Transmembrane structure and function of dctPQM encoding C4－dicarboxylate transport proteins from nitrogen－fixing P. stutzeri A1501

C4-dicarboxylate transport proteins of diazotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ,and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene encoding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii.Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively.The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobilization plasmid pSZ2L The resultant plasmid was named pSZY6.By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501,randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was constructed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20,10 and 5mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A150L This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.     

高原地区耐冷菌的分离鉴定

经过10℃下7天驯化,活性污泥系统对生活污水的COD去除率达到90%以上,污泥驯化完成,此时污泥中的优势菌种为耐冷菌;通过反复分离得到32株在10℃下生长良好的优势菌株;再通过初筛阶段测定平均OD值,复筛阶段测定模拟生活污水COD去除率,筛选出低温情况下生长良好且COD去除效率高的五株菌株;通过菌落特征、形态特征和生理生化特性,鉴定得出高原地区的优势低温耐冷菌分别为:革兰氏阴性好氧杆菌—假单胞菌属、革兰氏阳性菌好氧杆菌—嗜冷杆菌属、革兰氏阳性芽胞杆菌和球菌—芽胞杆菌属、革兰氏阴性好氧杆菌—黄杆菌属、革兰氏阴性杆菌—产碱杆菌属。     

好氧反硝化菌株X31的反硝化特性

对好氧反硝化菌株X31在好氧条件下的反硝化特性进行了研究．结果表明,反硝化主要发生在菌体的对数生长期,氮气是反硝化过程的最终产物．在反硝化过程中,pH值呈逐渐上升趋势,而氧化还原电位(ORP)呈逐渐降低趋势．菌株X31能够以硝酸盐或亚硝酸盐和氧气为电子受体进行协同呼吸,并且亚硝酸盐呼吸要较硝酸盐呼吸更容易进行．硝酸盐呼吸和亚硝酸盐呼吸都具有较高的脱氮效率．和其他已报道的好氧反硝化茵相比,X31菌株有着更高的氧耐受浓度．当培养液中初始的氧化态氮质量浓度为150mg／L左右时,溶解氧值对X31菌株的反硝化效果没有显著的影响．     

微氧条件下厌氧颗粒污泥和消化污泥特性研究

用12 5mL血清瓶作为批量处理反应器,对厌氧颗粒污泥和消化污泥在厌氧和微氧条件下的COD去除率、污泥产率、产甲烷活性、抗冲击负荷能力等进行对比实验研究。实验结果表明:厌氧颗粒污泥和消化污泥均在微氧条件下表现出高COD去除率、低污泥产率、高产甲烷活性和强抗冲击负荷能力,且厌氧颗粒污泥在COD去除率、污泥产率、产甲烷活性和抗冲击负荷等方面更具有优势;对于0 5 gCOD/LR·d的有机负荷,反应器内最佳加氧量为10mL(10 %添加的COD)。     

BluB cannibalizes flavin to form the lower ligand of vitamin B12

Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products, and the only vitamin synthesized exclusively by microorganisms. The biosynthesis of the lower ligand of vitamin B(12), 5,6-dimethylbenzimidazole (DMB), is poorly understood. Recently, we discovered that a Sinorhizobium meliloti gene, bluB, is necessary for DMB biosynthesis. Here we show that BluB triggers the unprecedented fragmentation and contraction of the bound flavin mononucleotide cofactor and cleavage of the ribityl tail to form DMB and D-erythrose 4-phosphate. Our structural analysis shows that BluB resembles an NAD(P)H-flavin oxidoreductase, except that its unusually tight binding pocket accommodates flavin mononucleotide but not NAD(P)H. We characterize crystallographically an early intermediate along the reaction coordinate, revealing molecular oxygen poised over reduced flavin. Thus, BluB isolates and directs reduced flavin to activate molecular oxygen for its own cannibalization. This investigation of the biosynthesis of DMB provides clarification of an aspect of vitamin B12 that was otherwise incomplete, and may contribute to a better understanding of vitamin B12-related disease.     

Methanotrophy below pH 1 by a new Verrucomicrobia species

Mud volcanoes, mudpots and fumaroles are remarkable geological features characterized by the emission of gas, water and/or semi-liquid mud matrices with significant methane fluxes to the atmosphere (10(-1) to 10(3) t y(-1)). Environmental conditions in these areas vary from ambient temperature and neutral pH to high temperatures and low pH. Although there are strong indications for biological methane consumption in mud volcanoes, no methanotrophic bacteria are known that would thrive in the hostile conditions of fumaroles (temperatures up to 70 degrees C and pH down to 1.8). The first step in aerobic methane oxidation is performed by a soluble or membrane-bound methane mono-oxygenase. Here we report that pmoA (encoding the beta-subunit of membrane-bound methane mono-oxygenase) clone libraries, made by using DNA extracted from the Solfatara volcano mudpot and surrounding bare soil near the fumaroles, showed clusters of novel and distant pmoA genes. After methanotrophic enrichment at 50 degrees C and pH 2.0 the most distant cluster, sharing less than 50% identity with any other described pmoA gene, was represented in the culture. Finally we isolated an acidiphilic methanotrophic bacterium Acidimethylosilex fumarolicum SolV belonging to the Planctomycetes/Verrucomicrobia/Chlamydiae superphylum, 'outside' the subphyla of the Alpha- and Gammaproteobacteria containing the established methanotrophs. This bacterium grows under oxygen limitation on methane as the sole source of energy, down to pH 0.8--far below the pH optimum of any previously described methanotroph. A. fumarolicum SolV has three different pmoA genes, with two that are very similar to sequences retrieved from the mudpot. Highly homologous environmental 16S rRNA gene sequences from Yellowstone Park show that this new type of methanotrophic bacteria may be a common inhabitant of extreme environments. This is the first time that a representative of the widely distributed Verrucomicrobia phylum, of which most members remain uncultivated, is coupled to a geochemically relevant reaction.     

Submerged culture of Magne-tospirillum gryphiswaldense under N2-fixing condition and regulation of activity of nitrogen fixation

A submerged culture technique for Magnetospirillum gryphiswaldense under the nitrogen-fixing condition (microaerobic and N-limited) was set up. In N-limited medium with Na-lactate as a sole carbon source, the optical density (A600 nm) and activity of nitrogen fixation of cells were 1.3 and 217 nmol of ethylene produced per hour per A600nm respectively within 21 h by three times of feeds. The pH and temperature were controlled at 7.2 and 30℃ respectively, and the oxygen concentration was controlled by sparging with N2 containing 0.4%-0.8% of O2. The activity of nitrogen fixation of cells was obviously inhibited by oxygen and ammonium. It indicated that the posttranslational regulation of nitrogenase existed in M. gryphiswaldense.     

碳源和盐度对好氧反硝化细菌脱氮特性的影响

采用16S rDNA序列分析对菌株LZX301进行了初步鉴定,在150 r/m摇瓶好氧培养,探讨了碳源及盐度对菌株好氧反硝化特性的影响. 结果表明,该菌株16S rDNA序列与Pseudomonas stutzeri ATTC 17594（AY905607.1）等3株施氏假单胞菌序列相似度为99%,系统发育树分析显示菌株LZX301与P.stutzeri 的关系比同属的P.aeruginosa 和P.putida更近,因此初步确定菌株LZX301为Pseudomonas stutzeri. 培养液初始含7 mg/L亚硝酸盐和28 mg/L硝酸盐、C/N比为10:1条件下,以葡萄糖、乙酸钠和蔗糖为碳源时无机氮去除率分别为79.1%、67.9%和38.8%,氨氮积累量分别为1.978、1.224、0.727 mg/L. 以葡萄糖为唯一碳源时,在5、15、25等3个盐度下无机氮总去除率分别为73.2%、85.8%和78.7%,其中硝酸盐去除率分别为89.8%、86.1%和76.5%,亚硝酸盐去除率分别为36.2%、94.7%和96.4%,氨氮质量浓度分别为2.117、0.691、0.595 mg/L. 研究结果表明菌株LZX301在盐度5~25 范围内具有较强的好氧反硝化能力,以葡萄糖为碳源脱氮效果最好,对该菌株的应用具有指导意义.     

偶氮染料及其还原产物的微生物降解

从印染厂排出水中分离出了两种细菌。其中的邻单胞菌Plesiomonas sp.X_(13)菌株对五种偶氮染料均有脱色作用。脱色率达75％以上;脱色产物中有芳香胺,但X_(13)菌株对苯胺在振荡培养或深层静止培养条件下均无降解能力。另一种,无色细菌Achromobacter sp.A_8菌株对脱色过程中产生的芳香胺则具有降解作用,其降解能力也因培养条件而异。在振荡培养条件下,其降解芳香胺的速率远比静止培养时为快。     

阿特拉津降解菌Enterobacter sp.的基因差异分析

筛选并分析生物降解菌Enterobacter sp.在阿特拉津作用下的差异基因。以Enterobacter sp.BIDMC 29基因组为参考,利用高通量测序技术,对阿特拉津降解菌株和对照菌株进行转录组测序分析。结果表明,共有368个基因表现出显著差异,其中上调基因231个,下调基因137个。在差异基因GO富集分析的前30个条目中,尿嘧啶分解代谢、氮利用、硝酸盐同化、硫化氢生物合成、裂解酶活性和过氧化物酶活性等相关基因表现富集。Pathway分析显示,差异基因涉及70条代谢途径,与氮代谢、氨基酸合成和代谢、ABC转运蛋白、丙酮酸代谢等过程有关。菌株通过调节基因表达来提高对阿特拉津的降解能力,为进一步解析阿特拉津的代谢机制奠定基础。     

高盐胁迫对坛紫菜(Pyropia haitanensis)叶状体光合作用的影响

坛紫菜(Pyropia haitanensis)具有极强的耐高盐胁迫能力,但其耐盐机理尚不明确。检测了不同高盐（100、110）胁迫处理不同时间（0,4,8,24 h）时坛紫菜Z-61藻体的最大光化学效率（Fv/Fm）和净光合作用,并选取高盐胁迫4 h后的藻体提取RNA,采用第二代高通量测序技术分析正常盐度（对照组,30）与高盐胁迫处理的坛紫菜叶状体转录组数据,探究坛紫菜响应高盐胁迫过程中的光合生理机制。结果发现:盐度100对坛紫菜藻体Fv/Fm无显著影响;而盐度110下,藻体Fv/Fm逐渐下降至0,但转移至对照海水后其仍能恢复到初始水平,可将110称之为“亚致死”（sub-lethal）盐度;当盐度增加至120时,藻体Fv/Fm急剧下降至0,且转移到对照海水中不能恢复,即120是致死的盐度。转录组数据显示,高盐胁迫下的转录本与对照组有很大差异,光合作用相关基因在高盐胁迫下显著上调表达,包括多条碳酸酐酶基因和天线蛋白基因,并且在盐度100胁迫下其上调趋势更为明显。以上结果说明:坛紫菜在耐受盐度100条件下可以通过积极提高光合作用相关基因的表达,维持光合活性,为藻体生长提供物质和能量;而在亚致死盐度110条件下,坛紫菜光合活性逐渐下降,光合基因表达受到抑制,活性氧的产生减少,避免藻体细胞遭受过氧化损伤。这说明坛紫菜可以通过积极响应调节光合系统来应答高盐胁迫。     

NADH氧化酶的研究进展

NADH氧化酶是一类催化NADH氧化为NAD+并消耗氧气的氧化还原酶,因其能够再生NAD+而成为人工调控微生物代谢流向的重要调控酶.文中综述了NADH氧化酶的分类、理化性质、反应机制,并分析NADH氧化酶清除细胞内氧毒性、介入细胞代谢过程,以及调节细胞生理和代谢等重要的生理功能.     

Red/ET重组AHBA生物合成基因簇打靶载体的构建与鉴定

为确定3-氨基-5-羟基-苯甲酸(AHBA)生物合成基因簇在链霉菌中与次生代谢产物的关系,运用PCR技术,从33株AHBA合酶基因阳性菌株扩增与AHBA生物合成基因簇中编码AHBA合酶(A)、氧化还原酶(O)、磷酸化酶(P)基因,获得24株AOP基因阳性菌株.根据靶基因A基因下游和P基因上游同源序列设计50 bp引物,中间插入卡那霉素抗性基因的DNA片段,进行PCR,获得外源DNA片段.经过电转化,将外源DNA片段和pKC1139-AOP重组质粒共转入含重组酶质粒大肠杆菌HS996/ pSC101-BAD-gba-(Tet).在Red重组酶的作用下,外源DNA片段与重组质粒pKC1139-AOP上的AHBA基因簇的同源区域重组,构建了AHBA基因簇打靶载体.研究显示了Red/ET重组工作效率高、操作简单、精确的优点,可大大缩短构建打靶载体的时间.     

有氧运动对青少年骨骼肌细胞葡萄糖氧化代谢的影响

研究有氧运动对青少年骨骼肌細胞葡萄糖代谢的影响,因为青少年骨骼肌細胞葡萄糖氧化代谢规律和青年小鼠是相同的。为了便于采集骨骼肌细胞將青年小鼠作为研究対象,选用3月龄健康雄性Sprague-Dawley小鼠30只,随机将其划分成安静组、中等强度有氧运动组和过度有氧运动组,每组10只洧氧运动方式为游泳。经7周有氧运动后,中等强度有氧运动组体重、体重变化和体重变化率均显著低于对照组(P0.05);过度有氧运动组各体重指标均低于对照组,差异具有非常显著性(P0.01)。经7周有氧运动后,中等强度有氧运动组血糖显著低于对照组(P0.05),血清胰岛素显著高于对照组(P0.05)过度有氧运动组血糖低于对照组血清胰岛素高于对照组差异均具有非常显著性(P0.01)。经7周有氧运动后,中等强度有氧运动组和过度有氧运动组GT4含量均显著高于对照组(P0.05)。经7周有氧运动后,中等强度有氧运动组和过度有氧运动组MEF2mRNA表达均高于对照组差异具有非常显著性(P0.01)。说明适宜有氧运动能够促进青少年骨骼肌細胞葡萄糖代谢,但过度有氧运动会使机体在一定程度上受到损伤。     

Cloning and expression profiles of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.)

WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identification, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame (ORF) encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except TaWRKY10 which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids.     

Screening and denitrification characteristics of a slight halophilic heterotrophic nitrobacteria

A slight halophilic heterotrophic nitrobacteria named gs1 was separated from the matured activated sludge. According to the morphological observation,physiological biochemical tests and sequence analysis of the 16S rDNA,strain gs1 was identified to be as Pseudomonas sp. Sodium acetate and ammonium chloride were used as carbon and nitrogen sources,respectively,to investi-gate the characteristics of the bacterium. When cultured for 24 h under aerobic conditions,with the removal rates of the NH4+-N and COD being 82.2% and 74.73%,respectively,strain gs1 will have a nitrification function of producing NO2--N. When cultured for 24 h under aerobic conditions in nitrite medium,the removal rate of the NO2--N became 100%,and when cultured for 24 h under aerobic conditions in nitrate medium,the removal rate of the NO3--N became 97%. The result shows that this strain functions for either nitrification or denitrification,i.e.,it can complete the full process of biological deoxidation.     

异养硝化菌Alcaligenes faecalis strain NR 的 硝 化 性 能 及 其 酶 活 性

从膜生物反应器的活性污泥中分离出一株异养硝化细菌Alcaligenes faecalis strain NR,采用柠檬酸三钠为碳源、氯化铵为氮源研究其硝化性能,通过超声波破碎法对调控其硝化过程的2个关键酶--氨单加氧酶（AMO）和羟胺氧化酶（HAO）进行粗提,考察影响其提取率的主要因素（功率、工作与间歇时间、菌液浓度和总工作时间）,并采用正交实验进行优化.结果表明：菌株NR具有产生亚硝酸盐和硝酸盐的能力;在30 °C和120 r/min的好氧条件下,当NH+4 N浓度为20、40和60 mg/L时,菌株NR在24 h内对NH+4 N的去除率分别为94.8%、93.5%和94.5%;粗酶提取的最佳工作条件为菌液光密度1.876,总工作时间600 s,工作和间歇时间4、6 s,功率300 W;在好氧条件下,测得AMO和HAO的酶比活力分别为0.011和0.016 U/mg protein.     

好氧颗粒污泥膜生物反应器处理畜禽废水

采用好氧颗粒污泥膜生物反应器处理畜禽废水,分别对COD、NH4 -N、NO2--N、NO3--N的去除效果和对膜通量的影响进行了研究。结果表明:在水力停留时间(HRT)为8h,进水COD浓度为600mg/L,NH4 -N浓度为40mg/L的条件下,出水COD、NH4 -N的浓度分别为46.6和4.8mg/L。NO2--N和NO3--N的去除率也可达90%以上。并且好氧颗粒污泥的加入减缓了膜的污染。