螺旋藻氢酶的制备及其特性的研究

氢是理想的不污染环境的能源,在世界能源日趋紧张的情况下,氢代谢这一研究课题受到了许多学者的重视。藻类中一些绿藻可以放氢已有不少研究;关于蓝藻的放氢,特别是不具有异形胞蓝藻的放氢研究目前报道得很少。螺旋藻是一种不能固氮的丝状蓝藻,但具有氢酶。顾天青等已进行了螺旋藻整体细胞放氢特性的研究,证明了氢酶与硝酸还原酶之间存在还原能力的竞争。在此基础上,我们提取了螺旋藻的氢酶,在分子水平上对它的放氢特性进行了研究,并以Na_2S_2O_4还原的甲基紫精作为催化放氢的试验。     

潮间带污泥中厌氧发酵产氢混合菌群组成与 Fe-氢酶基因多样性分析

以采自天津海水浴场潮间带的污泥为研究材料,将污泥热休克处理后富集混合菌群进行厌氧发酵产氢,测定混合菌群在发酵过程中累积产氢量和相关产氢指标(吸光度、酸碱度和氧化还原电位)的变化。结果表明,热休克处理后富集的产氢混合菌群产氢量为0.41mol H2/mol葡萄糖。产氢结束后,利用变性梯度凝胶电泳(DGGE)分析混合菌群组成和Fe-氢酶基因的多样性。混合菌群基因组DNA的16S rRNAV3区扩增产物经过DGGE电泳分离,结果得到6条丰度比较高的条带,测序结果表明优势菌为Clostridium sp.。利用梭菌氢酶基因保守性引物克隆混合产氢菌群中梭菌Fe-氢酶基因,扩增产物经DGGE电泳分离,电泳结果表明混合菌群中Fe-氢酶基因分为3个分类单元,NCBI-BLAST比对结果与Clostridium roseum和Clostridium perfringens的Fe-氢酶基因相似度分别为79%和98%。分析产氢混合菌群组成和Fe-氢酶基因多样性,可以为扩大产氢微生物种质资源奠定基础。     

红树林混合发酵产氢菌群中梭菌属Fe-氢酶基因(hydA)的克隆及序列分析

采用间歇产气试验方法对红树林淤泥中的混合菌群进行产氢菌群富集,并对混合产氢菌群发酵产氢过程中发酵液的pH值和氧化还原电位(ORP)的变化进行分析.此外,在产氢速率最高时段,发酵液中菌群经过常规的基因组提取,分别采用通用的梭菌属Fe-氢酶基因和16S rRNA基因引物进行PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分析.结果发现产氢菌群中至少有6种,而Fe-氢酶基因只含有一种.将Fe-氢酶基因的扩增片段切胶纯化之后,经PCR重新扩增、纯化,克隆测序.NCBI序列比对结果表明,该片段序列与产气荚膜梭菌Fe-氢酶基因的序列相似性达99%.根据已知产气英膜梭菌Fe-氢酶基因序列设计引物,经两轮PCR扩增获得Fe-氢酶基因全序列.混合菌群中Fe-氢酶基因GenBank数据库的登录号为EU590683.此外,采用Bioedit和Mega2软件构建了Fe-氢酶的NJ系统进化树,结果表明该Fe-氢酶基因与产气荚膜梭菌(Clostridium perfringens)聚为一类.推测的氨基酸序列与产气英膜梭菌的相应序列相似性达97%-99%.PfamHMM结构域查找结果发现,此氢酶含有五个结构域,分别为1个2Fe-2S铁硫簇结合区域、2个4Fe-4S结合区域、Fe-氢酶大亚基和Fe-氢酶小亚基.     

中国南海一株固氮类芽孢杆菌的筛选和分离鉴定

为了解中国南海海洋自生固氮菌的种类,作者对采集的南海海底淤泥样品进行了固氮微生物的分离、筛选及鉴定。经过土样沸水加热处理,无氮培养基平板初筛后,对分离获得的细菌固氮酶结构基因nif H进行扩增,并对其固氮酶活性进行检测,最终获得一株能够产芽孢的固氮细菌。对该菌株进行生理生化性状测定、16S r DNA序列分析(Gen Bank登录号KJ627376),并基于nif H、16S r DNA系统进化树分析,确定该菌为一株固氮类芽孢菌(Paenibacillus sp.)NH-1。本研究表明固氮类芽孢杆菌在海洋中确有分布,海洋自生固氮菌的多样性远远超出人们之前的认识。     

微藻制氢的研究进展

化石能源的不可再生性和使用过程中造成的环境污染使开发新能源变得非常迫切。氢是理想的能源,因此,利用微藻制氢具有诱人前景。本文综述了微藻光合制氢技术的研究历史、固氮酶和可逆产氢酶的产氢机制及研究进展;介绍了基于微藻的硫缺乏生理调控而发展起来的一步法与两步法光解制氢的方式;指出了利用微藻可逆产氢酶两步法间接光水解制氢最具开发潜力;分析了实现微藻光合制氢的限制因子和存在的问题;归纳了今后的主要研究方向。     

成团泛菌依赖型磷酸甘油酸变位酶基因的克隆、序列分析及产氢中的差异表达

为研究高效产氢菌株成团泛菌(Pantoea agglomerans)BH-18中依赖型磷酸甘油酸变位酶(cofactor-dependent phosphoglycerate mutase, dPGM)与产氢之间的关系,本研究根据GenBank上已登录的肠杆菌中编码 dPGM的基因序列设计一对引物,从细菌基因组 DNA中克隆得到编码 dPGM基因的完整开放阅读框,其长度为753 bp,编码250 aa。采用BLAST对其与NCBI GenBank中的核苷酸序列进行比较分析,结果表明该基因保守性相对较高,与肠杆菌科众多菌株中的 dPGM 基因相似性达100%。采用Bioedit和Mega4软件构建NJ系统进化树,结果表明成团泛菌BH-18的dPGM氨基酸序列与Enterobacter asburiae的dPGM聚为一类,而与成团泛菌属中其他菌株的该蛋白关系较远, dPGM的氨基酸序列在属内不保守。最后,根据已获得的基因序列设计引物,采用RT-PCR的方法分析了成团泛菌BH-18产氢过程中dPGM基因的转录情况,结果表明dPGM基因的转录与产氢呈正相关,依赖型磷酸甘油酸变位酶是产氢过程中的关键酶。     

雨生红球藻八氢番茄红素脱氢酶pds基因上游序列的分离和分析

在某些蓝藻、绿藻和高等植物中,八氢番茄红素脱氢酶是β-胡萝卜素合成过程中的一个关键酶之一,应用巢式PCR方法从雨生红球藻中克隆了八氢番茄红素脱氢酶基因约1kb的5’上游序列。通过生物信息学方法进行序列分析,发现pds基因上游序列中包含了一些可能的顺式元件,如ABRE调控元件、C-repeat/DRE调控元件等。同时,构建了由pds-启动子控制报告基因的重组载体,并转化到雨生红球藻细胞中,进行了报告基因瞬间表达的检测。结果表明,克隆的pds启动子区域具有启动子活性,可以驱使报告基因进行瞬间表达。     

转rhG-CSF基因鱼腥藻的重组基因及蛋白检测

提取质粒进行PCR和酶切鉴定人粒细胞集落刺激因子(rhG-CSF)基因成功转入鱼腥藻7120中,并用酶联免疫方法测得转基因鱼腥藻7120-G-CSF中G-CSF蛋白在第21d表达量达到最大。对转基因蓝藻的生长曲线、叶绿素a含量测定结果表明,rhG-CSF基因转化蓝藻后,对蓝藻前中期的生长影响不大,但21d后生长速度下降,同时外源蛋白表达也达到最大值,这有利于转基因藻的大规模培养。     

海洋固氮蓝藻Calothrix sp.与Lyngbya sp.固氮生理的研究

研究盐度、昼夜变化、温度及阿特拉津(光合系统Ⅱ抑制剂)对2种海洋固氮蓝藻Calothrixsp.strain(代号为MCT1)和Lyngbyasp.strain(代号为MCT6)固氮活性的影响。结果表明,MCT1在盐度10—48范围内具有相对较高的固氮活性,在盐度为30时固氮活性最高,达到0.687 2μmolC2H2.(g.h)-1;而MCT6随盐度改变固氮活性变化幅度较大,盐度为24时具有最高固氮活性,其活性为0.876 8μmolC2H2.(g.h)-1。MCT1和MCT6固氮活性的昼夜变化明显不同,具有异型胞的海洋固氮蓝藻MCT1白天的固氮活性明显高于夜晚;而无异型胞的MCT6最高固氮活性发生在晚上,白天固氮活性较低。一定浓度的阿特拉津通过抑制光合作用阻断能量和还原剂的提供,使藻体在较短时间内丧失固氮能力。加入阿特拉津后2种藻体的固氮活性与对照组相比有明显的变化,实验第3天开始MCT1的所有经阿特拉津处理的样品固氮活性丧失;MCT6经(50—1 000)×10-6阿特拉津处理的样品从实验第3天开始固氮活性完全消失。     

海洋生物固氮速率与影响因素的研究进展

海洋生物固氮是指固氮生物利用固氮酶将氮气转化为生物可利用铵盐的海洋氮元素输入过程,和反硝化及厌氧氨氧化等氮流失途径一起制约着大洋氮收支平衡。而固氮速率的测定是研究海洋生物固氮的最直接方式。自发现海洋生物固氮作用以来,固氮速率的测定方法在不断更新改进,但总体来说仍存在较大不确定性。最近用¹⁵N₂同位素示踪法及其他相关数据综合得到全球海洋固氮量为196.1 Tg N∙a⁻¹,最高固氮速率发生在南太平洋热带地区。但分布受到多种因素的影响。其中,物理因素中的光照和温度是全球范围固氮速率分布的最佳预测因子,光照为固氮过程提供能量,温度通过影响固氮酶活性而发挥作用。在化学因素中,铁元素的缺乏成为固氮的重要限制因子。除此之外,还有生物因素,如浮游植物和异养固氮生物等,对固氮量的贡献影响较大。最近有研究对以往固氮作用区域和反硝化作用空间相互耦合的观点表示质疑,提出二者分布空间分离的新格局。研究多控制因素对固氮生物的耦合效应、明确不同物种对固氮总量的相对贡献以及进一步建立固氮速率的原位测定方法是未来海洋固氮作用研究的主要工作。     

Diel N2 fixation in an intertidal marine cyanobacterial mat community

Diurnal and diel rates of nitrogen fixation (acetylene reduction) were measured in an intertidal marine mat community consisting of Microcoleus chthonoplastes, Lyngbya aestuarii, and an underlying layer of anaerobic bacteria (probably photosynthetic, fermentative, and sulfate reducing bacteria). The community is located on Shackleford Banks, a barrier island off the coast of North Carolina. Rates of nitrogenase activity during the diurnal period were generally highest in the morning. Rates measured during the day, but incubated in the dark, at times equalled those of samples incubated continuously in the light. Fixation also occurred at night. Mat samples deprived of light prior to the acetylene reduction assay continued to reduce acetylene during the night and into the following day, although at lower rates than samples exposed to light. The persistence of dark nitrogenase activity serves as evidence that reducing power initially generated through photosynthesis can be stored and subsequently utilized through heterotrophic metabolism. Although cyanobacteria are responsible for light-mediated nitrogenase activity, non-photosynthetic bacterial contributions to dark-mediated nitrogenase activity cannot be ruled out.     

Intercellular localization of nitrogenase in a non-heterocystous cyanobacterium (cyanophyte), <Emphasis Type="Italic">Trichodesmium</Emphasis> sp. NIBB1067

The nitrogenase activities of cyanobacteria of the genus Trichodesmium are strictly light-dependent, although they do not develop heterocysts. Recently, the development of heterocyst-equivalent cells (diazocytes) was suggested in Trichodesmium spp. However, no cells with a similar appearance to diazocytes could be found in Trichodesmium sp. NIBB1067. An immunocytochemical analysis of nitrogenase in individual cells was performed using polyclonal antibodies generated from DNA fragments of genes encoding the Fe-protein and the α-and the β-subunit of the MoFe-protein of nitrogenase of Trichodesmium sp. NIBB1067. Visualization of the antibody binding was carried out using a horseradish peroxidase-conjugated secondary antibody and a chromogenic substrate, 3,3′-diaminobenzidine, to avoid the masking effects caused by the bright autofluorescence emitted by Trichodesmium cells. Nitrogenase proteins were detected in almost all the cells (always higher than 95% of total cells) grown under nitrogen-limited conditions. These results indicate that Trichodesmium sp. NIBB1067 does not differentiate heterocyst-equivalent cells.     

Detection of nitrogenase in individual cells of a natural population of <Emphasis Type="Italic">trichodesmium</Emphasis> using immunocytochemical methods for fluorescent cells

The intercellular distribution of nitrogenase was detected in a natural population of Trichodesmium spp. collected from Shitaba Bay, Uwajima, Ehime Prefecture, Japan. Polyclonal antibodies raised against the Fe-protein and the MoFe-protein (α-subunit) of nitrogenase were used as probes. Visualization of the antibody binding to the nitrogenase was performed using horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate, 3,3′-diaminobenzidine tetrahydrochloride, to avoid the masking effects caused by the bright autofluorescence emitted by the Trichodesmium cells. Nitrogenase proteins were detected in 66 to 81% of the trichomes. More than 77% of the cells were immunostained in individual nitrogenase-containing trichome. The data show natural populations of Trichodesmium spp. do not developed heterocyst-equivalent cells.     

黄海冷水团海域微型异养鞭毛虫对异养细菌和蓝细菌摄食作用的初步研究

2006年10月在黄海冷水团海域的三个站点开展了微型异养鞭毛虫、异养细菌和蓝细菌的密度和生物量调查,进行了微型异养鞭毛虫的现场摄食实验,通过荧光标记细菌法和消化系数法获得该海区微型异养鞭毛虫对异养细菌和蓝细菌的摄食率,并估算了微型异养鞭毛虫对异养细菌和蓝细菌现存量及生产力的摄食压。结果显示,微型异养鞭毛虫、异养细菌和蓝细菌的密度分别为0.36×103~1.13×103,0.39×106~1.13×106和0.04×104~3.74×104cells/cm3,温跃层以上明显高于底层。微型异养鞭毛虫对异养细菌的摄食率为5.33~14.89个/(HF·h),对蓝细菌的摄食率为0.26×102~23.10×10-2cells/(HF·h),摄食率随深度而下降。微型异养鞭毛虫每天能消耗9.27%~33.08%的异养细菌现存量和8.12%~16.09%的蓝细菌现存量。同时,微型异养鞭毛虫对异养细菌和蓝细菌的日摄食量各占它们生产力的2.66%~13.10%和8.12%~16.09%。研究表明微型异养鞭毛虫的摄食可能不是秋季黄海冷水团海域浮游细菌及其生产力的主归宿。     

深海热液区化能自养菌Caminibacter profundus氢酶对环境因子的响应特点

以深海热液区化能自养菌Caminibacter profundus为对象,采用扫描电镜和化学能谱分析,研究了菌株表面形态和矿物元素沉积;同时研究了菌株生长、甲基紫精(MV)还原的氢酶活性及膜结合的类型I NiFe氢酶基因(hynL)表达对盐度、pH和温度几种环境因子变化的响应特点。结果表明,菌株表面被Si、Ca、S和Fe等多种元素组成的矿物质层所包裹。不同条件下,菌株生长、MV还原的氢酶活性和hynL的表达趋势相一致,其最适条件为盐度30、pH5.5和55℃培养温度。研究结果表明,在环境改变时,C.profundus通过调控hynL的表达,以调整菌株的能量代谢,维系菌株的生长繁殖。     

Influence of the vertical distribution of cyanobacteria in the water column on the remote sensing signal

An increased intensity of cyanobacterial blooms and their potentially harmful effects have attracted the attention of environmental agencies, water authorities and the general public worldwide. Reliable operational monitoring methods of coastal waters, lakes and ponds are needed. Mapping of the surface extent of cyanobacterial blooms with remote sensing is straightforward, but recognizing waters dominated by cyanobacteria throughout the water column and quantitative mapping of cyanobacterial biomass with remote sensing is more complicated. Unlike most algae, cyanobacteria can regulate their buoyancy and move vertically in the water column. We used the Hydrolight 4.2 radiative transfer model and the specific optical properties of three species of cyanobacteria to study the impact of vertical distribution of cyanobacteria on the remote sensing signal. The results show that the vertical distribution of cyanobacteria in the water column has a significant impact on the remote sensing signal. This result indicates that developing remote sensing methods for quantitative mapping of cyanobacterial biomass is much more complex than quantitative mapping of an algal biomass that is uniformly distributed in the top mixed layer of water column.     

Formation and functional significance of storage products in cyanobacteria

The most common storage products of cyanobacteria are polyphosphate as a phosphorus storage compound, cyanophycin or phycobilin protein pigment as nitrogen storage products, and glycogen as a storage product of both carbon and energy. Nutrient uptake kinetics are regulated by the storage pools, and the patterns of regulation have a feedback effect on the amount of accumulated nutrient in the cells. Besides having a storage function the nutrient storage products are likely to act as metabolic sinks during conditions of energy stress. Regulation of storage products is especially strict in light‐limited cultures. By increasing the rate of polysaccharide formation during growth with short photoperiods, cyanobacteria are able to sustain relatively high growth rates. This effect is enhanced by keeping respiratory losses very low.     

Physiological changes in the coastal marine cyanobacterium Synechococcus sp. PCC 7002 exposed to low ferric ion levels

Cyanobacteria are ubiquitous in marine waters. These prokaryotic cells are of particular interest in areas of the ocean where the availability of iron may be limiting for cell growth since these organisms commonly excrete iron-specific organic ligands (siderophores) in response to low levels of iron. It is generally considered that the production of siderophores provides a competitive advantage over the competing microorganisms that do not produce these ligands.In order to ascertain the influence of iron availability on the physiology of picoplanktonic cyanobacteria we performed a series of experiments on the coastal coccoid cyanobacterium, Synechococcus sp. PCC 7002. Physiological responses were examined in cells grown in a continuous cuture system with influx media containing a range of iron concentrations (from 4.2 × 10⁻⁵ to 5.1 × 10⁻⁹ M FeCl₃). Steady-state growth rates, combined with growth data from batch cultures demonstrated a non-linear response between iron availability and cell proliferation: cell yields were considerably higher in the lowest-iron chemostats than predicted based on the yields in the higher-iron chemostats. The higher yields during low-iron growth corresponded with the production of the extracellular siderophores and the induction of the specific iron-siderophore membrane transport proteins. A comparison of iron transport and carbon acquisition rates between the low-iron grown cells and the high-iron grown cells indicates that under low-iron growth conditions, iron and carbon acquisition meets the growth demands of the cells, whereas growth at higher iron levels enabled excessive (luxury) carbon acquisition and storage. We conclude that cyanobacteria are efficiently adapted to grow in low-iron environments (providing sufficient light for photosynthesis is available) and the luxury-uptake of carbon may serve as the source material for the extracellular ligands released by these cells. Since the release of siderophores was at iron levels in excess of the levels that induce the siderophore-mediated transport of iron, cyanobacteria growing in an environment with varying levels of iron may contribute substantial amounts of their stored carbon reserves into the DOC as iron-specific ligands.