Study On Response Regulators In Deinococcus Radiodurans

Deinococcus radiodurans R1 is one of the most radioresistant bacteria in the world.This particular species could stand a wide variety of DNA-damage agents such as gamma-radiation,UV,H₂O₂ and desiccation.For many years,it has been an ideal model organism to investigate the integrity and stability of the genome.However,the molecular mechanisms underlying this phenotype remain poorly understood.Microorganisms have developed diverse mechanisms for sensing and acclimating to changes in vitro or in vivo.Prokaryotes usually response to specific environmental stimuli through two-component systems(TCS).A typical TCS consists of a histidine kinase(HK)and a partner response regulator(RR).The HK containing an invariant histidine residue is autophosphorylated in response to external or internal signals.Then,the phosphoryl group is transferred to the aspartate residue on the RR. The RR is generally composed of a conserved receiver domain and a variable effector domain.This particular modular architecture of RRs enables them to function as versatile components that couple a series of cellular behaviors.As the toughest bacteria in the earth,Deinococcus radiodurans must be armed with strong signaling pathways for sensing and response to the ambient condition,the exploration of which might be helpful to reveal the basis of the extreme radioresistance of this bacterium.It has been shown in our previous work that one of the predicted response regulator, DR2418,is likely contributive to the extreme radioresistance of D.radiodurans.So we have laid emphasis on this interesting component.The work is detailed below.1.To compile a whole list of D.radiodurans response regulators(RRs),an extensive search using the current databases such as GenBank was conducted.More than twenty response regulators were hit,eight of which possess DNA-binding domains.Among them,DR0781 and DR0743 reflect the highest similarity with the predicted response regulator DR2418.2.The RRs above and the other genes located in the same operon as DR2418 were deleted or disrupted instead of a drug cassette for detection.It was shown that DR0743 is required for the metabolism,whereas DR0781 is not vital for the radioresistance of D.radiodurans.However,the viability of DR2418-deficient strain is greatly reduced after ionizing radiation.Besides,neither DR2419 nor DR2420 that shares the same operon with DR2418 is essential for the extreme radioresistance of D. radiodurans.In addition,the double mutantâ–³DR2418â–³PprI is more radiosensitive than either of the single mutant.3.The mutantâ–³DR2418 was then characterized.The growth rate and natural mutation frequency of the mutant is accordant to the wild-type R1.However,the mutant strain displays extreme sensitivity to various DNA-damage agents such as gamma-radiation,hydrogen peroxide and desiccation.Therefore,the anti-oxidation activity was tested by chemiluminescence,and it turned out that the ROS scavenging activity is reduced under either non-stress or irradiation condition in the mutant strain compared to the wild-type.Further investigation revealed that the activities of two essential enzymes involved in antioxidation,catalases(KAT)and superoxide dismutases(SOD),are weakened either under radiation stress or not in the mutant by PAGE gel activity-staining assays and total activity assays.Furthermore,the different levels of two vital DNA repair-related enzymes,RecA and PprA,were detected by Western Blotting assays.Finally,the resistance of the double mutantâ–³DR2418â–³PprI to various DNA-damage agents was also determined,and it seemed that these two genes are functionally additive.4.DR2418 is predicted as a RR with DNA-binding activity to regulate the transcription of a serial of downstream genes upon internal or external stimuli.To gain insights into the downstream pathways regulated by DR2418,the transcriptional profile of the disruptant strain under no-stress or gamma-radiation condition was analyzed and compared with that of wild-type strain at exponential growth phase.An array of genes display changes in their expression levels at least 2-fold statistically. Interestingly,the genes down-regulated in the mutant under non-stress condition overlap with nearly one third of those genes which are up-regulated in the wild-type strain after 2 kGy radiation treatment.Changes in transcript abundance were verified by quantitative real-time PCR(Q-RT-PCR).From characterization of the mutant and microarray data above,we were aware that disruption of DR2418 might affect DNA repair process in D.radiodurans.To testify this,we performed the pulsed-field gel electrophoresis(PFGE)assay.The result demonstrates that deletion of DR2418 retard the process of DNA repair in D.radiodurans.5.To test whether DR2418 is a response regulator,we mutated the Asp groups suspected to be involved in the signal transduction and complemented the site-mutated DNA fragments to the DR2418 mutant strain.Just as the DR2418 null mutant strain,the mutation DR2418^D54Nresults in significant decrease of its radioresistance.Because Asp⁵⁴within the receiver domain is highly conserved and is the predicted phosphorylation site in nearly all response regulators,we propose that DR2418 acts as a response regulator.To see whether DR2418 possesses DNA-binding activity,the recombinant DR2418 was purified and mixed with FITC-labeled promoter DNA fragments.A band of retarded mobility appeared when it was incubated with DR0997 promoter,indicating that this protein is capable of binding to the specific promoter directly.The relative expression of DR0997 was basically reduced in the mutant compared with the wild-type strain under either normal condition or gamma-radiation stress.6.Tandem affinity purification(TAP)is a generic method to obtain pure natural protein complex under native condition.It is predicted that one third of genes in the genome are unknown or unique to D.radiodurans.Constructing an effective network for protein interaction might be a good choice.Therefore,the plasmid pBS1479 was reconstructed to pBS1479::Kana by changing the selective marker and adding the mutiple cloning sites.Thus,the TAP tag could be directly integrated into the C-terminal of RecA or DR2418 forming the fusion RecA-CTAP or DR2418-CTAP in vivo.The former has been verified by Western blot.On the other hand,the N-terminal and C- terminal TAP tags with mutiple cloning sites were fused to the shuttle vector pRADZ3 to form pRADN and pRADC,respectively.Thus,genes are convenient to be insertd into the plasmids to transform the corresponding mutants. The radioresistance of the complemented strain carrying pRADN-PprI has been detected.These work laid a foundation for unveiling the relationship among RecA, PprI and DR2418. Cumulatively,DR2418 is essential for the extreme radioresistance of D. radiodurans.It is a response regulator with specific DNA-binding activity,and functions mainly through antioxidant and DNA repair pathways.It seems that DR2418 and PprI are partially additive.The relationship between essential proteins is under investigation.