# Structural and Biochemical Characterization of Thioredoxin-2 from _Deinococcus radiotrans_
## 3 Results and Discussion  
### Gene Expression and Phenotype Assay under H\({}_{2}\)O\({}_{2}\) Stress  
The _D. radiodurans_ genome encodes two Trx proteins, DR0944 (DrTrx1) and DRA0164 (DrTrx2). To evaluate the expression profile of _drtrx1_ and _drtrx2_, we performed qRT-PCR, using total RNA isolated from the wild-type _D. radiodurans_, which had been exposed to 20, 40, and 60 mM H\({}_{2}\)O\({}_{2}\) (Figure 1A). Transcriptional levels of _drtrx1_ and _drtrx2_ increased more than 2-fold following exposure to 40 mM H\({}_{2}\)O\({}_{2}\) and almost 17-fold and 9-fold for _drtrx1_ and _drtrx2_, following exposure to 60 mM H\({}_{2}\)O\({}_{2}\) treatment, respectively. To further investigate the roles of DrTrx1 and DrTrx2 in the resistance to oxidative stress, we constructed _\(\Delta\)drtrx1_ and _\(\Delta\)drtrx2_ disruption mutants and examined the resistance of these mutants toward H\({}_{2}\)O\({}_{2}\). Wild-type _\(\Delta\)drtrx1_, and _\(\Delta\)drtrx2_ were treated with 20, 40, 60, and 80 mM H\({}_{2}\)O\({}_{2}\) (Figure 1B). The resistance of \(\Delta\)drtrx1 and _\(\Delta\)drtrx2_ towards H\({}_{2}\)O\({}_{2}\) decreased over a thousand-fold compared to that of the wild-type when the cells were treated with 60 mM of H\({}_{2}\)O\({}_{2}\) for 1 h. Moreover, _\(\Delta\)drtrx2_ did not survive when the concentration of H\({}_{2}\)O\({}_{2}\) exceeded 60 mM (Figure 1B), indicating that _\(\Delta\)drtrx2_ was more sensitive to H\({}_{2}\)O\({}_{2}\) than _\(\Delta\)drtrx1_. We constructed expression plasmids producing DrTrx1 and DrTrx2 to complement _\(\Delta\)drtrx1_ and _\(\Delta\)drtrx2_. Complementation by plasmid-borne _drtrx1_ and _drtrx2 in trans_ showed the restoration of the sensitive phenotype of _\(\Delta\)drtrx1_ and _\(\Delta\)drtrx2_ (Figure S1). In _E. coli_, it was reported that _trx2_ acts as a member of the OxyR regulon and that the expression of _trx2_ is induced by H\({}_{2}\)O\({}_{2}\). In contrast, _trx1_ expression is not affected by H\({}_{2}\)O\({}_{2}\), indicating that _trx1_ is not regulated by OxyR. In fact, _trx1_ expression in _E. coli_ is known to be regulated by guanosine 3\({}^{\prime}\),5\({}^{\prime}\)-bispyrophosphate (ppGpp) [16]. The transcription levels of _drtrx1_ and _drtrx2_ in _D. radiodurans oxyR_ mutant (\(\Delta\)droxx\(R\)) showed a similar pattern with those of the wild-type after being exposed to 0, 20, and 40 mM H\({}_{2}\)O\({}_{2}\). Even though the expression of _drtrx1_ and _drtrx2_ was induced by H\({}_{2}\)O\({}_{2}\), the defect in _droxyR_ did not affect the  
Figure 1: Transcription and phenotype of _\(\Delta\)drtrx1_ and _\(\Delta\)drtrx2_ under H\({}_{2}\)O\({}_{2}\) stress. (**A**) _D. radiodurans_ was exposed to H\({}_{2}\)O\({}_{2}\) at the indicated concentrations (0, 20, 40, 60 mM), and qRT-PCR analysis was performed to determine _drtrx1_ and _drtrx2_ transcription level. The fold increase of transcription was determined by setting the expression level of the non-treated control to 1. (**B**) Survival curves of _D. radiodurans_ wild-type, _\(\Delta\)drtrx1_, and _\(\Delta\)drtrx2_ treated with different concentrations of H\({}_{2}\)O\({}_{2}\) (0, 20, 40, 60, and 80 mM). The cells spotted on TGY plates and colonies were counted after 3 d. Error bars indicate the standard deviation for three experimental replicates. expression of \(dtrx1\) and \(dtrx2\). This result shows that the mechanisms of the expression regulation of \(dtrx1\) and \(dtrx2\) and the correlation between these two genes and \(droxyR\) may differ from those reported in _E. coli_ (Figure S2). However, it should be noted that the DrOxyR is an atypical 1-cysteine, containing OxyR [51], and thus further investigation may be required in order to elucidate the exact role and regulon genes of DrOxyR. In a previous report, the deletion of _E.