D.radiodurans紫外辐射损伤修复机制及环境应用研究
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摘要
一、研究背景
耐辐射奇球菌(D.radiodurans)于1956年分离自γ射线辐照后的肉罐头,其对高强度离子辐射的抵抗力备受学者关注。近年来,围绕该菌耐辐射机制的研究成为人类探索生物体极端辐射耐受机制的突破口。
D.radiodurans属于非致病性革兰氏阳性微球菌,与芽胞菌依赖其芽孢结构抵抗外界因素干扰不同,D.radiodurans并不具有芽孢结构,而且其胞壁结构和组成与革兰氏阴性菌较为相近,具有外膜,而不具有革兰氏阳性菌特有的磷壁酸。扫描电子显微镜结果发现,在D.radiodurans菌稳定生长阶段,每个D.radiodurans菌均由两条垂直线分隔成四分裂体结构,4个室里面含有相等数量的DNA,每个室内含有一条完整的环状染色体,在辐射损伤前期(1h以内),所有基因组均被不同程度打断,而四个相对独立的空间保证了在一定范围内单个基因组的独立性和完整性,在辐照1小时后,环状的染色体转换为S形结构,并通过膜孔将两个室的DNA进行渐进式扩散。因此,其成熟体的四联体结构形态保证了即使在多组基因组受到损伤后,其损伤的DNA链始终能够在其他基因组中找到合适的退火延伸模板片段。目前研究结果表明,其独特的高剂量辐射抗性能力更多地来自于本身DNA修复和蛋白调控机制。当前较为明确的DNA损伤修复机制主要有以下几种:
(一)同源重组(homologous recombination, HR):其主要原理是利用同源的序列来修复自身的双链断裂,是dsDNA(double-stransded DNA)损伤修复的主要方式。此种方式在D. radiodurans离子辐射后发生较为普遍,由于RecBCD途径在D.radiodurans中的缺失,RecFOR在同源重组途径中具有重要作用。
(二)非同源末端连接(non-homologous end-joining, NHEJ):NHEJ修复机制与HR最大的差异在于完全不需要任何模板的帮助,致密的类核体结构保证了辐射致DNA损伤时断裂端彼此邻近,在PprA的作用下,抑制核酸外切酶III的活性,促进由DNA连接酶催化的DNA末端连接反应。因此,在辐射损伤早期阶段,断裂基因组片段可能以非同源末端连接修复方式开始早期修复;
(三)染色体内和染色体间单链退火(intra- and interchromosomal single-strandannealing):是指在DNA双链断裂处开始,在单链特异性核酸外切酶的作用下,断裂点两侧逐渐形成DNA单链区域,直至两个断点处出现互补的序列,完成单链缺口修复连接;
(四)合成依赖单链退火(Synthesis dependent strand annealing, SDSA):SDSA是一种不依赖RecA的无错双链断裂修复模式,由产生DNA双链断裂的其中一条链的3’末端入侵未损伤的姊妹双链同源区开始DNA合成,并扩展姊妹双链形成的D- loop环作为模板或其他3’末端DNA合成的引物;
(五)延伸合成依赖式退火(Extended synthesis-dependent strand annealing, ESDSA):如果未发现末端游离的互补断裂片段,则利用RecA蛋白丝识别同源片段,利用同源片段的序列进行延伸,直至发现互补断裂末端,进行单链退火修复。
既往研究表明,在大肠杆菌中,rec基因决定了其重组修复系统的主要途径,对D.radiodurans菌的rec基因研究主要集中于recA等基因,Kim J等研究显示,D. radiodurans菌的RecA比E.coli的更容易结合DSBs(double-strand breaks)而促进快速高效的DNA修复。对E.coli重组修复途径研究发现,其修复途径启动阶段包括一条涉及recB、recC、recD基因的RecBCD途径和一条包括recF、recO、recR基因的RecF途径。在大部分野生株中,RecBCD(或者其功能相似基因AddAB)启动DSBs的修复。然而,在耐辐射奇球菌基因组中,没有找到RecB和RecC的同源物,如果对照大肠杆菌中同源重组的几种途径,按照经典遗传学理论,在RecBCD途径不能运作的情况下,基因组仍然具有重组活性,RecFOR途径必然发挥非常重要的作用。
本研究以D. radiodurans为研究对象,从四个方面开展耐辐射奇球菌紫外辐射抗性和应用前景研究:1.以RecFOR途径的recF基因为切入点,通过recF基因突变株的构建,以野生株为参照,研究recF突变株与野生株的生长周期和辐射抗性差异,探讨recF在D.radiodurans紫外辐射抗性中的作用;2.通过分子生物学手段克隆表达并纯化RecFOR途径的关键蛋白,为功能蛋白的体外研究打下基础。3.通过选择性紫外辐射压力驯化D.radiodurans,经过25代的驯化,得到紫外辐射抗性得到明显增强的D.radiodurans,利用二维蛋白电泳技术,通过对800 J/m2辐照后eD.radiodurans和D.radiodurans蛋白差异点的鉴定和分析,探讨紫外辐射抗性的可能机制和途径;4.鉴于D.radiodurans超强的环境耐受能力,利用基因工程改良的生物有机体对抗生素污染环境进行生物修复是一种具有良好应用前景的方法。本部分实验旨在通过对重庆市水体环境中抗生素水平检测、D.radiodurans耐药谱检测、穿梭载体的转化效率等方面的研究,为D.radiodurans基因工程菌的构建提供必要的实验支持,为D.radiodurans应用于抗生素污染环境有毒物质的降解修复打下基础。
二、材料和方法
(一)利用同源重组双交换方法,将插入groEL promoter+卡那霉素抗性基因的同源臂载体通过电转法转入D.radiodurans,替换recF基因,通过卡那霉素抗生素TGY平板筛选得到阳性克隆子,利用多对引物对突变株同源臂进行PCR扩增,扩增产物进行测序验证。绘制recF突变株与标准株生长曲线和紫外辐射抗性生存曲线,从而评价recF在D.radiodurans紫外辐射抗性中的作用。
(二)以pET32a(+)为载体,设计带酶切位点的引物对D. radiodurans RecFOR途径关键基因recF、recO、recR、SSB、reA进行PCR扩增,将PCR产物连接至pGEM-T,限制性内切酶双酶切载体和连接至pGEM-T的PCR产物,T4连接酶连接后转化至E. coli BL21。通过IPTG浓度、培养温度、培养时间的摸索,探讨各蛋白诱导表达的最佳条件,镍柱纯化蛋白产物用Western-blot进行鉴定。
(三)通过800 J/m2紫外辐射压力驯化25代,筛选到D.radiodurans紫外辐射能力得到明显增强的进化株,绘制紫外辐照生长曲线,比较进化株和野生株紫外生存能力。运用2-DE技术对进化株和野生株辐照后蛋白表达进行差异比较,筛选差异蛋白,质谱分析差异蛋白并对分析结果在NCBI上进行序列相似性比对,文献查阅差异蛋白的功能,针对有意义的差异蛋白点设计引物,利用RT-PCR技术对功能蛋白蛋白转录水平进行半定量分析,探讨紫外辐射抗性增强的可能机制。
(四)利用在线固相萃取法和液相色谱电喷雾质谱法对重庆市医院排放污水、养殖场和屠宰场、地表水、污水处理厂进出口水中乙酰红霉素、水合乙酰红霉素、罗红霉素、泰乐菌、林可霉素、甲氧苄啶、环丙沙星、洛美沙星、氟哌酸、氧氟沙星、磺胺嘧啶、磺胺二甲氧嘧啶、磺胺甲嘧啶、磺胺甲基异恶唑、iso-氯四环素、epi-iso-氯四环素、氯四环素、四环素等水平进行检测,参照CLSI2008标准对D.radiodurans耐药谱进行检测,采用Kirby-Bauer法进行阿米卡星(AK, 30μg)、氨曲南(ATM, 30μg)、哌拉西林/他唑巴坦(TZP, 100/10μg)、头孢哌酮/舒巴坦(75/10μg)、头孢噻肟(CTX, 30μg)、替考拉宁(TEC, 30μg)、头孢曲松(CRO, 30μg)、阿奇霉素(AZM, 30μg)、阿莫西林/克拉维酸(20/10μg)、万古霉素(VA, 30μg)药敏试验。采用VITEK-Ⅱ药敏分析系统进行抑菌直径和最低抑菌浓度(Minimal Inhibitory Concentration, MIC)检测,选用以下抗生素:环丙沙星(CIP)、褐霉素(FD)、四环素(TET)、庆大霉素(CN)、红霉素(E)、亚胺培能(IPM)、磷霉素(FOS)、利奈唑烷(ZYVOX)、莫西沙星(MF)、利福平(RD)、替考拉宁(TEC)、复方新诺明(SXT)、氨苄西林/舒巴坦(SAM),将抑菌圈大小换算成MIC。将检测结果与水体抗生素水平进行比较,探讨D.radiodurans的环境应用前景。通过化学转化法和电转化法对pRADZ3载体转化D. radiodurans的能力进行研究,探讨载体转化的最适条件。
三、结果
(一)pUC18质粒作为同源交换臂的载体,能够递呈目的片段至D. radiodurans,并发生同源交换,通过电转法,用卡那霉素抗性平板成功筛选到阳性克隆,并通过PCR对recF突变株进行了确认;recF突变株生长曲线表现为对数生长期的延迟,且生长高峰菌落数低于野生株,recF可能对D. radiodurans的生长繁殖具有影响;recF突变株的紫外抗性能力降低显著,其F10值低于野生株将近3倍,recF转化至E. coli能够增强其紫外辐射抗性2.5倍左右。
(二)以D. radiodurans为模板,扩增得到RecFOR途径关键基因recF、recO、recR、ssb、recA,双酶切后连接至PET32a(+)载体,CaCl2化学转化法转化至E. coli BL21,IPTG成功诱导5个蛋白的表达,并通过Western-blot进行了验证。其中,SSB和RecR以可溶性蛋白形式表达,RecF、RecO、RecA以包涵体形式表达;RecF、RecO、RecR、SSB、RecA纯度均>90%,浓度分别为0.5 mg/ml、0.5 mg/ml、0.6 mg/ml、0.5 mg/ml、0.6mg/ml,蛋白的浓度和纯度均能满足体外实验要求。
(三)紫外驯化法能够筛选到紫外辐射抗性明显增强的进化株,二维蛋白差异蛋白分析能够对辐照后野生株和进化株的差异蛋白进行筛选,其中,TerD、Maf、LEA76干燥抗性蛋白以及黄色相关蛋白尤为值得引起关注;针对有意义的功能差异蛋白点,利用RT-PCR技术在蛋白转录水平上对其表达的上调进行了确认,Maf转录因子与压力信号感应,转录调控以及细胞增殖相关;黄色相关蛋白在辐射后的再生过程或者在类胡萝卜素表达调控方面具有重要作用;LEA76干燥相关蛋白不仅与D.radiodurans干燥抗性机制相关,可能在紫外辐射抗性机制中也具有重要作用,辐射抗性机制和干燥抗性机制可能具有相同的修复途径;TerD与氧化应激相关,对包括重金属阳离子、甲磺酸甲酯、丝裂霉素C、紫外线等在内的环境因素对生物体造成的影响具有保护作用。
(四)在所采水样中,污水处理厂抗生素含量和种类最高,其次为医院污水和养殖屠宰场。D. radiodurans对25种抗生素的耐受水平检测结果表明,D. radiodurans对庆大霉素、亚胺培能、莫西沙星、替考拉宁、复方新诺明、氨苄西林/舒巴坦、阿米卡星、头孢吡肟、哌拉西林/他唑巴坦、万古霉素、头孢曲松、利奈唑烷、阿莫西林/克拉维酸等敏感。D. radiodurans对抗生素的耐受能力远远高于水环境中的抗生素含量水平。D. radiodurans在电压2.2 kV,电容25μF,电阻200欧姆,2μg/ml pRADZ3载体浓度时,电转转化率最高,适宜于应用与基因工程菌的构建。
四、结论
(一)本实验通过对recF突变株的构建成功,摸索出一套行之有效的突变株构建方法,为后续关键基因蛋白的功能研究打下基础;
(二)recF的缺失能够导致D.radiodurans生长复制减缓,紫外辐射抗性的降低,其具体机制有待进一步的研究;
(三)RecFOR途径的关键蛋白构建至pET32a(+)后能够在IPTG的诱导下表达,关键蛋白的成功纯化为体外实验的开展奠定了基础,同时,蛋白的纯化表达为相应抗体的制备打下基础,为将来western-blot应用于菌体RecFOR途径蛋白的表达调控检测提供了条件;
(四)通过对进化株和野生株紫外辐射后蛋白组学比较,14个差异蛋白功能涉及转录、光合作用、核酸/肽结合、蛋白折叠、氧化和干燥应激相关、离子转运、碳水化合物代谢及糖类代谢等;
(五)水环境中的抗生素浓度远远低于D. radiodurans对抗生素的耐受浓度,基因工程构建D. radiodurans可应用重庆市各种水环境中,pRADZ3的结构特点决定其可以电转化法将携带的功能基因转入D. radiodurans中。
五、创新点
(一)通过突变株的构建和recF在E. coli中的表达,首次报道了recF在紫外辐射防护方面具有重要作用,recF的缺失能够造成D. radiodurans生长复制的减缓。
(二)Maf、LEA76干燥抗性蛋白以及黄色相关蛋白表达调控参与了D. radiodurans的紫外辐射抗性机制,既往研究未见报道。
(三)首次报道了重庆三峡库区水体环境中的抗生素水平以及D. radiodurans的MIC水平,为D. radiodurans的环境应用研究打下基础。
六、缺点和不足
(一)recF的研究虽然从突变和导入E. coli设计实验进行了探讨,但是如果能够运用穿梭载体将D. radiodurans和E. coli的recF基因导入recF突变株,观察D. radiodurans的紫外抗性恢复情况,将对整个实验结论是个良好的补充;
(二)RecFOR途径蛋白克隆表达纯化后,有三个蛋白为包涵体的形式表达,要获得活性蛋白进行后续的功能研究,必须对这三个蛋白进行复性操作,这样会对蛋白的浓度造成损失,后续可通过降低诱导温度、降低IPTG浓度、延长诱导时间等方式来调整蛋白的表达,如果还是以包涵体的形式则通过多个载体和宿主细菌的选择来改善蛋白诱导表达条件,尽量以可溶性表达功能蛋白;
(三)由于检测条件的限制,水环境中检测出的抗生素种类与耐药谱选择抗生素种类并不完全匹配。有较多水环境中检出抗生素在D. radiodurns最低抑菌浓度(MIC)分析时药敏分析仪器中并未包含,药敏纸片法的纸片也未有此类抗生素纸片。(四)本研究在D. radiodurans的应用研究仅为初步探讨,未将具有降解功能的基因通过穿梭载体导入D. radiodurans进行基因工程菌的构建和功能验证研究。
七、下一步研究计划
(一)鉴于RecFOR途径在辐照抗性的重要作用,有必要开展实验对其具体作用机制进行深入研究。由于各种环境压力(包括离子辐射、紫外辐射、氧化损伤等)对D. radiodurans作用靶点各有不同,目前对该途径作用于单链断裂、双链断裂均有报道,更多的报道倾向于修复单链断裂。那么,对单或双链修复的偏好程度研究将解决其修复效能作用靶点的疑问。同时,该途径的修复作用起始位点是5’端还是3’端,其具体作用起始位点将决定后期对其具体作用机制假说的推导和验证。
(二)干燥抗性基因(如DR1172)在辐射抗性中是否具有重要作用?通过本研究所建立的突变株构建方法和穿梭载体的应用,可以从正反两方面进行该研究的验证,通过此研究,可推测D. radiodurans超强辐射抗性可能的起源,为起源假说提供参考。如果干燥基因同时具有辐射抗性的作用,将为基因工程菌株的构建提供良好的功能基因选择。
(三)能量代谢在生物体的复制、生长、凋亡中具有重要的作用,Kamble VA的文章(2010)表明,当D. radiodurans受到紫外辐射后,cAMP、ATP和蛋白磷酸化水平升高。在本研究中,通过二维蛋白研究,发现了多种能量代谢相关酶水平的升高,从蛋白水平上为能量代谢在辐射后修复中具有重要作用提供了佐证,下一步需要进一步研究的是:1)能量代谢和功能蛋白磷酸化的关系;2)高剂量辐照无论对DNA还是蛋白,均能导致其断裂和失活,而DNA的断裂损伤修复一定是需要通过活性蛋白来介导的,这些活性蛋白的保护或迅速恢复是否与能量代谢变化相关?如果研究表明,能量代谢与蛋白磷酸化水平或保护相关,将对于临床放疗病人的辐射防护和高剂量离子或紫外暴露人群保护提供重要的研究价值,同时为军民两用辐射防护药物的研发提供研究思路。
The extremely radioresistant bacterium, Deinococcus radiodurans (D. radiodurans), is characterized by its unusual capability to tolerate numerous DNA damaging agents, including ionizing radiation, ultraviolet radiation, and desiccation. D. radiodurans is capable of surviving 15,000 grays (Gy) of ionizing radiation, whereas doses below 10 Gy are lethal to all other organisms. Studies on mechanisms of radioresistance of D. radiodurans were thought to be the point to breakthrough the ability of organisms to surviving in extreme environment in recent years.
D. radiodurans belongs to non-pathogenic gram-positive micrococcus. The ability of radioresistant of Bacillus spp. attribute to the characteristics of spore structure. Considering the critical role of spore in Bacillus spp., the mechanisms of DNA repair and modulation of proteins ought to be further investigated.
Transmission electron microscopy studies showed that a membranous framework demarcates the four compartments of a single D. radiodurans cell, but orifices were evident in the internal membranes of both growing and stationary-state cells. Chromatin was present in all compartments, and it adopted a distinctive toroidal shape. DNA labeling revealed that, whereas DNA toroids are present in all compartments of stationary-state bacteria, the nucleoid in one or two compartments of growing cells is dispersed. The mechanisms of radioresistance included several pathway as follows:
1.Homologous recombination at the fragment ends: In this way, the strain utilize recombination sequences as template to manipulate the gap DNA. dsDNA damage was mainly repaired by this way. In D. radiodurans, the RecFOR pathway plays a critical role in DNA damage repair due to a deficiency of RecBCD.
2.Non-homologous end-joining (NHEJ): One major mechanism of DSB repair in eukaryotes is non-homologous end-joining (NHEJ), in which the two ends of a broken chromosome are rejoined directlyusing only the base-pairing information of the ends themselves to guide ligation. In the early phase after irradiation, the broken fragments maybe repaired by NHEJ.
3.Intra- and interchromosomal single-strand annealing: The single strand DNA could be formed by the specific exonucleace at the dsDNA broken site until the occurrence of complement strand DNA. And then the single strand gap was repaired.
4.Synthesis dependent strand annealing (SDSA): Recent research has suggested a compelling model for genome restitution in this species, in which the related but more efficient process called synthesis-dependent singlestrand annealing (SDSA) plays a necessary part. During SDSA, the 3’end of a strand derived from a DNA double-strand break invades the homologous region of a sister duplex. The invading 3′end is used to prime DNA synthesis, unwinding the sister duplex and enlarging the D-loop. The displaced strand in the undamaged complex anneals to the remaining free 3’end created by the double-strand break.
5.Extended synthesis-dependent strand annealing (ESDSA): How does ESDSA differ from SDSA? Crucially, it requires at least two genome copies that are broken at different positions. Once overlapping fragments have aligned, a single-round multiplex PCR-like step- a variant of PCR that simultaneously amplifies different target sequences by using multiple primer pairs occurs to produce long single-stranded overhangs that anneal accurately to produce reassembled chromosomal segments. Last, RecA-mediated homologous recombination using long intermediates synthesized by DNA-polymerase-I produces full-length chromosomes.
In order to investigate the mechanisms of radioresistance of D. radiodurans, our studies were divided into four parts:
1.The function of recF in UV-C resistance was characterized by the construction of the recF mutant D. radiodurans R1 strain and the transformation of D. radiodurans recF into E. coli BL21.
2.The critical genes of RecFOR pathway were cloned and expressed in E. coli BL21. The purified proteins were further confirmed by Western-blot.
3.To investigate the mechanisms of UVC radiation resistance in D. radiodurans, we produced an enhanced UVC-resistant strain (eD. radiodurans) by selective pressure of direct UVC radiation. We then examined proteome changes between the wild-type strain and eD. radiodurans following UVC irradiation using 2-DE and silver-staining. Genes encoding proteins that may play important roles in UVC radiation resistance were further investigated by RT-PCR.
4.To investigate the application of gene engineering in D. radiodurans, the levels of antibiotics in Three Gorge Reservoir of Chongqing region were tested. The MIC values were determined by Kirby-Bauer disc diffusion method and VITEK-II. In addition and the frequency of transformation were analyzed by chemical transformation and electro transformation with pET32a(+).
Materials and methods
1.recF mutant construction
The kanamycin resistance gene was obtained from the pET30b(+) plasmid using primers K1 and K2 (containing BamHI and PstI restriction sites) and ligated to groEL promoter which was obtained from the D. radiodurans genome using primers G1 and G2 (containing KpnI and BamHI restriction sites). A 994-bp DNA fragment corresponding to the immediate upstream portion of the dr1089 (recF D. radiodurans) initial codon was amplified using primers MF1 and MF2 (containing EcoRI and KpnI restriction sites), and a 1063-bp DNA fragment corresponding to the immediate downstream region of the dr1089(recF D. radiodurans) stop codon was amplified using primers MF3 and MF4 (containing PstI and HindIII restriction sites). The PCR products were digested with their respective enzymes listed above and then ligated to a groEL promoter-kanamycin cassette. The tripartite ligation products were amplified using L1 and R2, and the resulting fragments were ligated to pUC18 that had been digested with EcoRI and HindIII. The fragments were then transformed into D. radiodurans R1 as follows: 2 ml mid-exponential phase cultures were harvested at room temperature by successive 30 sec. centrifugations at 16,000×g, resuspended in 0.6 ml water at 4oC, and placed on ice; the cells were then centrifuged for 30 sec. at 4oC, washed once more in 0.6 ml water on ice, washed once in 0.3 ml 10% (v/v) glycerol on ice, and resuspended in 50μL 10% glycerol. The recF::GK mutant strain was selected on TGYF (0.5% tryptone, 0.1% glucose, 0.3% yeast extract, and 15 mM fructose) agar containing 10μg/ml kanamycin.
2.UV-C irradiation experiments
D. radiodurans, recF-mutant strain, E.coli-recF, and E.coli BL21 cells were grown to early stationary phase and used for determining cell survival rate following UV-C irradiation (254nm). About 108 CFU cells of D. radiodurans, recF-mutant strain and E. coli BL21were used to UV-C treatment. These cells were washed twice with phosphate buffered saline (PBS) containing 0.02% Triton X-100 to ensure the separation of individual CFU and then irradiated in phosphate suspension with a mercury low pressure lamp (Shenxing Company, Jiangyin, Jiangsu, China) at a rate of 1.0J/m2/s with a main emission line of 254 nm. During irradiation, the strain suspension was stirred continuously to ensure homogeneous exposure at room temperature. The spectral irradiance was monitored by use of a UV Light Meter (TAINA, TN-2254). After UV-C radiation at defined fluences, 100μl from the aqueous suspension was taken for further analysis. Survival was determined from appropriate dilutions in PBS as colony forming ability (CFA) after cells were plated in TGY (D. radiodurans and recF-mutant strain) or LB (E. coli BL21and E.coli-recF) and incubated in the dark overnight at 30°C (D. radiodurans and recF-mutant strain) or 37°C (E. coli BL21 and E.coli-recF) for 2 to 4 days.
3.Numerical and statistical analysis
The surviving fractions of these cells were determined from the quotient N/N0, with N = colony formers after UV-C irradiation and N0 = colony formers without UV-C irradiation (i.e. the non-treated controls). Inactivation curves were obtained by plotting the logarithm of survival versus the applied UV fluence. The characteristics of the survival curves F10, extrapolation number, and inactivation constant IC, were determined according to Moeller. All experiments were repeated at least three times.
4.2-DE.
Samples were prepared as described for the comparison of the UVC resistance between D. radiodurans and eD.radiodurans. Approximately 150μg of protein was loaded onto an IPG strip (pH 4-7, 18 cm) and subjected to isoelectric focusing in IPGphor. The treated strips were subjected to 12% SDS-PAGE in the Ettan DALT II System for secondary electrophoresis. The proteins on the 2-DE gels were visualized using silver staining. All 2-DE gels were scanned with Image Scanner. Mass spectra of peptides were obtained using an Ultraflex MALDI- TOF MS.
5.Semi-quantitative RT-PCR
For semi-quantitative RT-PCR analysis, 16S rRNA which has relatively constant transcription levels in bacteria, was chosen as an internal control. cDNA from each sample was amplified with primers for 16S rRNA, and the cDNA samples were normalized by differential dilutions according to the quantity of 16S rRNA product. Selected genes were amplified from normalized cDNA samples with specific primers. The PCR products were analyzed on a 1.2% agarose gel and visualized with ethidium bromide staining.
6.Antibiotics analytical methods Samples from 4 hospitals, 1 nursery, 1 slaughter house, 1 wastewater treatment plant and source water samples of Chongqing region of Three Gorge Reservoir were analyzed for macrolide, lincosamide, trimethoprim, fluorouinolone, sulfonamide and tetracycline antibiotics by on-line solid-phase extraction and liquid chromatography- tandem mass spectrometry.
7.Antibiotic resistance profies of D. radiodurans
Antibiotic susceptibility tests (AST) were performed with Kirby-Bauer disc diffusion method recommended by CLSI (2009) and VITEK-II.
8.The optimality condition of electroporation
To study the optimality condition of electrporation, different levels of pRADZ3 were transformed into D. radiodurans through electroporation and chemical transformation.
Results and Discussion
1.A DNA fragment containing the D. radiodurans groEL gene promoter and a kanamycin resistance gene fusion was cloned by PCR amplification and used to displace the recF gene in the wild-type strain R1. The method to construct a mutant strain were established and we could performed further investigation on other function genes by this way.
2.recF is needed for the replication of D. radiodurans. Furthermore, as a part of the RecFOR pathway in D. radiodurans, disruption of recF could dramatically decrease the UV-C resistance of D.radiodurans and recF could increase the UV-C resistance of E.coli BL21 cells.
3.These purified proteins of RecFOR pathway were got by clone and expression in E. coli BL21 by pET32a(+) vector. We could study the interaction of these proteins in vitro. Furthermore, we could get the antibody of these proteins by animal immune and detect the expression levels of the proteins by Western-blot.
4.We generated an extremely UVC-radioresistant strain of D. radiodurans (eD. radiodurans) by directed evolution using UVC radiation. We looked for proteins differentially expressed between D. radiodurans and eD. radiodurans following UVC- irradiation using two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver-staining. The eD. radiodurans strain showed 1.64-fold more UVC resistance than wild-type strains. Furthermore, the expression levels of 25 protein spots showed significant changes under UVC radiation stress. Of these spots, 14 (including 10 up-regulated and 4 down-regulated proteins) were identified with peptide mass fingerprinting (PMF) using matrix-assisted laser desorption/ ionizationtime of flight mass spectrometry (MALDI- TOF MS) after tryptic in-gel digestion. These proteins are involved in various cellular functions, including 1) transcription, 2) photosynthetic reactions, 3) nucleotide/peptide binding, 4) PRM (protein turnover), 5) protein against oxidative and desiccation stress, 6) ion transport, 7) carbohydrate metabolism, and 8)proteolysis enzymes. Maf protein, v-type ATP synthase, amino acetyltransferase, TerD, DR1172 (LEA76 family desiccation resistance protein) and DR0392 were detected in eD. radiodurans but not in D. radiodurans following UVC radiation. Most of these proteins have not previously been reported to be relevant to UVC resistance. eD. radiodurans also had higher levels of Yellow-related protein, carbohydrate kinase, serine protease and DR0972. Maf protein, Yellow-related protein, TerD and a LEA76 family desiccation resistance protein were found to be involved in UVC resistance in D.radiodurans. Genes encoding proteins that may play important roles in UVC radiation resistance were further investigated by RT-PCR. These data provide an in-depth analysis of the response of the D. radiodurans proteome to UVC resistance, and they reveal protein alterations that are associated with UVC resistance. TerD may play an important protective role against oxidation and desiccation to ensure the repair of general stress-induced damage. LEA76 family desiccation resistance protein ) in eD.radiodurans following UVC radiation indicates a potential relationship between UV radiation resistance and desiccation resistance. Considering the role of yellow locus in Drosophila and the up-regulation of Yellow-related protein in D. radiodurans, this protein could function in the regeneration process, or it maybe involved in regulation of carotenoid expression. In D. radiodurans, maf may have critical roles in various cellular processes, including stress signaling, regulation of transcription and cell proliferation.
5.The concentration of antibiotics in water environment of Chongqing was far below the antibiotic resistance of D.radiodurans. pRADZ3 could be transformed between E. coli and D. radiodurans. The optimality condition of electroporation was 2.2 kV, 25μF, 200 ?,
2μg/ml vector. Point of innovation
1.Metholds
1.1 Methold of homologous recombinant was applied to investigate the construction of gene mutant strain. pHMR186 vector was used to construct the mutant D. radiodurans. Nucleotide fragment which contained the homologous arm was transformed into receipt strain directly to construct gene mutant strain. In this study, homologous arm was lingaged into pUC18 and the positive strain could be got on the antibiotic resistant plate. The positive strains were further confirmed by PCR.
1.2 Until now, plate process was usually used to test the UV resistant ability of strain, but agar surfaces have pits and fissures at the microscopic level. Bacteria inside these microcrevices will be exposed to less, or even no, UV and, hence, appear to be resistant. If one bacterium in a thousand was shielded in this manner, it could limit the linear portion of the survival curve to the -3 log level after which tailing would begin. To avoid these surface effects, many researchers irradiated the bacteria in dilute solutions that were vigorously stirred.
2.Results
The critical role of recF in UV resistance of D. radiodurans was the first time to reported. recF could induced growth defect of D. radiodurans. Maf protein, Yellow-related protein, TerD and a LEA76 family desiccation resistance protein were found to be involved in UVC resistance in D.radiodurans. this was the first time to report the antibiotics levels in Three Gorge Reservoir of Chongqing region and the antibiotic resistance profiles of D. radiodurans.
3.Application
The transformation condition of pRADZ3 was optimized and the antibiotics levels in aquatic environment was far from inhibition of D. radiodurans surviving.
Shortcoming
1.If we used shuttle vector which comprised recF gene to complement the deficient of recF in recF mutant strain, it would be more convincing than current results to elucidate the role of recF in UV resistance.
2.RecF, RecO, RecA expressed by pET32a(+) belongs to inclusion body. To get the active proteins, the incubation time, incubation temperature and concentration of IPTG ought ot optimized. If the process described above has negative effect on inclusion body, the expression vector should be changed.
3.The category of antibiotics selected in water environment wasn’t coincidence with antibiotics selected in antibiotic resistance profiles restricted by detection condition.
4.We haven’t cloned functional gene into D. radiodurans and this research ought to be finished in the future.
Project in the future
1.The specific mechanism ought to be investigate considering the critical role of RecFOR pathway in UV resistance of D. radiodurans. Several matter which include ionizing radiation, UV radiation and oxidation damage could incure single-strand break and double-strand break. Which damage status could this repair pathway favored to? Which terminal did this repair action started?
2.If we could testify the critical role in UV resistance of DR1172, we could deduce the origin of radiation resistance of D.radiodurans.
3.Energy metabolism played an important role in replication, surviving and apoptosis. In cells treated with g radiation, the levels of cyclic AMP (cAMP) and ATP increased rapidly by differentially regulating adenylyl cyclase (AC) and cAMP phosphodiesterase. In this study, energy metabolism related proteins exhibit high expression after UV treatment. Further study may included: 1) The relationship between energy metabolism and phosphorylation of proteins; 2) Is the recovery of proteins following radiation related to energy metabolism? If we could verify the relationship between energy metabolism and phosphorylation of proteins, we could explore a newly method or drug to prevente patient from radiation damage after radiotherapy.
耐辐射奇球菌(D.radiodurans)于1956年分离自γ射线辐照后的肉罐头,其对高强度离子辐射的抵抗力备受学者关注。近年来,围绕该菌耐辐射机制的研究成为人类探索生物体极端辐射耐受机制的突破口。
D.radiodurans属于非致病性革兰氏阳性微球菌,与芽胞菌依赖其芽孢结构抵抗外界因素干扰不同,D.radiodurans并不具有芽孢结构,而且其胞壁结构和组成与革兰氏阴性菌较为相近,具有外膜,而不具有革兰氏阳性菌特有的磷壁酸。扫描电子显微镜结果发现,在D.radiodurans菌稳定生长阶段,每个D.radiodurans菌均由两条垂直线分隔成四分裂体结构,4个室里面含有相等数量的DNA,每个室内含有一条完整的环状染色体,在辐射损伤前期(1h以内),所有基因组均被不同程度打断,而四个相对独立的空间保证了在一定范围内单个基因组的独立性和完整性,在辐照1小时后,环状的染色体转换为S形结构,并通过膜孔将两个室的DNA进行渐进式扩散。因此,其成熟体的四联体结构形态保证了即使在多组基因组受到损伤后,其损伤的DNA链始终能够在其他基因组中找到合适的退火延伸模板片段。目前研究结果表明,其独特的高剂量辐射抗性能力更多地来自于本身DNA修复和蛋白调控机制。当前较为明确的DNA损伤修复机制主要有以下几种:
(一)同源重组(homologous recombination, HR):其主要原理是利用同源的序列来修复自身的双链断裂,是dsDNA(double-stransded DNA)损伤修复的主要方式。此种方式在D. radiodurans离子辐射后发生较为普遍,由于RecBCD途径在D.radiodurans中的缺失,RecFOR在同源重组途径中具有重要作用。
(二)非同源末端连接(non-homologous end-joining, NHEJ):NHEJ修复机制与HR最大的差异在于完全不需要任何模板的帮助,致密的类核体结构保证了辐射致DNA损伤时断裂端彼此邻近,在PprA的作用下,抑制核酸外切酶III的活性,促进由DNA连接酶催化的DNA末端连接反应。因此,在辐射损伤早期阶段,断裂基因组片段可能以非同源末端连接修复方式开始早期修复;
(三)染色体内和染色体间单链退火(intra- and interchromosomal single-strandannealing):是指在DNA双链断裂处开始,在单链特异性核酸外切酶的作用下,断裂点两侧逐渐形成DNA单链区域,直至两个断点处出现互补的序列,完成单链缺口修复连接;
(四)合成依赖单链退火(Synthesis dependent strand annealing, SDSA):SDSA是一种不依赖RecA的无错双链断裂修复模式,由产生DNA双链断裂的其中一条链的3’末端入侵未损伤的姊妹双链同源区开始DNA合成,并扩展姊妹双链形成的D- loop环作为模板或其他3’末端DNA合成的引物;
(五)延伸合成依赖式退火(Extended synthesis-dependent strand annealing, ESDSA):如果未发现末端游离的互补断裂片段,则利用RecA蛋白丝识别同源片段,利用同源片段的序列进行延伸,直至发现互补断裂末端,进行单链退火修复。
既往研究表明,在大肠杆菌中,rec基因决定了其重组修复系统的主要途径,对D.radiodurans菌的rec基因研究主要集中于recA等基因,Kim J等研究显示,D. radiodurans菌的RecA比E.coli的更容易结合DSBs(double-strand breaks)而促进快速高效的DNA修复。对E.coli重组修复途径研究发现,其修复途径启动阶段包括一条涉及recB、recC、recD基因的RecBCD途径和一条包括recF、recO、recR基因的RecF途径。在大部分野生株中,RecBCD(或者其功能相似基因AddAB)启动DSBs的修复。然而,在耐辐射奇球菌基因组中,没有找到RecB和RecC的同源物,如果对照大肠杆菌中同源重组的几种途径,按照经典遗传学理论,在RecBCD途径不能运作的情况下,基因组仍然具有重组活性,RecFOR途径必然发挥非常重要的作用。
本研究以D. radiodurans为研究对象,从四个方面开展耐辐射奇球菌紫外辐射抗性和应用前景研究:1.以RecFOR途径的recF基因为切入点,通过recF基因突变株的构建,以野生株为参照,研究recF突变株与野生株的生长周期和辐射抗性差异,探讨recF在D.radiodurans紫外辐射抗性中的作用;2.通过分子生物学手段克隆表达并纯化RecFOR途径的关键蛋白,为功能蛋白的体外研究打下基础。3.通过选择性紫外辐射压力驯化D.radiodurans,经过25代的驯化,得到紫外辐射抗性得到明显增强的D.radiodurans,利用二维蛋白电泳技术,通过对800 J/m2辐照后eD.radiodurans和D.radiodurans蛋白差异点的鉴定和分析,探讨紫外辐射抗性的可能机制和途径;4.鉴于D.radiodurans超强的环境耐受能力,利用基因工程改良的生物有机体对抗生素污染环境进行生物修复是一种具有良好应用前景的方法。本部分实验旨在通过对重庆市水体环境中抗生素水平检测、D.radiodurans耐药谱检测、穿梭载体的转化效率等方面的研究,为D.radiodurans基因工程菌的构建提供必要的实验支持,为D.radiodurans应用于抗生素污染环境有毒物质的降解修复打下基础。
二、材料和方法
(一)利用同源重组双交换方法,将插入groEL promoter+卡那霉素抗性基因的同源臂载体通过电转法转入D.radiodurans,替换recF基因,通过卡那霉素抗生素TGY平板筛选得到阳性克隆子,利用多对引物对突变株同源臂进行PCR扩增,扩增产物进行测序验证。绘制recF突变株与标准株生长曲线和紫外辐射抗性生存曲线,从而评价recF在D.radiodurans紫外辐射抗性中的作用。
(二)以pET32a(+)为载体,设计带酶切位点的引物对D. radiodurans RecFOR途径关键基因recF、recO、recR、SSB、reA进行PCR扩增,将PCR产物连接至pGEM-T,限制性内切酶双酶切载体和连接至pGEM-T的PCR产物,T4连接酶连接后转化至E. coli BL21。通过IPTG浓度、培养温度、培养时间的摸索,探讨各蛋白诱导表达的最佳条件,镍柱纯化蛋白产物用Western-blot进行鉴定。
(三)通过800 J/m2紫外辐射压力驯化25代,筛选到D.radiodurans紫外辐射能力得到明显增强的进化株,绘制紫外辐照生长曲线,比较进化株和野生株紫外生存能力。运用2-DE技术对进化株和野生株辐照后蛋白表达进行差异比较,筛选差异蛋白,质谱分析差异蛋白并对分析结果在NCBI上进行序列相似性比对,文献查阅差异蛋白的功能,针对有意义的差异蛋白点设计引物,利用RT-PCR技术对功能蛋白蛋白转录水平进行半定量分析,探讨紫外辐射抗性增强的可能机制。
(四)利用在线固相萃取法和液相色谱电喷雾质谱法对重庆市医院排放污水、养殖场和屠宰场、地表水、污水处理厂进出口水中乙酰红霉素、水合乙酰红霉素、罗红霉素、泰乐菌、林可霉素、甲氧苄啶、环丙沙星、洛美沙星、氟哌酸、氧氟沙星、磺胺嘧啶、磺胺二甲氧嘧啶、磺胺甲嘧啶、磺胺甲基异恶唑、iso-氯四环素、epi-iso-氯四环素、氯四环素、四环素等水平进行检测,参照CLSI2008标准对D.radiodurans耐药谱进行检测,采用Kirby-Bauer法进行阿米卡星(AK, 30μg)、氨曲南(ATM, 30μg)、哌拉西林/他唑巴坦(TZP, 100/10μg)、头孢哌酮/舒巴坦(75/10μg)、头孢噻肟(CTX, 30μg)、替考拉宁(TEC, 30μg)、头孢曲松(CRO, 30μg)、阿奇霉素(AZM, 30μg)、阿莫西林/克拉维酸(20/10μg)、万古霉素(VA, 30μg)药敏试验。采用VITEK-Ⅱ药敏分析系统进行抑菌直径和最低抑菌浓度(Minimal Inhibitory Concentration, MIC)检测,选用以下抗生素:环丙沙星(CIP)、褐霉素(FD)、四环素(TET)、庆大霉素(CN)、红霉素(E)、亚胺培能(IPM)、磷霉素(FOS)、利奈唑烷(ZYVOX)、莫西沙星(MF)、利福平(RD)、替考拉宁(TEC)、复方新诺明(SXT)、氨苄西林/舒巴坦(SAM),将抑菌圈大小换算成MIC。将检测结果与水体抗生素水平进行比较,探讨D.radiodurans的环境应用前景。通过化学转化法和电转化法对pRADZ3载体转化D. radiodurans的能力进行研究,探讨载体转化的最适条件。
三、结果
(一)pUC18质粒作为同源交换臂的载体,能够递呈目的片段至D. radiodurans,并发生同源交换,通过电转法,用卡那霉素抗性平板成功筛选到阳性克隆,并通过PCR对recF突变株进行了确认;recF突变株生长曲线表现为对数生长期的延迟,且生长高峰菌落数低于野生株,recF可能对D. radiodurans的生长繁殖具有影响;recF突变株的紫外抗性能力降低显著,其F10值低于野生株将近3倍,recF转化至E. coli能够增强其紫外辐射抗性2.5倍左右。
(二)以D. radiodurans为模板,扩增得到RecFOR途径关键基因recF、recO、recR、ssb、recA,双酶切后连接至PET32a(+)载体,CaCl2化学转化法转化至E. coli BL21,IPTG成功诱导5个蛋白的表达,并通过Western-blot进行了验证。其中,SSB和RecR以可溶性蛋白形式表达,RecF、RecO、RecA以包涵体形式表达;RecF、RecO、RecR、SSB、RecA纯度均>90%,浓度分别为0.5 mg/ml、0.5 mg/ml、0.6 mg/ml、0.5 mg/ml、0.6mg/ml,蛋白的浓度和纯度均能满足体外实验要求。
(三)紫外驯化法能够筛选到紫外辐射抗性明显增强的进化株,二维蛋白差异蛋白分析能够对辐照后野生株和进化株的差异蛋白进行筛选,其中,TerD、Maf、LEA76干燥抗性蛋白以及黄色相关蛋白尤为值得引起关注;针对有意义的功能差异蛋白点,利用RT-PCR技术在蛋白转录水平上对其表达的上调进行了确认,Maf转录因子与压力信号感应,转录调控以及细胞增殖相关;黄色相关蛋白在辐射后的再生过程或者在类胡萝卜素表达调控方面具有重要作用;LEA76干燥相关蛋白不仅与D.radiodurans干燥抗性机制相关,可能在紫外辐射抗性机制中也具有重要作用,辐射抗性机制和干燥抗性机制可能具有相同的修复途径;TerD与氧化应激相关,对包括重金属阳离子、甲磺酸甲酯、丝裂霉素C、紫外线等在内的环境因素对生物体造成的影响具有保护作用。
(四)在所采水样中,污水处理厂抗生素含量和种类最高,其次为医院污水和养殖屠宰场。D. radiodurans对25种抗生素的耐受水平检测结果表明,D. radiodurans对庆大霉素、亚胺培能、莫西沙星、替考拉宁、复方新诺明、氨苄西林/舒巴坦、阿米卡星、头孢吡肟、哌拉西林/他唑巴坦、万古霉素、头孢曲松、利奈唑烷、阿莫西林/克拉维酸等敏感。D. radiodurans对抗生素的耐受能力远远高于水环境中的抗生素含量水平。D. radiodurans在电压2.2 kV,电容25μF,电阻200欧姆,2μg/ml pRADZ3载体浓度时,电转转化率最高,适宜于应用与基因工程菌的构建。
四、结论
(一)本实验通过对recF突变株的构建成功,摸索出一套行之有效的突变株构建方法,为后续关键基因蛋白的功能研究打下基础;
(二)recF的缺失能够导致D.radiodurans生长复制减缓,紫外辐射抗性的降低,其具体机制有待进一步的研究;
(三)RecFOR途径的关键蛋白构建至pET32a(+)后能够在IPTG的诱导下表达,关键蛋白的成功纯化为体外实验的开展奠定了基础,同时,蛋白的纯化表达为相应抗体的制备打下基础,为将来western-blot应用于菌体RecFOR途径蛋白的表达调控检测提供了条件;
(四)通过对进化株和野生株紫外辐射后蛋白组学比较,14个差异蛋白功能涉及转录、光合作用、核酸/肽结合、蛋白折叠、氧化和干燥应激相关、离子转运、碳水化合物代谢及糖类代谢等;
(五)水环境中的抗生素浓度远远低于D. radiodurans对抗生素的耐受浓度,基因工程构建D. radiodurans可应用重庆市各种水环境中,pRADZ3的结构特点决定其可以电转化法将携带的功能基因转入D. radiodurans中。
五、创新点
(一)通过突变株的构建和recF在E. coli中的表达,首次报道了recF在紫外辐射防护方面具有重要作用,recF的缺失能够造成D. radiodurans生长复制的减缓。
(二)Maf、LEA76干燥抗性蛋白以及黄色相关蛋白表达调控参与了D. radiodurans的紫外辐射抗性机制,既往研究未见报道。
(三)首次报道了重庆三峡库区水体环境中的抗生素水平以及D. radiodurans的MIC水平,为D. radiodurans的环境应用研究打下基础。
六、缺点和不足
(一)recF的研究虽然从突变和导入E. coli设计实验进行了探讨,但是如果能够运用穿梭载体将D. radiodurans和E. coli的recF基因导入recF突变株,观察D. radiodurans的紫外抗性恢复情况,将对整个实验结论是个良好的补充;
(二)RecFOR途径蛋白克隆表达纯化后,有三个蛋白为包涵体的形式表达,要获得活性蛋白进行后续的功能研究,必须对这三个蛋白进行复性操作,这样会对蛋白的浓度造成损失,后续可通过降低诱导温度、降低IPTG浓度、延长诱导时间等方式来调整蛋白的表达,如果还是以包涵体的形式则通过多个载体和宿主细菌的选择来改善蛋白诱导表达条件,尽量以可溶性表达功能蛋白;
(三)由于检测条件的限制,水环境中检测出的抗生素种类与耐药谱选择抗生素种类并不完全匹配。有较多水环境中检出抗生素在D. radiodurns最低抑菌浓度(MIC)分析时药敏分析仪器中并未包含,药敏纸片法的纸片也未有此类抗生素纸片。(四)本研究在D. radiodurans的应用研究仅为初步探讨,未将具有降解功能的基因通过穿梭载体导入D. radiodurans进行基因工程菌的构建和功能验证研究。
七、下一步研究计划
(一)鉴于RecFOR途径在辐照抗性的重要作用,有必要开展实验对其具体作用机制进行深入研究。由于各种环境压力(包括离子辐射、紫外辐射、氧化损伤等)对D. radiodurans作用靶点各有不同,目前对该途径作用于单链断裂、双链断裂均有报道,更多的报道倾向于修复单链断裂。那么,对单或双链修复的偏好程度研究将解决其修复效能作用靶点的疑问。同时,该途径的修复作用起始位点是5’端还是3’端,其具体作用起始位点将决定后期对其具体作用机制假说的推导和验证。
(二)干燥抗性基因(如DR1172)在辐射抗性中是否具有重要作用?通过本研究所建立的突变株构建方法和穿梭载体的应用,可以从正反两方面进行该研究的验证,通过此研究,可推测D. radiodurans超强辐射抗性可能的起源,为起源假说提供参考。如果干燥基因同时具有辐射抗性的作用,将为基因工程菌株的构建提供良好的功能基因选择。
(三)能量代谢在生物体的复制、生长、凋亡中具有重要的作用,Kamble VA的文章(2010)表明,当D. radiodurans受到紫外辐射后,cAMP、ATP和蛋白磷酸化水平升高。在本研究中,通过二维蛋白研究,发现了多种能量代谢相关酶水平的升高,从蛋白水平上为能量代谢在辐射后修复中具有重要作用提供了佐证,下一步需要进一步研究的是:1)能量代谢和功能蛋白磷酸化的关系;2)高剂量辐照无论对DNA还是蛋白,均能导致其断裂和失活,而DNA的断裂损伤修复一定是需要通过活性蛋白来介导的,这些活性蛋白的保护或迅速恢复是否与能量代谢变化相关?如果研究表明,能量代谢与蛋白磷酸化水平或保护相关,将对于临床放疗病人的辐射防护和高剂量离子或紫外暴露人群保护提供重要的研究价值,同时为军民两用辐射防护药物的研发提供研究思路。
The extremely radioresistant bacterium, Deinococcus radiodurans (D. radiodurans), is characterized by its unusual capability to tolerate numerous DNA damaging agents, including ionizing radiation, ultraviolet radiation, and desiccation. D. radiodurans is capable of surviving 15,000 grays (Gy) of ionizing radiation, whereas doses below 10 Gy are lethal to all other organisms. Studies on mechanisms of radioresistance of D. radiodurans were thought to be the point to breakthrough the ability of organisms to surviving in extreme environment in recent years.
D. radiodurans belongs to non-pathogenic gram-positive micrococcus. The ability of radioresistant of Bacillus spp. attribute to the characteristics of spore structure. Considering the critical role of spore in Bacillus spp., the mechanisms of DNA repair and modulation of proteins ought to be further investigated.
Transmission electron microscopy studies showed that a membranous framework demarcates the four compartments of a single D. radiodurans cell, but orifices were evident in the internal membranes of both growing and stationary-state cells. Chromatin was present in all compartments, and it adopted a distinctive toroidal shape. DNA labeling revealed that, whereas DNA toroids are present in all compartments of stationary-state bacteria, the nucleoid in one or two compartments of growing cells is dispersed. The mechanisms of radioresistance included several pathway as follows:
1.Homologous recombination at the fragment ends: In this way, the strain utilize recombination sequences as template to manipulate the gap DNA. dsDNA damage was mainly repaired by this way. In D. radiodurans, the RecFOR pathway plays a critical role in DNA damage repair due to a deficiency of RecBCD.
2.Non-homologous end-joining (NHEJ): One major mechanism of DSB repair in eukaryotes is non-homologous end-joining (NHEJ), in which the two ends of a broken chromosome are rejoined directlyusing only the base-pairing information of the ends themselves to guide ligation. In the early phase after irradiation, the broken fragments maybe repaired by NHEJ.
3.Intra- and interchromosomal single-strand annealing: The single strand DNA could be formed by the specific exonucleace at the dsDNA broken site until the occurrence of complement strand DNA. And then the single strand gap was repaired.
4.Synthesis dependent strand annealing (SDSA): Recent research has suggested a compelling model for genome restitution in this species, in which the related but more efficient process called synthesis-dependent singlestrand annealing (SDSA) plays a necessary part. During SDSA, the 3’end of a strand derived from a DNA double-strand break invades the homologous region of a sister duplex. The invading 3′end is used to prime DNA synthesis, unwinding the sister duplex and enlarging the D-loop. The displaced strand in the undamaged complex anneals to the remaining free 3’end created by the double-strand break.
5.Extended synthesis-dependent strand annealing (ESDSA): How does ESDSA differ from SDSA? Crucially, it requires at least two genome copies that are broken at different positions. Once overlapping fragments have aligned, a single-round multiplex PCR-like step- a variant of PCR that simultaneously amplifies different target sequences by using multiple primer pairs occurs to produce long single-stranded overhangs that anneal accurately to produce reassembled chromosomal segments. Last, RecA-mediated homologous recombination using long intermediates synthesized by DNA-polymerase-I produces full-length chromosomes.
In order to investigate the mechanisms of radioresistance of D. radiodurans, our studies were divided into four parts:
1.The function of recF in UV-C resistance was characterized by the construction of the recF mutant D. radiodurans R1 strain and the transformation of D. radiodurans recF into E. coli BL21.
2.The critical genes of RecFOR pathway were cloned and expressed in E. coli BL21. The purified proteins were further confirmed by Western-blot.
3.To investigate the mechanisms of UVC radiation resistance in D. radiodurans, we produced an enhanced UVC-resistant strain (eD. radiodurans) by selective pressure of direct UVC radiation. We then examined proteome changes between the wild-type strain and eD. radiodurans following UVC irradiation using 2-DE and silver-staining. Genes encoding proteins that may play important roles in UVC radiation resistance were further investigated by RT-PCR.
4.To investigate the application of gene engineering in D. radiodurans, the levels of antibiotics in Three Gorge Reservoir of Chongqing region were tested. The MIC values were determined by Kirby-Bauer disc diffusion method and VITEK-II. In addition and the frequency of transformation were analyzed by chemical transformation and electro transformation with pET32a(+).
Materials and methods
1.recF mutant construction
The kanamycin resistance gene was obtained from the pET30b(+) plasmid using primers K1 and K2 (containing BamHI and PstI restriction sites) and ligated to groEL promoter which was obtained from the D. radiodurans genome using primers G1 and G2 (containing KpnI and BamHI restriction sites). A 994-bp DNA fragment corresponding to the immediate upstream portion of the dr1089 (recF D. radiodurans) initial codon was amplified using primers MF1 and MF2 (containing EcoRI and KpnI restriction sites), and a 1063-bp DNA fragment corresponding to the immediate downstream region of the dr1089(recF D. radiodurans) stop codon was amplified using primers MF3 and MF4 (containing PstI and HindIII restriction sites). The PCR products were digested with their respective enzymes listed above and then ligated to a groEL promoter-kanamycin cassette. The tripartite ligation products were amplified using L1 and R2, and the resulting fragments were ligated to pUC18 that had been digested with EcoRI and HindIII. The fragments were then transformed into D. radiodurans R1 as follows: 2 ml mid-exponential phase cultures were harvested at room temperature by successive 30 sec. centrifugations at 16,000×g, resuspended in 0.6 ml water at 4oC, and placed on ice; the cells were then centrifuged for 30 sec. at 4oC, washed once more in 0.6 ml water on ice, washed once in 0.3 ml 10% (v/v) glycerol on ice, and resuspended in 50μL 10% glycerol. The recF::GK mutant strain was selected on TGYF (0.5% tryptone, 0.1% glucose, 0.3% yeast extract, and 15 mM fructose) agar containing 10μg/ml kanamycin.
2.UV-C irradiation experiments
D. radiodurans, recF-mutant strain, E.coli-recF, and E.coli BL21 cells were grown to early stationary phase and used for determining cell survival rate following UV-C irradiation (254nm). About 108 CFU cells of D. radiodurans, recF-mutant strain and E. coli BL21were used to UV-C treatment. These cells were washed twice with phosphate buffered saline (PBS) containing 0.02% Triton X-100 to ensure the separation of individual CFU and then irradiated in phosphate suspension with a mercury low pressure lamp (Shenxing Company, Jiangyin, Jiangsu, China) at a rate of 1.0J/m2/s with a main emission line of 254 nm. During irradiation, the strain suspension was stirred continuously to ensure homogeneous exposure at room temperature. The spectral irradiance was monitored by use of a UV Light Meter (TAINA, TN-2254). After UV-C radiation at defined fluences, 100μl from the aqueous suspension was taken for further analysis. Survival was determined from appropriate dilutions in PBS as colony forming ability (CFA) after cells were plated in TGY (D. radiodurans and recF-mutant strain) or LB (E. coli BL21and E.coli-recF) and incubated in the dark overnight at 30°C (D. radiodurans and recF-mutant strain) or 37°C (E. coli BL21 and E.coli-recF) for 2 to 4 days.
3.Numerical and statistical analysis
The surviving fractions of these cells were determined from the quotient N/N0, with N = colony formers after UV-C irradiation and N0 = colony formers without UV-C irradiation (i.e. the non-treated controls). Inactivation curves were obtained by plotting the logarithm of survival versus the applied UV fluence. The characteristics of the survival curves F10, extrapolation number, and inactivation constant IC, were determined according to Moeller. All experiments were repeated at least three times.
4.2-DE.
Samples were prepared as described for the comparison of the UVC resistance between D. radiodurans and eD.radiodurans. Approximately 150μg of protein was loaded onto an IPG strip (pH 4-7, 18 cm) and subjected to isoelectric focusing in IPGphor. The treated strips were subjected to 12% SDS-PAGE in the Ettan DALT II System for secondary electrophoresis. The proteins on the 2-DE gels were visualized using silver staining. All 2-DE gels were scanned with Image Scanner. Mass spectra of peptides were obtained using an Ultraflex MALDI- TOF MS.
5.Semi-quantitative RT-PCR
For semi-quantitative RT-PCR analysis, 16S rRNA which has relatively constant transcription levels in bacteria, was chosen as an internal control. cDNA from each sample was amplified with primers for 16S rRNA, and the cDNA samples were normalized by differential dilutions according to the quantity of 16S rRNA product. Selected genes were amplified from normalized cDNA samples with specific primers. The PCR products were analyzed on a 1.2% agarose gel and visualized with ethidium bromide staining.
6.Antibiotics analytical methods Samples from 4 hospitals, 1 nursery, 1 slaughter house, 1 wastewater treatment plant and source water samples of Chongqing region of Three Gorge Reservoir were analyzed for macrolide, lincosamide, trimethoprim, fluorouinolone, sulfonamide and tetracycline antibiotics by on-line solid-phase extraction and liquid chromatography- tandem mass spectrometry.
7.Antibiotic resistance profies of D. radiodurans
Antibiotic susceptibility tests (AST) were performed with Kirby-Bauer disc diffusion method recommended by CLSI (2009) and VITEK-II.
8.The optimality condition of electroporation
To study the optimality condition of electrporation, different levels of pRADZ3 were transformed into D. radiodurans through electroporation and chemical transformation.
Results and Discussion
1.A DNA fragment containing the D. radiodurans groEL gene promoter and a kanamycin resistance gene fusion was cloned by PCR amplification and used to displace the recF gene in the wild-type strain R1. The method to construct a mutant strain were established and we could performed further investigation on other function genes by this way.
2.recF is needed for the replication of D. radiodurans. Furthermore, as a part of the RecFOR pathway in D. radiodurans, disruption of recF could dramatically decrease the UV-C resistance of D.radiodurans and recF could increase the UV-C resistance of E.coli BL21 cells.
3.These purified proteins of RecFOR pathway were got by clone and expression in E. coli BL21 by pET32a(+) vector. We could study the interaction of these proteins in vitro. Furthermore, we could get the antibody of these proteins by animal immune and detect the expression levels of the proteins by Western-blot.
4.We generated an extremely UVC-radioresistant strain of D. radiodurans (eD. radiodurans) by directed evolution using UVC radiation. We looked for proteins differentially expressed between D. radiodurans and eD. radiodurans following UVC- irradiation using two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver-staining. The eD. radiodurans strain showed 1.64-fold more UVC resistance than wild-type strains. Furthermore, the expression levels of 25 protein spots showed significant changes under UVC radiation stress. Of these spots, 14 (including 10 up-regulated and 4 down-regulated proteins) were identified with peptide mass fingerprinting (PMF) using matrix-assisted laser desorption/ ionizationtime of flight mass spectrometry (MALDI- TOF MS) after tryptic in-gel digestion. These proteins are involved in various cellular functions, including 1) transcription, 2) photosynthetic reactions, 3) nucleotide/peptide binding, 4) PRM (protein turnover), 5) protein against oxidative and desiccation stress, 6) ion transport, 7) carbohydrate metabolism, and 8)proteolysis enzymes. Maf protein, v-type ATP synthase, amino acetyltransferase, TerD, DR1172 (LEA76 family desiccation resistance protein) and DR0392 were detected in eD. radiodurans but not in D. radiodurans following UVC radiation. Most of these proteins have not previously been reported to be relevant to UVC resistance. eD. radiodurans also had higher levels of Yellow-related protein, carbohydrate kinase, serine protease and DR0972. Maf protein, Yellow-related protein, TerD and a LEA76 family desiccation resistance protein were found to be involved in UVC resistance in D.radiodurans. Genes encoding proteins that may play important roles in UVC radiation resistance were further investigated by RT-PCR. These data provide an in-depth analysis of the response of the D. radiodurans proteome to UVC resistance, and they reveal protein alterations that are associated with UVC resistance. TerD may play an important protective role against oxidation and desiccation to ensure the repair of general stress-induced damage. LEA76 family desiccation resistance protein ) in eD.radiodurans following UVC radiation indicates a potential relationship between UV radiation resistance and desiccation resistance. Considering the role of yellow locus in Drosophila and the up-regulation of Yellow-related protein in D. radiodurans, this protein could function in the regeneration process, or it maybe involved in regulation of carotenoid expression. In D. radiodurans, maf may have critical roles in various cellular processes, including stress signaling, regulation of transcription and cell proliferation.
5.The concentration of antibiotics in water environment of Chongqing was far below the antibiotic resistance of D.radiodurans. pRADZ3 could be transformed between E. coli and D. radiodurans. The optimality condition of electroporation was 2.2 kV, 25μF, 200 ?,
2μg/ml vector. Point of innovation
1.Metholds
1.1 Methold of homologous recombinant was applied to investigate the construction of gene mutant strain. pHMR186 vector was used to construct the mutant D. radiodurans. Nucleotide fragment which contained the homologous arm was transformed into receipt strain directly to construct gene mutant strain. In this study, homologous arm was lingaged into pUC18 and the positive strain could be got on the antibiotic resistant plate. The positive strains were further confirmed by PCR.
1.2 Until now, plate process was usually used to test the UV resistant ability of strain, but agar surfaces have pits and fissures at the microscopic level. Bacteria inside these microcrevices will be exposed to less, or even no, UV and, hence, appear to be resistant. If one bacterium in a thousand was shielded in this manner, it could limit the linear portion of the survival curve to the -3 log level after which tailing would begin. To avoid these surface effects, many researchers irradiated the bacteria in dilute solutions that were vigorously stirred.
2.Results
The critical role of recF in UV resistance of D. radiodurans was the first time to reported. recF could induced growth defect of D. radiodurans. Maf protein, Yellow-related protein, TerD and a LEA76 family desiccation resistance protein were found to be involved in UVC resistance in D.radiodurans. this was the first time to report the antibiotics levels in Three Gorge Reservoir of Chongqing region and the antibiotic resistance profiles of D. radiodurans.
3.Application
The transformation condition of pRADZ3 was optimized and the antibiotics levels in aquatic environment was far from inhibition of D. radiodurans surviving.
Shortcoming
1.If we used shuttle vector which comprised recF gene to complement the deficient of recF in recF mutant strain, it would be more convincing than current results to elucidate the role of recF in UV resistance.
2.RecF, RecO, RecA expressed by pET32a(+) belongs to inclusion body. To get the active proteins, the incubation time, incubation temperature and concentration of IPTG ought ot optimized. If the process described above has negative effect on inclusion body, the expression vector should be changed.
3.The category of antibiotics selected in water environment wasn’t coincidence with antibiotics selected in antibiotic resistance profiles restricted by detection condition.
4.We haven’t cloned functional gene into D. radiodurans and this research ought to be finished in the future.
Project in the future
1.The specific mechanism ought to be investigate considering the critical role of RecFOR pathway in UV resistance of D. radiodurans. Several matter which include ionizing radiation, UV radiation and oxidation damage could incure single-strand break and double-strand break. Which damage status could this repair pathway favored to? Which terminal did this repair action started?
2.If we could testify the critical role in UV resistance of DR1172, we could deduce the origin of radiation resistance of D.radiodurans.
3.Energy metabolism played an important role in replication, surviving and apoptosis. In cells treated with g radiation, the levels of cyclic AMP (cAMP) and ATP increased rapidly by differentially regulating adenylyl cyclase (AC) and cAMP phosphodiesterase. In this study, energy metabolism related proteins exhibit high expression after UV treatment. Further study may included: 1) The relationship between energy metabolism and phosphorylation of proteins; 2) Is the recovery of proteins following radiation related to energy metabolism? If we could verify the relationship between energy metabolism and phosphorylation of proteins, we could explore a newly method or drug to prevente patient from radiation damage after radiotherapy.
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