奇古菌亚硝酸盐还原酶基因相似基因的多样性

摘要:【目的】氨氧化古菌(Ammonia-oxidizing archaea,AOA)是奇古菌门中的唯一类群,广泛分布于各个生态系统中,对氮素生物地球化学循环起着重要作用。亚硝酸还原酶是反硝化作用的关键酶,目前关于AOA反硝化作用的研究较少,对AOA 亚硝酸盐还原酶基因多样性的研究有利于揭示AOA在反硝化中的作用。【方法】本研究以水体、沉积物和土壤为研究对象,构建了奇古菌样的nirK基因克隆文库,研究了这些环境中nirK相似基因的多样性。【结果】对奇古菌样的nirK基因文库及其序列分析表明:湖水及其沉积物的 nirK基因克隆文库得到10个OTUs,菜田土壤和水样则有8个OTUs;系统发育进化树表明这些nirK氨基酸序列和Candidatus Nitrosopumilus koreensis AR1,Nitrosopumilus maritimus SCM1最为相似,但相似度较低(53%－68%)。克隆文库多样性指数分析表明:所有样品都存在不同类型的nirK基因,水体样品nirK基因类型的多样性和均匀度高于土壤样品,菜田土壤的nirK基因类型多样性最高,分布最均匀。【结论】本研究表明土壤和淡水环境中奇古菌门nirK基因也具有较高的多样性,并且这些基因型与海洋样品差异非常大,这些基因编码的亚硝酸盐还原酶可能对这些环境中的反硝化作用有重要意义。     

Cd1-型亚硝酸盐还原酶脱氮工程菌的构建与表达

目的用基因工程技术将铜绿假单胞菌PAO1中nirS基因定向克隆至表达载体pQE-30上,使nirS基因得到高效表达。方法根据GenBank公布的nirS碱基序列和表达载体pQE-30的多克隆位点设计引物,以铜绿假单胞菌PAO1的基因组DNA为模板,应用PCR技术扩增目的片段nirS;之后经过BamH I和HindⅢ双酶切,定向克隆到pQE-30上,化学转化DH5α,构建含有重组质粒的转化子pQE30-nirS-DH5α;经酶切和测序鉴定,扩增产物的碱基序列与GenBank公布的序列完全吻合,再将重组质粒pQE30-nirS转化表达菌株SG13009构建脱氮基因工程菌pQE30-nirS-SG-13009（PNS）。最后用SDS-PAGE（IPTG浓度：0．05mmol／L;诱导温度：25℃;诱导时间：2～3h）和His-tag in-gel Stain鉴定cd1-型亚硝酸盐还原酶的分子量与特异性。结果构建了高效表达cd1-型亚硝酸盐还原酶的脱氮基因工程菌PNS。结论Cd1-型亚硝酸盐还原酶能够在脱氮基因工程菌PNS中得到正确和高效的表达。     

沼泽红假单胞菌偶氮还原酶相关基因的克隆及其生物信息学分析

脱色实验证明沼泽红假单胞菌(Rhdopsedomonas palustris)对偶氮染料有较强的降解能力,作者通过Gen Bank搜索,对所获得的所有偶氨还原酶基因在NCBI进行比对并设计引物,从沼泽红假单胞菌质粒中扩增获得了一条含471bp完整开放阅读框架的序列 GenBank搜索表明该基因为未登录的新基因。通过互联同数据库及生物信息学分析工具进行初步分析表明：该基因编码的蛋白是一种等电点为9.65的不稳定蛋白,定位于细胞质;由α螺旋、延伸带和随机卷曲三种形式组成,具有蛋白激酶C磷酸化等多个位点,并具有一个强跨膜疏水区。     

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     

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).     

过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化

【目的】为了体现并突出亚硝酸盐还原酶在污水脱氮以及短程硝化中的重要性,对过表达亚硝酸盐还原酶的大肠杆菌进行了污水脱氮的研究。【方法】通过转化带有亚硝酸盐还原酶基因的重组质粒,将亚硝酸盐还原酶在大肠杆菌中过表达,通过分析重组大肠杆菌的产物研究了该酶的表达及还原亚硝酸盐的情况,通过将该重组菌与已报道的硝化-反硝化细菌或生活污水进行混合培养,研究重组菌用于辅助氨氮去除的短程硝化能力。【结果】重组大肠杆菌能正确表达亚硝酸盐还原酶,OD600=2.0的菌悬液在2 h内还原约1 mmol/L的亚硝酸盐,并产生几乎等量的一氧化氮;重组大肠杆菌与Acinetobacter sp.YF14菌株等比例混合时,12 h能够提高氨氮脱氮效率约(36.0±7.4)%,且在4 h时,最大亚硝酸盐的积累量减少37%;重组大肠杆菌(OD600=1.0)12 h内能够提高污水厂活性污泥的脱氮效率约(31.0±5.7)%,且未检测到亚硝酸盐和硝酸盐的积累;溶氧水平对于亚硝酸盐还原酶重组菌辅助脱氮具有明显的影响,中等溶氧量[(6.4?0.7)mg/L]时脱氮效果最好。【结论】过表达亚硝酸盐还原酶的大肠杆菌可以提高污水脱氮的短程硝化能力。     

Pseudomonas alcaliphila AD-28的脱氮性能及其关键酶活性

【背景】异养硝化-好氧反硝化菌由于能够同时实现硝化反硝化作用而备受关注,但由于菌的种类不同,其脱氮途径不尽相同,研究菌株脱氮关键酶的种类及其活性可以推测菌株的脱氮途径,从而为菌株在生产上的应用提供技术支撑。【目的】研究Pseudomonas alcaliphila AD-28的脱氮性能及其关键酶的活性,为菌株脱氮分子机理研究奠定基础。【方法】以柠檬酸钠为碳源,以硫酸铵、亚硝酸钠、硝酸钾为氮源,研究菌株AD-28的脱氮性能并检测其关键酶氨单加氧酶(AMO)、羟胺氧化还原酶(HAO)、亚硝酸盐还原酶(NIR)、硝酸盐还原酶(NAR)的酶活性。【结果】菌株AD-28培养24h的菌密度(OD600)可达1.971,对初始浓度为18.85mg/L的氨氮、26.13mg/L的硝酸盐氮、19.47mg/L的亚硝酸盐氮、66.11 mg/L的总氮去除率均达到96%以上;关键酶AMO、HAO、NIR和NAR的比活力分别为0.028、0.003、0.011、0.027 U/mg。【结论】Pseudomonas alcaliphila AD-28能同时进行异养硝化-好养反硝化作用,该菌在AMO作用下将NH4+-N氧化为羟胺,然后由HAO氧化为NO2--N,NO2--N和NO3--N在NIR、NAR等酶的催化作用下脱氮。     

养虾池好氧反硝化细菌新菌株的分离鉴定及特征

利用间歇曝气选择性富集并对所获菌株的好氧反硝化活性进行检测。筛选到一株亚硝酸盐去除活性较高的好氧反硝化细菌。在溶解氧（D0）为3.80-5．21mg／L的培养条件下。该菌株10h内将亚硝态氮由26．18mg／L降至0;在盐度为0—25之间20h内均可达到同样的去除效果。通过形态学特征、生理生化反应及部分长度16SrDNA序列分析对筛选菌株进行鉴定,初步判定它为嗜麦芽寡养单胞菌Stertotrophomonas maltophilia。亚硝酸盐还原酶基因分析结果表明。该菌株只含亚硝酸盐还原酶nitS基因,其序列与Alcaligenes faecalis A15（后来被重新鉴定为Pseudomonas stutzeri）的nirS基因序列相似。     

耐冷亚硝酸盐型反硝化菌Pseudomonas tolaasii Y-11的鉴定及其脱氮特性

摘要:【目的】反硝化细菌在生物脱氮中具有重要作用,而耐冷亚硝酸盐型反硝化细菌研究较少,本文从长期淹水的冬水田泥土分离获得一株耐冷高效去除亚硝酸盐氮和总氮的好氧反硝化细菌Y-11,明确其分类地位以及除氮特性,以期为后续利用该菌在初冬到春末处理亚硝酸盐水体污染奠定基础。【方法】通过形态学特征、特异性磷脂脂肪酸以及16S rRNA基因测序分析对该菌株进行鉴定;在好氧条件下以亚硝酸钠为唯一氮源,分别研究不同初始温度、转速、pH、碳源、接种量以及亚硝酸盐氮浓度对该菌去除亚硝酸盐氮和总氮的影响,确定最适降解条件。【结果】分离得到的菌株Y-11,经鉴定归于托拉斯假单胞菌(Pseudomonas tolaasii);在国内外尚无该种菌具有反硝化作用的报道,是对亚硝酸盐型反硝化细菌的进一步补充。Y-11菌株的最适脱氮条件为15 ℃,200 r/min,pH7.0,100 mL反硝化培养基中最适接种量为1.5×108 CFU,最佳碳源为乙酸 钠,亚硝酸盐氮为10 mg/L;以乙酸钠为电子供体,15 ℃、初始pH为7.2、150 r/min 振荡培养,48 h对亚硝酸盐氮和总氮的去除率分别为100%和61.28%。【结论】Y-11是一株具有较高反硝化能力的托拉斯假单胞菌,能高效地去除亚硝酸盐氮和总氮,其最适温度是15 ℃左右,是一株耐冷反硝化细菌。     

Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).     

Electron transfer proteins of the purple phototrophic bacterium, Rhodopseudomonas rutila.

The soluble electron transfer protein content of Rhodopseudomonas rutila was found to consist of two basic cytochromes and a (4Fe-4S) ferredoxin. Cytochrome c' was easily identified by its characteristic high spin absorption spectra. The native molecular weight is 29,000 and the subunit is 14,000. Cytochrome c-550 has low spin absorption spectra and a high redox potential (376 mV) typical of cytochromes c2. The molecular weight is about 14,000. The ferredoxin is apparently a dimer (43,000) of approximately 18,000 Da subunits. There are 1.3 to 1.5 iron-sulfur clusters per monomer of 18- to 21-kDa protein. The N-terminal amino acid sequence is like the (7Fe-8S) ferredoxins of Rhodobacter capsulatus and Azotobacter vinelandii. Remarkably, there are only 2 or 3 out of 25 amino acid substitutions. Difference absorption spectra of Rps. rutila membranes indicate that there is not tetraheme reaction center cytochrome c, such as is characteristic of Rps. viridis. However, there are a high potential cytochrome c and a low potential cytochrome b in the membrane, which are suggestive of a cytochrome bc1 complex. Rps. rutila is most similar to Rps. palustris in microbiological properties, yet it does not have the cytochromes c-556, c-554, and c-551 in addition to c2 and c', which are characteristic of Rps. palustris. Furthermore, the Rps. rutila cytochrome c' is dimeric, whereas the same protein from Rps. palustris is the only one known to be monomeric. The cytochrome pattern is more like that of Rhodospirillum rubrum and Rb. capsulatus, which are apparently only able to make cytochromes c2 and c'.     

Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.     

Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

Purification and molecular properties of nitrite reductase from Anabaena sp. 7119

Ferredoxin-nitrite reductase (EC 1.7.7.1.) from the cyanobacteria Anabaena sp. 7119 has been purified 763-fold with a specific activity of 21.5 units/nig protein (0.358 μkatals/mg). The enzyme has a molecular mass of 52,000 daltons with a Stokes radius of 3.09 nm and a sedimentation coefficient of 4.07 S. The cellular level of nitrite reductase activity gradually increases in response to the addition of increasing amounts of iron to the culture medium.
When partially purified nitrite reductase preparations are subjected to sucrose-density-gradient centrifugation there is a dose correspondence between nitrite reductase activity and absorbance at 400 nm. This suggests the association of a heme chromophore with the enzyme. Furthermore, the presence of an iron-sulfur center is suggested by a close association of acid-labile sulfide with nitrite reductase activity. Carbon monoxide inhibits nitrite reductase activity. The nature and kinetics of this reaction are comparable to other siroheme-containing nitrite reductases.     

M?ssbauer and electron nuclear double resonance study of oxidized bidirectional hydrogenase from Clostridium pasteurianum W5

The bidirectional hydrogenase from Clostridium pasteurianum W5 is an iron-sulfur protein containing approximately 12 Fe atoms and 12 labile sulfides. We have studied oxidized samples of the enzyme with M?ssbauer and electron nuclear double resonance (ENDOR) spectroscopy to elucidate the nature of the center that gives rise to the EPR signal with principal g-values at 2.10, 2.04, and 2.01. The g = 2.10 center exhibits two well-resolved 57Fe ENDOR resonances. One is isotropic with A1 = 9.5 MHz; the other is nearly isotropic with A2 = 17 MHz. These magnetic hyperfine coupling constants are substantially (approximately 50%) smaller than those observed for [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. The M?ssbauer and ENDOR data, taken together, suggest that the g = 2.10 center contains at least two but not more than four iron atoms. Comparison of our data with recent results reported for Escherichia coli sulfite reductase and the ferricyanide-treated [4Fe-4S] cluster from Azotobacter vinelandii ferredoxin I suggests that the g = 2.10 center may possibly be formed, by oxidation, from a structure with a [4Fe-4S] core. The M?ssbauer spectra give evidence that at least 8 of the 12 Fe atoms of oxidized hydrogenase are organized in two ferredoxin-type [4Fe-4S] clusters, supporting conclusions derived previously from EPR studies of the reduced enzyme.