DrwH, a novel WHy domain-containing hydrophobic LEA5C protein from Deinococcus radiodurans, protects enzymatic activity under oxidative stress

Water stress and hypersensitive response (WHy) domain is typically found as a component of atypical late embryogenesis abundant (LEA) proteins closely associated with resistance to multiple stresses in numerous organisms. Several putative LEA proteins have been identified in Deinococcus bacteria; however their precise function remains unclear. This work reports the characterization of a Deinococcus-specific gene encoding a novel WHy domain-containing hydrophobic LEA5C protein (named DrwH) in D. radiodurans R1. The expression of the drwH gene was induced by oxidative and salinity stresses. Inactivation of this gene resulted in increased sensitivity to oxidative and salinity stresses as well as reduced activities of antioxidant enzymes. The WHy domain of the DrwH protein differs structurally from that of a previously studied bacterial LEA5C protein, dWHy1, identified as a gene product from an Antarctic desert soil metagenome library. Further analysis indicated that in E. coli, the function of DrwH is related to oxidative stress tolerance, whereas dWHy1 is associated with freezing-thawing stress tolerance. Under oxidative stress induced by H2O2, DrwH protected the enzymatic activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH). These findings provide new insight into the evolutionary and survival strategies of Deinococcus bacteria under extreme environmental conditions.


Construction of the plasmid-mediated complementation strain of the drwH mutant.
The plasmid carrying the wild-type drwH gene with its endogenous promoter was constructed using the E. coli-D. radiodurans shuttle plasmid pRADZ3 as a vector 9 (see Supplementary   Fig. S8 online) to complement the constructed R1-01 deletion strain. Briefly, an 835-bp DNA fragment of the wild-type drwH gene with its endogenous promoter and terminator regions was amplified from genomic DNA of strain R1 with the primer P11 with a HindIII site and P12 with a BamHI site (see Supplementary Table S3 online). The amplicon was double-digested with HindIII and BamHI, and ligated into the corresponding site of pRADZ3 to yield the complementation plasmid pRA-drwH. Correct recombination was checked by PCR, followed by nucleotide sequencing of the amplicon obtained. pRA-drwH was transformed into R1-01 deletion strain to generate the plasmid-mediated complementation strain, designated R1-11 (ΔdrwHcomp) (see Supplementary Table S2 online).
Abiotic stress-resistance assays. The D. radiodurans cells were grown in TGY medium with the appropriate antibiotics to the beginning of the exponential phase (OD600≈0.6) at 30°C. Cultures were then pelleted from 1 mL cultures by centrifugation to remove the growth medium. For H2O2 treatment, the pelleted cells were treated with fresh TGY medium containing various concentrations (20, 40, 60, 80, 100 mM) of H2O2 in the dark for 30 min.
For NaCl treatment, the prepared cells were resuspended in fresh TGY medium containing different concentrations (ranged from 0 to 5 M) of NaCl with shaking for 5 h. Desiccation stress assays were carried out as previously described 10 with some modifications. Briefly, 100 µL of cell suspension was placed inside a sealed desiccator at 25°C. Relative humidity within the desiccator was measured as less than 5% with a hygrometer. The desiccators were sealed, and the dried cultures were stored undisturbed at 25°C for 60 days. The samples were rehydrated by the addition of 1 mL TGY at regular intervals (ranged from 0 to 60 days) under sterilized conditions for 30 min. At the times indicated, 10 times serial dilutions were made, and 100 or 10 μL of each serial dilution of cell suspensions was spread/dripped onto TGY 4 agar plates. These plates were incubated at 30°C for 3 days before colony growth was observed and enumerated. The survival rate was expressed as the percentage of the number of colonies in the treated samples compared with those in the untreated controls. All the experiments are performed three times, and the values are shown as the mean ± standard deviation.
The wild-type and recombinant E. coli strains were grown in LB broth supplemented with kanamycin (50 μg/mL) and 0.1 mM isopropyl-thiogalactopyranoside (IPTG) at 37°C to an OD600 of 0.5. Cells were then pelleted from 1 mL cultures by centrifugation to remove the growth medium and resuspended in 1 mL fresh LB medium. For oxidative stress, 1.5 μL 30% H2O2 was added to a final concentration of 15 mM in the cell suspensions for 10 min. For freezing-thawing stress, 1 mL cell culture was frozen two times at -80°C for 20 min and thawed at room temperature for 20 min. After incubation, serial dilutions of 10 times were made. Ten microliters of each dilution was dripped onto LB agar plates at 37°C overnight. All assays were performed in triplicate.

Preparation of the Dr-WHy protein. The recombinant strain BL21-1 expressing
Dr-WHy protein was grown in LB broth supplemented with kanamycin (50 μg/mL) at 37°C to an OD600 of 0.5 and then induced with 0.5 mM IPTG at 16°C overnight. Then cells were harvested and resuspended in 20 mM Tris-HCl, pH 8.0, and 300 mM NaCl. Cells were lysed by sonication on ice, and the lysates were cleared by centrifugation at 13,000 g for 20 min at 4°C. The supernatant was then subjected to affinity chromatography on Ni 2+ -NTA agarose (Qiagen). After washing, the proteins were eluted with buffer (50 mM Tris-HCl, pH 8.0, 1 M NaCl) containing 300 mM imidazole and dialyzed overnight. The purity of purified Dr-WHy proteins was greater than 95%, as judged by electrophoresis analysis on a 15% polyacrylamide gel containing SDS that was then stained with Coomassie Blue (see

MDH and LDH enzymatic activities measurement under oxidative stress in vitro.
MDH from porcine heart (Sigma) and LDH from rabbit muscle (Sigma) were used to test the protective effects of the Dr-WHy (truncated-DrwH) protein against oxidative stress according to a previously published method 11,12 . Experiments were performed in Eppendorf tubes to avoid protein adsorption to glass. The enzymes and corresponding protein were diluted in 50 mM potassium phosphate buffer (pH 7.