A PerR-like protein involved in response to oxidative stress in the extreme bacterium Deinococcus radiodurans

Response and defense systems against reactive oxygen species (ROS) contribute to the remarkable resistance of Deinococcus radiodurans to oxidative stress induced by oxidants or radiation. However, mechanisms involved in ROS response and defense systems of D. radiodurans are not well understood. Fur family proteins are important in ROS response. Only a single Fur homolog is predicted by sequence similarity in the current D. radiodurans genome database. Our bioinformatics analysis demonstrated an additional guanine nucleotide in the genome of D. radiodurans that is not in the database, leading to the discovery of another Fur homolog DrPerR. Gene disruption mutant of DrPerR showed enhanced resistance to hydrogen peroxide (H₂O₂) and increased catalase activity in cell extracts. Real-time PCR results indicated that DrPerR functions as a repressor of the catalase gene katE. Meanwhile, derepression of dps (DNA-binding proteins from starved cells) gene under H₂O₂ stress by DrPerR point to its regulatory role in metal ions hemostasis. Thus, DrPerR might function as a Fur homolog protein which is involved in ROS response and defense. These results help clarify the complicated regulatory network that responds to ROS stress in D. radiodurans.     

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.     

Vicia root-mirconucleus and sister chromatid exchange assays on the genotoxicity of selenium compounds

Selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Excessive selenium in soil and water may contribute to environmental selenium pollution, and affect plant growth and human health. By using Vicia faba micronucleus (MN) and sister chromatid exchange (SCE) tests, possible genotoxicity of sodium selenite and sodium biselenite was evaluated in this study. The results showed that sodium selenite, at concentrations from 0.01 to 10.0mg/L, induced a 1.9-3.9-fold increase in MN frequency and a 1.5-1.6-fold increase in SCE frequency, with a statistically significantly difference from the control (P<0.05 and 0.01, respectively). Sodium selenite also caused mitotic delay and a 15-80% decrease in mitotic indices (MI), but at the lowest concentration (0.005mg/L), it slightly stimulated mitotic activity. Similarly, the frequencies of MN and SCE also increased significantly in sodium biselenite treated samples, with MI decline only at relatively higher effective concentrations. Results of the present study suggest that selenite is genotoxic to V. faba root cells and may be a genotoxic risk to human health.     

Ligand-induced effects on pyruvate dehydrogenase kinase isoform 2

Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: approximately 53%/3 microM for ATP; approximately 49%/15 microM for ADP; and approximately 71%/approximately 590 microM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and > or = 80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 microM pyruvate, > or = 150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2(red)) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2(red) was modestly hindered by 200 microM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 microM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2(red) may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.     

钩虾胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）的敏感性和特异性比较研究

研究了有机磷农药甲地基嘧啶流磷,有机氯农药林丹,菊酯类农药氯菊酯,表面活性剂直链苯磺酸钠和重金属Zn对钩虾（Gammarus pulex L.)胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）活性变化以及毒性影响,结果表明,在暴露24h和48h后,仅有机磷农药甲基嘧硫磷显著抑制胆碱酯的酶的活性,在暴露48h后,有机氯农药林丹和菊酯类农药氯菊酯能显著提高谷胱甘肽转硫酶活性,在暴露24h后,仅梵在导致谷胱甘肽转硫酶明显升高,作为生物标志物,胆碱酯酶比谷胱甘肽转硫酶具有更高的特异性,这两种生物标志物较毒性试验方法具有更高的敏感性。     

The key residue for SSB-RecO interaction is dispensable for Deinococcus radiodurans DNA repair in vivo

The RecFOR DNA repair pathway is one of the major RecA-dependent recombinatorial repair pathways in bacteria and plays an important role in double-strand breaks repair. RecO, one of the major recombination mediator proteins in the RecFOR pathway, has been shown to assist RecA loading onto single-stranded binding protein （SSB） coated single-stranded DNA （ssDNA）. However, it has not been characterized whether the protein-protein interaction between RecO and SSB contributes to that process in vivo. Here, we identified the residue arginine-121 of Deinococcus radiodurans RecO （drRecO-R121） as the key residue for RecO-SSB interaction. The substitution of drRecO-R121 with alanine greatly abolished the binding of RecO to SSB but not the binding to RecR. Meanwhile, SSB-coated ssDNA annealing activity was also compromised by the mutation of the residue of drRecO. However, the drRecO-R121A strain showed only modest sensitivity to DNA damaging agents. Taking these data together, arginine-121 of drRecO is the key residue for SSB-RecO interaction, which may not play a vital role in the SSB displacement and RecA loading process of RecFOR DNA repair pathway in vivo.     

细菌非编码小RNA的筛选及鉴定方法

细菌非编码小RNA（smallnon．codingRNAs,sRNAs）是一类长度为50～500nt、不编码蛋白质的功能RNA,在应对胁迫、毒力产生和新陈代谢等生命过程中起重要的调控作用。其主要通过碱基配对与靶mRNA发生作用,导致mRNA翻译和稳定性改变,从而在转录后水平调节基因的表达,最终影响细菌各种生命活动。近年来,利用生物信息学和分子生物学技术,已在细菌中筛选并鉴定得到了几百个sRNA。该文对细菌sRNA的筛选和鉴定方法作一简要综述。     

耐辐射奇球菌铁结合蛋白DRA0258的抗氧化功能

【目的】通过对极端环境耐受的耐辐射奇球菌Deinococcus radiodurans R1全基因组进行序列比对分析,获得具有铁储备蛋白Ferritin类似功能基序的未知功能蛋白DRA0258,采用分子生物学技术对该蛋白的功能和性质进行了验证和分析。【方法】首先对DRA0258进行克隆表达和纯化,并经络合物显色法测定蛋白上铁结合含量;通过三段连接敲除法构建dra0258突变株,检测突变株在双氧水协迫下的生存率、总抗氧化活性及过氧化氢酶活性;利用实时定量PCR检测突变株内抗氧化酶类及铁转运相关性调控蛋白的基因转录水平。【结果】经体内外蛋白铁含量检测证实DRA0258具有一定的铁结合能力;双氧水生存率实验表明dra0258的缺失导致细胞的抗氧化能力显著下降;过氧化氢酶活性、总抗氧化活性检测及抗氧化酶类的基因转录水平检测证实dra0258基因的缺失导致细胞内一些抗氧化基因转录水平下调,细胞的抗氧化应激系统受到损伤,并影响了一些铁调控网络蛋白的基因转录水平。【结论】本研究证实DRA0258是一种铁结合蛋白,该编码基因的缺失影响胞内铁转运系统并使细胞抗氧化能力下调。