沼泽红假单胞菌染色体的提取

近年来,由于工业产生的有机物对环境的污染越来越严重,推动了人们对能降解芳香物的沼泽红假单胞菌(Ｒ.ｐａｌｕｓｔｒｉｓ)的研究兴趣[1-8]。大多数芳香族化合物是严重危害人类健康的污染物质,为从基因水平研究沼泽红假单胞菌对芳香族化合物的生物降解机制,需要找到一种提取染色体的理想方法。泽沼红假单胞菌质膜构造比较特殊,且菌体富含蛋白质、色素、多糖等,给染色体提取造成一定难度。在本工作中采用了几种方法进行染色体的提取。1　材料与方法11　菌株来源:Ｙ6为本实验室分离、鉴定并保存的沼泽红假单胞菌。12　菌体培养121　沼泽红假单…     

沼泽红假单胞菌偶氮还原酶新基因的克隆表达

前期实验证明沼泽红假单胞菌（Rhodopsedomonas palustris）对偶氮染料有较强的隆解能力,通过PCR扩增,从该菌粒DNA中扩增获得了一条未登录新基因PAR-1。将该基因构建到融合蛋白表达载体pGEX-4T-1,通过IPTG诱导,进行原核蛋白表达,SDS-PAGE电泳显示,有约48kD融合蛋白表达。对经诱导的大肠杆菌（BL21）进行偶氮染料脱色试验,检测到轻微脱色活性。     

沼泽红假单胞菌培养液对铅中毒大鼠尿铅、血铅和粪铅的影响

目的研究沼泽红假单胞菌培养液对铅中毒大鼠的驱铅作用。方法采用自由饮用醋酸铅水溶液建立铅中毒模型。用石墨炉原子吸收光谱法检测沼泽红假单胞菌培养液对铅中毒大鼠尿铅、血铅和粪铅的影响。结果沼泽红假单胞菌培养液能显著增加铅中毒大鼠尿铅和粪铅的排出,并降低其血铅水平。结论沼泽红假单胞菌培养液对铅中毒大鼠具有显著的排铅作用。     

沼泽红假单胞菌Rhodopseudomonas palustris 2-8的亚硝酸盐还原酶基因克隆和序列分析

沼泽红假单胞菌2-8具有亚硝酸盐还原能力, 根据不同类型亚硝酸盐还原酶保守序列设计引物, 通过PCR扩增的方法对2-8菌株的亚硝酸盐还原酶类型进行鉴定, 发现该菌株的亚硝酸盐还原酶为Cu型亚硝酸盐还原酶。从2-8菌株基因组中克隆出编码该Cu型亚硝酸盐还原酶的基因(nirK), 该基因由1 154个碱基对组成, 在GenBank数据库的登录号为GU332847, 与沼泽红假单胞菌(Rhodopseudomonas palustris TIE和CGA009) 的nirK序列相似性为90%。互联网数据库及生物信     

沼泽红假单胞菌作为微生物细胞工厂的应用

沼泽红假单胞菌(Rhodopseudomonas palustris)是一种紫色非硫细菌,具有新陈代谢代谢方式多样、可利用多种碳源、天然产多种化合物等优点,作为微生物细胞工厂具有很大的研究潜力,目前主要应用于废水处理和水产养殖领域。本文中,笔者从三个方面对沼泽红假单胞菌在微生物细胞工厂领域的研究应用进行总结,包括碳源的多样性、基因工程策略以及微生物细胞工厂的应用,如在产燃料燃气和萜烯化合物、微生物燃料电池、微生物电合成和光催化合成等领域。目前沼泽红假单胞菌作为微生物细胞工厂处于发展的初级阶段,笔者对其目前面临的难点进行了总结,并对其进一步的研究方向进行了展望。     

光合细菌和小球藻对石生苔藓种源扩繁的作用

石生苔藓结皮可以从大气中吸收水分及养分,附着在岩石表面生长,具有促进矿物分化和植被演替等多种重要的生态功能,利用人工培育的苔藓结皮进行岩石工程创面生态修复具有广阔的市场前景,而针对特定地区的优势种探讨高效的种源扩繁技术则成为了首要任务。研究以秦岭北麓长势好、生物量大的石生匍匐型藓种羽枝青藓为研究材料,考虑沼泽红假单胞菌浓度(高浓度24 mL/L,低浓度12 ml/L,不添加)和小球藻浓度(高浓度250 mL/L,低浓度125 mL/L,不添加)开展双因素完全试验,观测人工气候箱条件下羽枝青藓的盖度、新发芽数和新发芽茎长,探究沼泽红假单胞菌和小球藻对羽枝青藓的作用效果、最适浓度及组合,为提高羽枝青藓扩繁效率提供科学依据。结果显示,(1)只添加低浓度沼泽红假单胞菌的处理羽枝青藓生长状况最优,相比于对照处理(不添加沼泽红假单胞菌和小球藻),可将盖度提高14.3%,新发芽数增加61.2%,新发芽茎长增长34.0%;(2)沼泽红假单胞菌浓度对羽枝青藓的盖度、新发芽数和新发芽茎长均有显著影响;(3)小球藻浓度对羽枝青藓各项生长指标均无显著影响,其与沼泽红假单胞菌的交互作用对羽枝青藓盖度有显著影响。...     

沼泽红假单胞菌对亚硒酸盐还原脱毒的研究

主要研究沼泽红假单胞菌对亚硒酸盐还原脱毒作用及其脱毒机理。通过单因子实验、正交试验, 对影响亚硒酸盐还原脱毒的因素进行研究, 得到沼泽红假单胞菌还原亚硒酸盐的最佳条件为: 亚硒酸钠添加量是25 mg/L, 培养的第5天接种接种量15% (质量比)。在该条件下, 对亚硒酸钠去除率可达98.2%。研究发现, 亚硒酸盐还原酶主要存在于细胞质, 分子量约为182 kD, 由4个亚基组成。通过透射电子显微镜观察, 菌体表面出现粒径在5 nm?200 nm之间的高电子密度颗粒, 初步表明亚硒酸盐在沼泽红假单胞菌体内被     

富纳米硒沼泽红假单胞菌的肝保护作用研究

目的:建立CCl4急性肝损伤动物模型,研究富纳米硒沼泽红假单胞菌对肝损伤昆明鼠的肝保护作用。方法:实验随机分为空白对照组、CCl4模型组、不同剂量富纳米硒沼泽红假单胞菌处理组等9个组别,在连续灌胃给药14 d后,分别进行1%的CCl4植物油溶液(10 mL/kg)腹腔注射,24 h后取材。结果:与空白对照组相比,CCl4模型组的肝脏指数显著增高(P0.01);与CCl4模型组相比,中、高剂量富纳米硒沼泽红假单胞菌处理组肝脏指数显著减小(P0.05,P0.01)。肝脏组织细胞形态学观察发现,富纳米硒沼泽红假单胞菌处理组与CCl4模型组相比,显著恢复了炎细胞浸润、细胞水肿、点状坏死等肝细胞结构损伤现象。结论:富纳米硒沼泽红假单胞菌具有较好的肝保护作用,可以作为保肝制剂用于畜禽养殖中肝疾病的预防。     

内蒙古碱湖中紫色非硫光合细菌的分离和鉴定

从内蒙古碱湖水样中分离得到一株紫色非硫光合细菌,命名为JH1-6.对该菌株进行了形态学观察、生理生化鉴定、活细胞吸收光谱以及16S rDNA序列分析.16S rDNA序列分析结果表明该菌株与沼泽红假单胞菌的16S rDNA序列同源性高达99%,结合形态特征和生理生化特性以及活细胞吸收光谱特征等,确定菌株JH1- 6在分类地位上属于沼泽红假单胞菌(Rhodopseudomonas palustris).     

丁香假单胞菌冰核基因inaQ变速箱启动子结构与活性分析

序列比对与结构预测显示丁香假单胞菌(Pseudomonas syringae)野生型菌株MB03的冰核基因inaQ启动子为一种在细菌中罕见的变速箱型启动子。通过克隆长度为522bp的inaQ基因启动子区(P522)并与绿色荧光蛋白基因gfp构建融合基因P522gfp后,在恶臭假单胞菌AB92019菌株中进行表达分析。结果表明,包含结构模块A-Box和B-Box的P522在该菌株中具有启动子活性,且在寡营养条件和较低温度下具有更高的活性,是一种可调控启动子。     

偶氮染料脱色菌株AZR偶氮还原酶基因的克隆及其序列分析

利用16S rRNA鉴定偶氮染料脱色菌株AZR属于葡萄球菌属(Staphylococcus)中的科氏葡萄球菌(Staphylococcus cohnii), 根据GeneBank上登录的葡萄球菌属的3个偶氮还原酶基因序列设计扩增引物, 从菌株AZR的基因组中扩增出偶氮还原酶基因, 其大小为567 bp, 编码188个氨基酸。GenBank搜索表明其为新基因, 递交GenBank数据库, 获得登录号为EU849488。通过互联网数据库及生物信息学分析工具进行初步分析表明, 该基因编码的蛋白属于黄素蛋白家族,     

Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737

A gene that encodes a protein with azoreductase activity was obtained by PCR amplification from Rhodobacter sphaeroides AS1.1737. The enzyme, with a molecular weight of 18.7 kD, was heterologously expressed in Escherichia coli and its azoreductase activity was characterized. Furthermore, the reduction mechanism of azo dyes catalyzed by the azoreductase was studied in detail. The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.     

Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.     

Molecular cloning and characterization of the gene coding for azoreductase from Bacillus sp. OY1-2 isolated from soil

Azo dyes are regarded as pollutants because they are not readily reduced under aerobic conditions. Bacillus sp. OY1-2 transforms azo dyes into colorless compounds, and this reduction is mediated by a reductase activity for the azo group in the presence of NADPH. A 1.2-kbp EcoRI fragment containing the gene that encodes azoreductase was cloned by screening the genomic library of Bacillus sp. OY1-2 with digoxigenin-labeled probe designed from the N-terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted from the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Southern blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the original Bacillus sp. OY1-2. In addition, the expressed azoreductase purified from the recombinant E. coli lysate by Red-Sepharose affinity chromatography showed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encoding the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its expression in E. coli.     

Molecular cloning and characterization of the gene coding for the aerobic azoreductase from Xenophilus azovorans KF46F

The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4'-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated.     

微生物对偶氮染料的脱色及其基因工程研究进展

偶氮染料广泛应用在纺织印染、造纸印刷等行业中。染料废水的排放将会导致严重的环境污染,使用微生物处理染料废水是解决此问题的有效方法。该文概述了微生物对偶氮染料的脱色的研究,包括细菌对偶氮染料的脱色,真菌对偶氮染料的脱色,脱色产生的芳香胺并进一步被降解,以及基因工程技术在微生物对偶氮染料脱色的研究进展。