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ABSTRACT: The extremely radioresistant bacterium Deinococcus radiodurans encodes a number of function-unknown genes, and some of them involve in the radioresistance. The radiation-inducible gene dr0171 has a recA-like expression pattern in the postirradiation recovery and was also supposed to encode a transcriptional regulator to contribute to the radioresistance. Here, we found that the EGFP-tagged DR0171 proteins gathered in the nucleoid regions after radiation. Further, we constructed a null mutant of dr0171 and found that the incapacitation of the dr0171 led to a significant decline in resistance to γ-rays, UV radiation, and hydrogen peroxide and delayed genomic DNA reconstruction after radiation, indicating that this gene is involved in the postirradiation recovery. The microarray assays showed that the disruption of dr0171 does not lead to a significant change in the transcriptional profile of D. radiodurans under normal conditions, except after the stress of 6 kGy γ radiation was applied. The transcript level of at least 153 genes decreased over twofold in the irradiated dr0171 mutant compared with those in the irradiated wild-type strain, indicating that DR0171 only functions after radiation damage. Taken together, the data presented here indicate that DR0171 serves as a regulator of the transcriptional response to radiation damage in D. radiodurans.
Archives of Microbiology 05/2011; 193(10):741-50. · 1.43 Impact Factor
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ABSTRACT: 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, OxyR(2) (DRA0336). In this study, we constructed the deletion mutant of oxyR(2) 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 oxyR(2) resulted in an obviously increased sensitivity to hydrogen peroxide, and the double mutant for oxyR and oxyR(2) 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 oxyR(2) was involved in antioxidation mechanisms. Site-direct mutagenesis and complementation analysis revealed that C(228) in OxyR(2) was essential. This is the first report of the presence of two OxyR in one organism. These results suggest that D. radiodurans OxyR and OxyR(2) function together to protect the cell against oxidative stress.
The Journal of Microbiology 08/2010; 48(4):473-9. · 1.10 Impact Factor
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ABSTRACT: The bacterium Deinococcus radiodurans is extremely resistant to the intense ionizing irradiation which causes extensive DNA double-strand breaks (DSBs). The deinococcal SbcCD complex (drSbcCD) is required for DSB repair. The drSbcC and drSbcD genes were cloned and overexpressed in Escherichia coli cells, respectively. The nearly homogeneous drSbcC and drSbcD proteins were purified and reconstituted to form a stable complex in vitro. The drSbcCD complex has an ATP-independent 3'-->5' exonuclease activity to cleave both dsDNA and ssDNA substrates in the presence of either Mn(2+) or Mg(2+) ion. The drSbcCD complex also has an ATP-independent endonuclease activity. It can cleave the circular ssDNA, nick the supercoiled circular dsDNA, cleave the 3' flap DNA substrate at the site of the single-strand branch adjacent to duplex DNA, and cleave the hairpin DNA taking no account of the DNA end free or not. It is a kind of secondary structure-specific endonuclease. The drSbcCD complex still has a 3'-->5' exonuclease activity when the DNA termini are blocked by the proteins. These results suggest that the drSbcCD complex takes part in the metabolism of DNA, and its nuclease activities may play important roles in DNA repair process.
Journal of biochemistry 11/2009; 147(3):307-15. · 1.95 Impact Factor
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ABSTRACT: Deinococcus radiodurans (Dr) withstands desiccation, reactive oxygen species, and doses of radiation that would be lethal to most organisms. Deletion of a gene encoding a homolog of mammalian nitric oxide synthase (NOS) severely compromises the recovery of Dr from ultraviolet (UV) radiation damage. The Deltanos defect can be complemented with recombinant NOS, rescued by exogenous nitric oxide (NO) and mimicked in the wild-type strain with an NO scavenging compound. UV radiation induces both upregulation of the nos gene and cellular NO production on similar time scales. Growth recovery does not depend on NO being present during UV irradiation, but rather can be manifested by NO addition hours after exposure. Surprisingly, nos deletion does not increase sensitivity to oxidative damage, and hydrogen peroxide does not induce nos expression. However, NOS-derived NO upregulates transcription of obgE, a gene involved in bacterial growth proliferation and stress response. Overexpression of the ObgE GTPase in the Deltanos background substantially alleviates the growth defect after radiation damage. Thus, NO acts as a signal for the transcriptional regulation of growth in D. radiodurans.
Proceedings of the National Academy of Sciences 10/2009; 106(43):18183-8. · 9.68 Impact Factor
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ABSTRACT: 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 gamma 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 gamma 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.
Molecular & Cellular Proteomics 11/2008; 8(3):481-94. · 7.40 Impact Factor
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ABSTRACT: The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of Pprl's crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. radiodurans R1 strain, pprf function-deficient mutant (YR1), and pprl function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence microscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells exhibit the tightly packed ring-like morphology, while the pprl function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprl completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprl partly function-complementary strain YR1002's nucleiods exhibit about 60% ring-like structure; (3) a Pprl C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensitive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.
Science in China Series C Life Sciences 09/2007; 50(4):525-9. · 1.61 Impact Factor
