Guangzhi Xu

A Novel OxyR Sensor and Regulator of Hydrogen Peroxide Stress with One Cysteine Residue in Deinococcus radiodurans

In bacteria, OxyR is a peroxide sensor and transcription regulator, which can sense the presence of reactive oxygen species and induce antioxidant system. When the cells are exposed to H2O2, OxyR protein is activated via the formation of a disulfide bond between the two conserved cysteine residues (C199 and C208). In Deinococcus radiodurans, a previously unreported special characteristic of DrOxyR (DR0615) is found with only one conserved cysteine. dr0615 gene mutant is hypersensitive to H2O2, but only a little to ionizing radiation. Site-directed mutagenesis and subsequent in vivo functional analyses revealed that the conserved cysteine (C210) is necessary for sensing H2O2, but its mutation did not alter the binding characteristics of OxyR on DNA. Under oxidant stress, DrOxyR is oxidized to sulfenic acid form, which can be reduced by reducing reagents. In addition, quantitative real-time PCR and global transcription profile results showed that OxyR is not only a transcriptional activator (e.g., katE, drb0125), but also a transcriptional repressor (e.g., dps, mntH). Because OxyR regulates Mn and Fe ion transporter genes, Mn/Fe ion ratio is changed in dr0615 mutant, suggesting that the genes involved in Mn/Fe ion homeostasis, and the genes involved in antioxidant mechanism are highly cooperative under extremely oxidant stress. In conclusion, these findings expand the OxyR family, which could be divided into two classes: typical 2-Cys OxyR and 1-Cys OxyR.

DrRRA: a novel response regulator essential for the extreme radioresistance of Deinococcus radiodurans

Two‐component systems are predominant signal transduction pathways in prokaryotes, and also exist in many archaea as well as some eukaryotes. A typical TCS consists of a histidine kinase and a cognate response regulator. In this study, a novel gene encoding a response regulator (we designate it drRRA) is identified to be essential for the extreme radioresistance of Deinococcus radiodurans. DrRRA null mutant (we designate it MR) is sensitive to gamma‐radiation compared with the wild‐type strain. Transcriptional assays show that numerous genes are changed in their transcriptional levels in MR at exponential growth phase under normal or gamma‐radiation condition. Most of them are related to stress response and DNA repair. Antioxidant activity assays exhibit that both superoxide dismutases and catalases are decreased in the mutant, whereas Western blotting assays show that RecA and PprA are also reduced in MR, verifying the microarray and quantitative real‐time PCR data. Furthermore, pulsed‐field gel electrophoresis assay demonstrates that deletion of drRRA results in the delay of genome restitution. These data support the hypothesis that DrRRA contributes to the extreme radioresistance of D. radiodurans through its regulatory role in multiple pathways such as antioxidation and DNA repair pathways.

Deinococcus radiodurans PprI Switches on DNA Damage Response and Cellular Survival Networks after Radiation Damage

Preliminary findings indicate that PprI is a regulatory protein that stimulates transcription and translation of recA and other DNA repair genes in response to DNA damage in the extremely radioresistant bacterium Deinococcus radiodurans. To define the repertoire of proteins regulated by PprI and investigate the in vivo regulatory mechanism of PprI in response to γ radiation, we performed comparative proteomics analyses on wild type (R1) and a pprI knock-out strain (YR1) under conditions of ionizing irradiation. Results of two-dimensional electrophoresis and MALDI-TOF MS or MALDI-TOF/TOF MS indicated that in response to low dose γ ray exposure 31 proteins were significantly up-regulated in the presence of PprI. Among them, RecA and PprA are well known for their roles in DNA replication and repair. Others are involved in six different pathways, including stress response, energy metabolism, transcriptional regulation, signal transduction, protein turnover, and chaperoning. The last group consists of many proteins with uncharacterized functions. Expression of an additional four proteins, most of which act in metabolic pathways, was down-regulated in irradiated R1. Additionally phosphorylation of two proteins was under the control of PprI in response to irradiation. The different functional roles of representative PprI-regulated genes in extreme radioresistance were validated by gene knock-out analysis. These results suggest a role, either directly or indirectly, for PprI as a general switch to efficiently enhance the DNA repair capability and extreme radioresistance of D. radiodurans via regulation of a series of pathways.

DdrB stimulates single-stranded DNA annealing and facilitates RecA-independent DNA repair in Deinococcus radiodurans

The bacterium Deinococcus radiodurans can survive extremely high exposure to ionizing radiation. The repair mechanisms involved in this extraordinary ability are still being investigated. ddrB is one gene that is highly up-regulated after irradiation, and it has been proposed to be involved in RecA-independent repair in D. radiodurans. Here we cloned, expressed and characterized ddrB in order to define its roles in the radioresistance of D. radiodurans. DdrB preferentially binds to single-stranded DNA. Moreover, it interacts directly with single-stranded binding protein of D. radiodurans DrSSB, and stimulates single-stranded DNA annealing even in the presence of DrSSB. The post-irradiation DNA repair kinetics of a ddrB/recA double mutant were compared to ddrB and recA single mutants by pulsed-field gel electrophoresis (PFGE). DNA fragment rejoining in the ddrB/recA double mutant is severely compromised, suggesting that DdrB-mediated single-stranded annealing plays a critical role in the RecA-independent DNA repair of D. radiodurans.

RecO Is Essential for DNA Damage Repair in Deinococcus radiodurans

Here we present direct evidence for the vital role of RecO in Deinococcus radioduranss radioresistance. A recO null mutant was constructed using a deletion replacement method. The mutant exhibited a growth defect and extreme sensitivity to irradiation with gamma rays and UV light. These results suggest that DNA repair in this organism occurs mainly via the RecF pathway.

Identification and evaluation of the role of the manganese efflux protein in Deinococcus radiodurans

BackgroundDeinococcus radiodurans accumulates high levels of manganese ions, and this is believed to be correlated with the radiation resistance ability of this microorganism. However, the maintenance of manganese ion homeostasis in D. radiodurans remains to be investigated.ResultsIn this study, we identified the manganese efflux protein (MntE) in D. radiodurans. The null mutant of mntE was more sensitive than the wild-type strain to manganese ions, and the growth of the mntE mutant was delayed in manganese-supplemented media. Furthermore, there was a substantial increase in the in vivo concentration of manganese ions. Consistent with these characteristics, the mntE mutant was more resistant to H2O2, ultraviolet rays, and γ-radiation. The intracellular protein oxidation (carbonylation) level of the mutant strain was remarkably lower than that of the wild-type strain.ConclusionsOur results indicated that dr1236 is indeed a mntE homologue and is indispensable for maintaining manganese homeostasis in D. radiodurans. The data also provide additional evidence for the involvement of intracellular manganese ions in the radiation resistance of D. radiodurans.

DR2539 is a novel DtxR-like regulator of Mn/Fe ion homeostasis and antioxidant enzyme in Deinococcus radiodurans

Transcriptional regulators of the diphtheria toxin repressor (DtxR) family control the expression of genes involved in the uptake of iron and manganese, which is not only necessitous nutrients but also was suggested to be essential for intracellular redox cycling of microorganisms. We identified a unique DtxR homologue (DR2539) with special characteristics from Deinococcus radiodurans, which is known for its extreme resistance to radiation and oxidants. The dr2539 mutant showed higher resistance to hydrogen peroxide than the wild-type strain R1. Intracellular catalase activity assay and semiquantitative PCR analysis demonstrated that this DtxR is a negative regulator of catalase (katE). Furthermore, quantitative real-time PCR, global transcription profile and inductively coupled plasma-mass spectrometry analysis showed that the DtxR is involved in the regulation of antioxidant system by maintaining the intracellular Mn/Fe ion homeostasis of D. radiodurans. However, unlike the other DtxR homologues, the DtxR of D. radiodurans acts as a negative regulator of a Mn transporter gene (dr2283) and as a positive regulator of Fe-dependent transporter genes (dr1219, drb0125) in D. radiodurans.

DNA binding is essential for PprI function in response to radiation damage in Deinococcus radiodurans

The extremely radioresistant bacterium Deinococcus radiodurans possesses a rapid and efficient but poorly known DNA damage response mechanism that mobilizes one-third of its genome to survive lethal radiation damage. Deinococcal PprI serves as a general switch to regulate the expression of dozens of proteins from different pathways after radiation, including the DNA repair proteins RecA, PprA and SSB. However, the underlying mechanism is poorly understood. In this study, we analyzed the dynamic alteration in global transcriptional profiles in wildtype and pprI mutant strains by combining microarrays and time-course sampling. We found that PprI up-regulated transcription of at least 210 genes after radiation, including 21 DNA repair and replication-related genes. We purified PprI and a helix-turn-helix (HTH) domain mutant and found that PprI specifically bound to the promoters of recA and pprA in vitro but did not bind nonspecific double-strand DNA. Chromatin immunoprecipitation (ChIP) assays confirmed that PprI specifically interacted with the promoter DNA of recA and pprA after radiation. Finally, we showed that a DNA-binding activity-deficient pprI mutant only partially restored resistance of the pprI mutant strain to γ radiation, UV radiation, and mitomycin C. Taken together, these results indicate that DNA-binding activity is essential for PprI to program the DNA repair process and cellular survival of D. radiodurans in response to radiation damage.

DRA0336, another OxyR homolog, involved in the antioxidation mechanisms in Deinococcus radiodurans

A novel OxyR (DR0615) with one conserved cysteine that senses hydrogen peroxide in Deinococcus radiodurans had been identified in our previous work. Comparative genomics revealed that D. radiodurans possesses another OxyR homolog, OxyR2 (DRA0336). In this study, we constructed the deletion mutant of oxyR2 and the double mutant of both the OxyR homologs to investigate the role of OxyR in response to oxidative stress in D. Radiodurans. Deletion of oxyR2 resulted in an obviously increased sensitivity to hydrogen peroxide, and the double mutant for oxyR and oxyR2 was significantly more sensitive than any of the two single mutants. The total catalase activity of the double mutant was lower than that of any of the single mutants, and reactive oxygen species (ROS) accumulated to a greater extent. DNA microarray analysis further suggested that oxyR2 was involved in antioxidation mechanisms. Site-direct mutagenesis and complementation analysis revealed that C228 in OxyR2 was essential. This is the first report of the presence of two OxyR in one organism. These results suggest that D. radiodurans OxyR and OxyR2 function together to protect the cell against oxidative stress.

Regulation of MntH by a dual Mn(II)- and Fe(II)-dependent transcriptional repressor (DR2539) in Deinococcus radiodurans.

The high intracellular Mn/Fe ratio observed within the bacteria Deinococcus radiodurans may contribute to its remarkable resistance to environmental stresses. We isolated DR2539, a novel regulator of intracellular Mn/Fe homeostasis in D. radiodurans. Electrophoretic gel mobility shift assays (EMSAs) revealed that DR2539 binds specifically to the promoter of the manganese acquisition transporter (MntH) gene, and that DR0865, the only Fur homologue in D. radiodurans, cannot bind to the promoter of mntH, but it can bind to the promoter of another manganese acquisition transporter, MntABC. β-galactosidase expression analysis indicated that DR2539 acts as a manganese- and iron-dependent transcriptional repressor. Further sequence alignment analysis revealed that DR2539 has evolved some special characteristics. Site-directed mutagenesis suggested that His98 plays an important role in the activities of DR2539, and further protein-DNA binding activity assays showed that the activity of H98Y mutants decreased dramatically relative to wild type DR2539. Our study suggests that D. radiodurans has evolved a very efficient manganese regulation mechanism that involves its high intracellular Mn/Fe ratio and permits resistance to extreme conditions.