to be expanded in ex-vivo condition to achieve a sufficient number for the controlling of the disease. Our group previously established ex-vivo Treg expansion condition with 25 mer oligonucleotide to maintain the stability and function of Tregs. However, non-biased investigations on the perturbation of transcriptomic and epigenetic changes during the culture condition has not been explored. Since cutting edge multiomics tools which can simultaneously profile epigenetic and transcript signatures in a single cell resolution has been commercialized, we adopt 10x multiome technique to investigate our frozen ex-vivo expanded Tregs(eTreg) compared to freshly isolated frozen human Treg(fTreg) from the same donor. Compared to fTreg cells, we have identified a new subset that exists only in the eTreg cells and named it eeTreg. We found that the eeTreg resembles the phenotype of both effector Tregs and tumor-infiltrated Tregs. eeTreg is characterized by a significantly increased expression and chromatin accessibility score of FOXP3, STAT5B, ETS1 and RUNX1, which are known to be the essential transcription factors of regulatory T cell function. In summary, we suggest a novel human treg subset which might be manufactured by manipulating ex-vivo culture condition.

DNA-protecting proteins (Dps) are crucial for safeguarding chromosomal DNA in starved cells during the stationary phase under stressful conditions. In previous research, the two Dps proteins in Deinococcus geothermalis, Dgeo_0257 (Dps3) and Dgeo_0281 (Dps1), were found to complement each other in protecting DNA from oxidative damage. This study investigates the physiological changes and transposition of insertion sequences (ISs) in a double-knockout (DK) mutant lacking both dps genes. Comparisons between the wild-type and mutant strains revealed significant phenotypic differences in viability under oxidative stress conditions induced by hydrogen peroxide and ferrous ions, particularly during the stationary phase. Notably, oxidative stress triggered the transposition of the IS families IS701 and IS5, with IS66 being transposed exclusively in the DK mutant into a gene encoding phytoene desaturase. Transcriptomic analysis using RNA-seq revealed substantial fold changes in gene expression across the genome. For example, the dgeo_1459–1460 gene cluster, which encodes a DUF421 domain-containing protein and a hypothetical protein, was highly upregulated under both oxidative and non-oxidative conditions. Interestingly, catalase, encoded by a single gene in D. geothermalis, was upregulated in the DK mutant during the stationary phase, with expression levels exceeding those observed in the single dps gene-deficient mutants. Conversely, a prominent downregulation of the Fur family regulator was detected. These findings highlight the growth phase-dependent physiological adaptation of the dps-DK mutant and reveal a novel IS transposition event of the ISBst12 group involving the IS66 family. Therefore, this study provides new observations into the influence of DNA-protective protein deficiency on oxidative stress responses and IS transposition in D. geothermalis, as well as the regulatory mechanisms of the catalase induction pathway, raising the need for further investigation into the role of OxyR.