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The Site-Directed A184S Mutation in the HTH Domain of the Global Regulator IrrE Enhances Deinococcus radiodurans R1 Tolerance to UV Radiation and MMC Shock
The Site-Directed A184S Mutation in the HTH Domain of the Global Regulator IrrE Enhances Deinococcus radiodurans R1 Tolerance to UV Radiation and MMC Shock
Journal of Microbiology and Biotechnology. 2015. Dec, 25(12): 2125-2134
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : July 02, 2015
  • Accepted : September 01, 2015
  • Published : December 28, 2015
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About the Authors
Chen Zhang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Zhengfu Zhou
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Wei Zhang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Zhen Chen
College of Biological Sciences, China Agricultural University, Beijing 100083, P.R. China
Yuan Song
College of Biological Sciences, China Agricultural University, Beijing 100083, P.R. China
Wei Lu
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Min Lin
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
Ming Chen
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China
chenming01@caas.cn

Abstract
IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad-IrrE in the D. radiodurans irrE -deletion mutant (Δ irrE ), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the Δ irrE mutant complemented strain with Drad-irrE , designated Δ irrE-E . The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA , and ddrB , were remarkably induced after UV radiation and MMC shock in the Δ irrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.
Keywords
Introduction
The Deinococcus species are famous for their ability to survive extreme stresses, such as ultraviolet radiation, ionizing radiation, desiccation, and oxidative stress [2 , 8 , 9 , 20] . IrrE is a novel regulator protein (DR0167, inducer of pleiotropic proteins promoting DNA repair, also named pprI ) and was discovered in the analysis of a DNA damage-sensitive strain [8 , 10] . Compared with wild-type D. radiodurans , the Δ irrE mutant displayed significantly altered phenotypes, including increased sensitivity to UV and γ radiations [23] . IrrE, as previously considered, is not only playing an important role in DNA repair probably by activating the expression of recA and pprA , but is also likely involved with transcriptional regulation, signal transduction, and proteolysis [15 , 16] . Lu et al. [15] revealed that the IrrE HTH domain can bind directly to the recA and pprA promoters, and disruption of irrE led to increased sensitivity to UV radiation.
D. gobiensis I-0 was isolated by our laboratory from the upper sand layers of the Gobi Desert, Xinjiang, China, where the environment rapidly changes and the bacteria are exposed to cycles of high and low temperatures and prolonged desiccation. Phylogenetic analysis of D. gobiensis I-0 ( Fig. 1 ) indicated that it belongs to the genus Deinococcus . Interestingly, this strain exhibited stronger resistance to γ and UV radiations than D. radiodurans [26] . IrrE in D. gobiensis I-0 is highly conserved with the IrrE sequences in other Deinococcus strains and bears 66% identity to D. radiodurans . Moreover, Dgob-irrE exhibits stronger radiation tolerance than Drad-irrE when used to complement the D. radiodurans Δ irrE mutant.
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Unrooted neighbor-joining phylogenetic tree from the alignment of the D. gobiensis I-0 IrrE protein with related proteins.

The bootstrap values based on 1,000 replications are listed as percentages at the branching point. The bar indicates 0.05 substitutions per position. The species containing the IrrE protein and the IrrE access numbers in brackets are listed as shown. The phylogenetic tree was constructed by MEGA ver. 5.0.

Crystal structure analysis of IrrE from D. deserti demonstrated three domains: an N-terminal peptidase-like domain, a central helix-turn-helix motif, and a GAF-like putative sensor domain [23] . Current research has shown that IrrE in D. deserti is a metalloprotease that cleaves the repressor protein DdrO [16] , which is in accord with the N-terminal function. A previous study showed that expression of HTH domain-deficient Drad-IrrE resulted in increased sensitivity to γ radiation, UV radiation, and mitomycin C stress. Therefore, the IrrE HTH domain is essential to program the DNA repair process and cellular survival of D. radiodurans in response to radiation damage [15] . Previous research on the HTH domain focused on the consensus sequence using site-directed mutagenesis to mutate specific residues, such as Y160, but failed to identify any key residue [23] . However, their research methodology for examining critical sites provided us with an idea.
From a multisequence alignment of IrrE in combination with some fundamental experiments, we hypothesized that three different sites in the Dgob-IrrE HTH domain may be critical for its stronger radiation tolerance. Here, we found that the S131A mutation greatly decreased the radiation tolerance, and the corresponding A184S mutant displayedhigher resistance against UV radiation and desiccation. Taken together, these results suggest that the serine residue is the critical site in the Dgob-IrrE HTH domain, which may contribute to improving the affinity of DNA binding and the enhanced UV radiation tolerance of Drad-IrrE. Moreover, the modified A184S strain possesses a series of remarkable traits, which can be applied in the agricultural field and aid in the production of high-quality crops.
Materials and Methods
- Bacterial Strains, Plasmids, and Growth Conditions
The bacterial strains and plasmids used in this study are listed in Table 1 . The E. coli strain and its variants were maintained in Luria-Bertani (LB) medium at 37℃ in the presence of the appropriate antibiotics as required. The Deinococcus strains were grown in TGY (1% tryptone, 0.5% yeast extract, 0.1% glucose) medium at 30℃ in the presence of the appropriate antibiotics as required.
Bacterial strains and plasmids used in this study.
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Bacterial strains and plasmids used in this study.
- Cloning and Site-Directed Mutagenesis
Plasmid constructions were carried out according to standard procedures. The D. radiodurans and D. gobiensis I-0 irrE genes ( Drad-irrE and Dgob-irrE , respectively) based on the Genbank sequences were amplified with pMD18T- Drad-irrE and pMD18-T Dgob-irrE , respectively, as the template and ligated into pRADZ3 using the restriction sites Nde I and Spe I. The resulting recombinant plasmids were named as pRADZ3- E and pRADZ3- IE . Mutagenesis was performed to introduce the following designed changes: A122P, S131A, and A184S. Six primers for each mutation were designed to contain the corresponding nucleotide changes (see Table 2 ). These primers and the pRADZ3- E and pRADZ3- IE templates were used to introduce mutations using the polymerase chain reaction (PCR) under the following conditions: one cycle of denaturation at 95℃ for 5 min, 30 cycles of denaturation at 95℃ for 30 sec, annealing at 60℃ for 30 sec, and extension at 72℃ for 50 sec, followed by an extra extension at 72℃ for 10 min. The PCR products were purified using the TianGen PCR purification kit and were ligated into the pARDZ3 vector. The nucleotide changes were then sequenced by the Beijing Genomics Institute.
Mutagenic primer sequences.
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Bold residues indicate the mutation sites.
- Transformation of the Constructed Plasmids intoD. radioduransΔirrEMutant Competent Cells
For the transformation, the Δ irrE mutant was grown to mid-exponential phase (optical density at 0.6-0.8) in the presence of spectinomycin (at 350 μg/ml) and then centrifuged at 6,000 × g for 3 min. The pellet was suspended in 180 μl of fresh TGY liquid, and 20 μl of 0.3 M CaCl 2 was added, and the mixture was incubated at 30℃ for approximately 90 min. A total of 50 μl of the resuspended pellet was mixed with 10 μl (0.03 pM) of the constructed plasmids and was incubated under the same conditions (at 30℃ for approximately 90 min). The total 60 μl volume was pipetted into 2 ml of TGY medium and cultivated with no antibiotics at 30℃ with shaking at 220 rpm overnight. A total of 200 μl of the overnight suspension culture was plated on TGY medium in the presence of chloromycetin (at 3.4 μg/ml) and then incubated for 3 days at 30℃ to select the targeted strains.
- Abiotic Stress Resistance Experiments
The strains listed in Table 1 (except for E. coli strains grown in LB medium) were grown in TGY medium at 30℃ to an OD 600 of 6. Then 20 ml of the bacteria suspension was centrifuged at 6,000 × g for 5 min and washed twice with PBS, and then after 20 ml of PBS buffer was added, the mix was treated with UV radiation (254 nm, 5 min 380 J/m 2 , 10 min 760 J/m 2 , 15 min 1.14 kJ/m 2 , 20 min 1.52 kJ/m 2 ). In the desiccation stress experiments, we collected cells when the OD 600 reached 6-8 and divided them into many equal parts. We removed the same batch from the desiccator every 10 days and counted the surviving cells. In mitomycin C (MMC) shock experiments, the cells were collected when the OD 600 reached 0.6-0.8, added to TGY liquid medium containing MMC (20 μg/ml) for 10 min, and then a diluted suspension was spotted onto TGY plates. All experiments were performed in triplicate.
- Quantitative RT-PCR (qRT-PCR)
The bacteria were grown to an OD 600 of 6, treated with UV radiation for approximately 10 min, and then harvested. Total RNA was isolated using an RNA extraction kit (FastRNA Pro Blue RNA; Qbiogene). The RNA samples were digested and reverse-transcribed using a cDNA synthesis kit (PrimeScriptRT reagent kit with gDNA Eraser; TaKaRa) according to the manufacturer’s protocol. The expression of recA, pprA, uvrA , and ddrB was determined using the ABI PRISM 7500 system. The primers were designed and optimized using primer software (primer length ranged from 18 to 24 nucleotides and the annealing temperature varied from 55℃ to 60℃). PCR amplification was detected using a SYBR fluorescence dye (TaKaRa). The 16S RNA gene served as an internal control to normalize for the differences in total RNA quantity. The qRT-PCR primers are shown in Table 3 .
List of primers used in the qRT-PCR study.
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List of primers used in the qRT-PCR study.
- Sequence Alignment and Molecular Modeling
The sequences of Drad-IrrE, Dgob-IrrE, and other IrrE proteins were aligned using ClustalW ver. 2.0 and ESPript software [10] . A protein model was built using the online Swiss-Model program after input of the aligned sequences [19] based on the amino acid sequence homology. The three-dimensional (3D) structure of Dgob-IrrE was modeled, based on the reported crystal structure of IrrE from D. deserti (PDB ID 3DTI), which was visualized using the PyMOL software [7] . We also used site-directed mutagenesis and interaction bond manipulation to explain the different phenotypes in detail.
Results
- Dgob-IrrE Enhanced the Tolerance of the ΔirrEMutant Against UV Radiation
A previous study revealed that D. gobiensis I-0 exhibited strong resistance against γ and UV radiations [26] . Owing to the significant contribution of IrrE in the protection against γ and UV radiations, we carried out an experiment to investigate whether Dgob-IrrE improves resistance to UV radiation in the Δ irrE mutant compared with the wild-type DR and the complemented Δ irrE-E strain. We constructed two strains in the Δ irrE mutant background containing the shuttle plasmid pRADZ3 carrying the Dgob-irrE gene and Drad-irrE gene. Four strains were analyzed in this assay: wild-type DR, ΔirrE, ΔirrE-E , and ΔirrE-IE . The four strains were treated under high-intensity UV radiation (254 nm) for 5 min and then grown in TGY plates as shown in Fig.2 A . Clearly, ΔirrE-IE displayed higher resistance to UV radiation than the others, and the survival curve ( Fig.2 B) was in agreement with this phenotype. We also examined other abiotic stresses, such as mitomycin C shock and desiccation; there was also stronger phenotype in ΔirrE-IE compared with Δ irrE -E and DR strains under MMC shock, but no differences under desiccation (data not shown).
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Effect of UV radiation on growth and survival curve of wild-type DR and its variant strains.

() The growth of DR and its variant strains in TGY medium after exposure to UV (254 nm) radiation; () The survival curves of DR (rhombuses), Δ (squares), Δ (triangles), and (circles) strains under UV (254 nm) radiation.

- Multiple Alignment of IrrE and Computerized Analysis of Site-Directed Mutations
Based on the multiple alignment of the IrrE proteins shown in Fig. 3 , there are three different sites in the HTH domain between Dgob-IrrE and Drad-IrrE. Both IrrE 3D structures were built on the basis of homology modeling and aligned and visualized using PyMOL software. The three different sites are marked with an orange sphere in Fig. 4 A . The HTH domain of Drad-IrrE ranges from residues 163 to 200, and the region spanning 172-200 is conserved. We analyzed the conformational change of the site-directed A184S mutant ( Fig. 4 B). The results showed that 184S added double interaction bonds compared with A184, causing the conformational change that stably fastened the lysine residue. Lysine is involved the interaction with the DNA minor groove [18 , 21] . Therefore, we speculated that the conformational change of the lysine residue may increase the DNA-binding activity. The analysis of the other two sites is shown in Fig. S1.
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Multiple alignment of the IrrE proteins.

The multiple alignment of the IrrE proteins from , R1, I-0, and is shown. The alignment was generated with ClustalW, and the secondary structure elements of IrrE were aligned at the top with the ESPript program. The regions of sequence identity are shown in red. The HTH domain is marked by a thin rectangle, and the region selected for mutagenesis is marked by an arrowhead below the sequence.

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3D structure of the IrrE proteins and the computer analysis of the site-directed mutagenesis.

() An aligned 3D model of Dgob-IrrE and Drad-IrrE (N-terminal residues1-162; HTH domain residues163-200; C-terminal residues201-328). () Interaction bond changes between A184 and 184S. The yellow dash lines represent the interaction bonds, the salmon-labeled residues represent interaction sites, and the white residues are the mutagenesis sites.

- The A184S Mutant Enhanced Resistance to UV Radiation and MMC Shock
The A184S mutant displayed stronger resistance against UV radiation and MMC shock than the wild-type DR strain. The results in time courses demonstrated that the A184S mutant had significantly increased tolerance against UV radiation compared with the Δ irrE-E strain ( Fig.5 A). The survival curve of A184S (Fig. S2A) was in accordance with the growth on TGY plate under UV radiation. A184S also performed stronger tolerance with the treatment of MMC ( Fig. 6 A). The corresponding site of A184 in Dgob-IrrE i s S131 a nd w as m utated i nto S131A. We also generated the A122P mutant, which is another different site in the Dgob-IrrE HTH domain. Both the A122P and S131A mutants lost resistance against UV radiation ( Fig.5 B) in the first 5 min coinciding with the survival curve (Fig. S2B). The S131A mutant also showed a dramatic loss of MMC shock ( Fig. 6 B) and at 50 days desiccation (Fig.S4 ). All of these results indicated that the serine residue is critical for the HTH domain and may cause the conformational change leading to the higher DNA-binding efficiency to improve the expression level of IrrE-dependent genes.
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A184S mutation effect on resistance to UV radiation.

() The growth of DR, Δ, Δ, and A184S after UV radiation in a time course on a TGY plate. () The growth of Δ, Δ, A122P, and S131A in the first 5 min on TGY plate.

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A184S mutation effect on resistance to MMC shock.

() The growth of Δ, Δ, and A184S on a TGY plate under MMC shock (20 μg/ml) for 10 min. () The growth of Δ, Δ, S131A, A122P, and V184A on a TGY plate under MMC shock (20 μg/ml) for 10 min.

- Quantitative RT-PCR Revealed that Crucial Genes of Excision and Recombination Repair Pathways were Induced Significantly in the ΔirrE-IE and A184S Mutants
It was previously reported that both the excision and recombination repair pathways were the main pathways employed by D. radiodurans against UV radiation [24] . Some of the crucial genes involved in both pathways are recA (DR_2340), pprA (DR_A0346), and uvrA (DR_1771). RecA is required for extended synthesis-dependent strand annealing (EMSA) and is essential for radioresistance [3 , 6 , 20 , 27] . PprA was implicated in oxidative stress protection and interacted with double-stranded DNA and DNA gyrase to facilitate the radioresistance of D. radiodurans [13 , 14] . UvrA is one of the key components of the nucleotide excision repair (NER) pathway responsible for the repair and removal of damaged DNA lesions caused by UV radiation [22] . In addition, DdrB has been reported to play a critical role at early times after radiation [25] . qRT-PCR results showed that all four genes were induced after 10 min UV radiation ( Fig. 7 ). The recA gene in the Δ irrE-IE and A184S mutants were expressed greater than 1.5-fold in DR and Δ irrE-E ( Fig. 7 A). It is evident that these four genes in the A184S mutant displayed higher induction than in the Δ irrE -E mutant. qRT-PCR results also revealed that recA was also induced in A184S and suppressed in S131A under MMC shock, as shown in Fig. S5, indicating the importance of the serine residue.
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Relative expression of recA (A), pprA (B), uvrA (C), and ddrB (D) after 10 min UV radiation.

, and represent the , and gene expression changes after UV radiation (grey bar) compared with the untreated samples (black bar), respectively.

Discussion
A previous study by our laboratory revealed that D. gobiensis I-0 displayed a stronger tolerance than D. radiodurans against UV radiation [26] , but the cause of this D. gobiensis I-0 phenotype has not been demonstrated. IrrE, as a general switch, plays a significant role in the extreme radioresistance of Deinococcus , including gamma radiation and UV radiation. Both stresses cause the similar impairment of DNA [12] . The IrrE protein sequence and biological function among the Deinococcus genus is highly conserved [23] . Our work revealed that Dgob-IrrE was not only fully restored but also demonstrated stronger radioresistance in the Δ irrE mutant than Drad-IrrE did against UV radiation. The previous work of Lu et al. [15] showed that the HTH domain of Drad-IrrE specifically bound to the promoters of recA and pprA ; both are important elements involved in the DNA repair pathway. The HTH domain-deficient mutant only partially restored resistance of the Δ irrE mutant to γ radiation, UV radiation, and mitomycin C. These results indicated that the HTH domain is essential for IrrE in response to radiation damage [15] . Therefore, we speculated that the stronger resistance of Dgob-IrrE was possibly due to different amino acids in the HTH domain. We undertook a series of biological experiments and bioinformatics analyses to identify the crucial sites.
The crystal structure of D. deserti IrrE was previously solved and was shown to contain an N-terminal peptidase-like domain, a central helix-turn-helix motif, and a GAF-like putative sensor domain [23] . We established the 3D structure of Drad-IrrE and Dgob-IrrE using the online Swiss-Model program based on homology modeling, and we improved the model quality using Chiron and SAVEs software to remove the clashes, as shown in Fig. 4 A. The three different sites, A122, S131, and T140, were marked with spheres. Compared with the A184 site, the mutated S184 site added two interaction bonds with the K183 residue, as shown Fig.4 B. A recent report showed that 80% of all protein interactions with the DNA minor groove at AT-tracts are mediated by arginine or lysine [18] . The additional interaction bonds of S184 with K183 may cause a conformational change and increase DNA-binding activity. Conversely, serine is a hydrophilic amino acid and alanine is a hydrophobic residue, and this site-directed mutagenesis also increased the DNA-binding affinity. The third site, T140 of Dgob-IrrE and S193 of Drad-IrrE, had similar chemical properties (Fig. S1), which were not processed by site-directed mutagenesis.
Based on the work of Lu et al. [15] , we performed qRT-PCR to identify the regulation of IrrE, as shown in Fig. 7 . The genes recA, pprA, uvrA , and ddrB are important in the DNA repair pathway and displayed significant downregulation in the S131A mutant and up-regulation in the A184S mutant under UV radiation and MMC shock. These results verified the importance of the serine residue for the HTH domain. Current research showed that the N-terminal region is a metalloprotease, and it can cleave the repressor protein DdrO, leading to the transcriptional induction of various genes required for repair and survival after exposure of Deinococcus to radiation [16] . We found that the G19 site of the N-terminal region is different among the conserved α2-helix region and has yet to be characterized. The C-terminal domain, also named GAF domain, is a small molecule-binding domain present in many signaling and sensory proteins [11] . The GAF domain contains a V184 site, which is different from the conserved sequences. The site-directed mutant V184A displayed no phenotypic changes (Fig.S3 ) against UV radiation and MMC shock, indicating that V184 may not be the key residue in the GAF domain and is not the reason for the stronger resistance of Dgob-IrrE. Identifying the change in the IrrE-DNA interaction by NMR may provide further information about the HTH domain residues.
Drad-IrrE has been investigated for several years. The tolerance of E. coli harboring Drad-IrrE to osmotic stress, heat stress, and oxidative stress was significantly enhanced [4] . Z. Zhou’s early work revealed that the Drad-IrrE protein can enhance E. coli salt tolerance, and his team transformed the irrE gene into Brassica napus , which improved growth of the plant in high-salinity soil [17] . Interestingly, E. coli strains carrying variants of IrrE selected from a library of randomly generated mutants, such as E1, E28, E30, E79, and E80, conferred enhanced tolerance toward ethanol, butanol, and acetate [5] . These results demonstrate the importance of Drad-IrrE application, so it is meaningful and necessary to generate IrrE variants with increased activity against extreme environmental conditions. The A184S mutant displayed stronger resistance, providing new insights into modifications of the IrrE protein. Therefore, we can potentially apply this excellent IrrE variant to engineered crops to enhance growth traits under extreme environmental conditions.
Acknowledgements
This work was supported by grants from the National Basic Research (973) Program of China (2015CB755700), the National High-Tech (863) Program of China (No. 2012AA02A703), the National Research and Development Project of Transgenic Crops of China (2014ZX0800301B), and the National Natural Science Foundation of China (No. 31370126).
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