一株具有氢自养能力的固氮粘质沙雷氏菌的分离鉴定、代谢特征与功能基因检测

氢氧化细菌（HOB）是能利用氧气氧化氢气供能以固定二氧化碳进行自养生长的一类细菌,但是目前对能够固氮的HOB研究甚少.在自然界中部分HOB常作为植物根际促生细菌（PGPR）存在,而固氮是可能的促生机制,有望从功能菌群中得到更多的固氮HOB,是否存在氢自养且可固氮的沙雷氏菌未知.从固氮氢氧化混合菌群中分离出一株产色素的固氮菌NF-HOB1,经16S rRNA基因测序鉴定为粘质沙雷氏菌（Serratia marcescens）(GenBank:OQ625884),通过碳源或氮源限制培养实验验证其具有异养固氮生长、氢自养固碳生长能力,氢气氧化速率为0.187 5 mmol/d.聚合酶链式反应（PCR）扩增发现其质粒DNA编码固氮酶铁蛋白结构基因nifH,基因组编码种特异性的核酮糖-1,5-二磷酸羧化酶（Rubisco）和氢化酶3的结构基因.基因组框架图测序进一步揭示了菌株的基因组特征,基因组长4 919 531 bp,蛋白编码基因4 778个.注释到氢化酶2、碳酸酐酶、GlnK型PII蛋白等,提示存在二氧化碳浓缩机制和以NtrB-GlnK-amtB/nifA为特征的全局氮调控机制.基因本体论...     

不同C/N值下亚硝酸盐氧化菌和异养菌混合体系的微生物多样性

采用PCR-RFLP技术研究了不同C/N比下亚硝酸盐氧化菌及异养菌混合体系的微牛物多样性,并探讨了微生物菌群结构与其功能(硝化件能)的关系.C/N=0时,混合体系主要由自养菌和寡营养菌(85.1%)组成,包括亚硝酸盐氧化菌(NOB)、拟杆菌门、α-变形菌纲、浮霉菌门和绿色非硫细菌中的一些菌株.C/N=0.44时,混合体系中的自养菌减少,异养菌(主要是γ-变形菌纲的成员)大量出现.C/N=8.82时,γ-变形菌纲的菌株尤其是反硝化菌Pseudomonas sp.占主导(93.8%),与此同时,随着C/N升高,该混合体系的硝化性能也由专一的亚硝酸盐氧化过程转变为同时硝化反硝化过程.微生物菌群结构的转变较好地解释了其硝化性能的改变.本研究揭示了微生物菌群结构与其功能的内在联系,同时表明PCR-RFLP技术与化学分析相结合是研究微生物菌群结构与功能的有力工具.图3表2参13     

石油烃降解酶及其基因的结构、功能和表达调控

研究烃降解酶及其基因是进行石油微生物分子检测和工程菌构建的重要基础.本文对目前烃降解酶及其基因的结构、功能和调控机制的最新研究进展进行了总结.催化烷烃好氧降解的起始酶有几类加氧酶,膜整合甲烷单加氧酶、萘-1,2-双加氧酶和异丙苯双加氧酶的晶体结构已经被解析.烷基或芳基琥珀酸合酶催化烃厌氧代谢的主要起始反应,而Azoarcus sp.乙苯厌氧代谢起始反应由乙苯脱氢酶催化.在细菌中,烃代谢相关基因主要通过形成操纵子进行表达调控,基因转录受烃或类似物诱导,并受细胞全局调控.一些微生物由于存在多种烃代谢途径而可能具有复杂的基因调控机制.此外,生态学研究表明,环境中烃降解基因的诱导动态与实验室内纯培养分析不同.在分析石油降解工程菌构建有待解决问题的基础上,提出了烃代谢综合调控和环境中相关酶及基因诱导研究的重要性,并对未来烃降解酶及其基因在有毒物降解理论研究和生物修复上的应用进行了展望.     

亚硝酸细菌amoA基因的克隆、测序与表达

采用PCR技术对亚硝酸细菌的特异性amoA基因进行克隆、测序 ,以确定其准确性 ;然后采用基因重组技术构建亚硝酸细菌的基因工程菌 ,并进行鉴定与功能初步评价 .结果表明 ,克隆得到的 4种亚硝酸细菌的amoA基因 ,与标准菌株Nitrosomonassp.GH2 2的amoA基因同源性达到 98%～ 99% ;构建了一种含有特异性amoA基因的大肠杆菌基因工程菌株 ,并采用直接比色法对重组细菌的氨氧化活性进行测定 ,发现基因工程菌的氧化氨氮速率明显高于分离的野生菌株 .研究表明 ,利用基因重组技术对亚硝酸细菌等自养菌进行改造 ,构建高效基因工程菌在水污染治理领域具有可行性 .图 6表 1参 12     

刺参(Apostichopus japonicus)养殖环境中可培养细菌与理化参数的动态及其相关性分析

随着刺参(Apostichopus japonicus)人工养殖业的快速发展,刺参病害日渐突出.养殖环境微生物和理化参数作为养殖生态系统的重要组成部分,其变化规律及其相关性研究对防治刺参病害具有重要意义.本研究于2012年4月至2013年4月,对山东省即墨市刺参养殖池塘水体中4种可培养细菌(总异养菌、弧菌、硝化细菌、硫化细菌)、4项理化参数(温度、pH、盐度、溶解氧)和沉积环境中6种可培养细菌(总异养菌、弧菌、硝化细菌、硝酸盐还原菌、硫化细菌、硫酸盐还原菌)、6项理化参数(温度、pH、氧化还原电位、硫化物、有机碳、总氮)的动态变化进行了监测,并分析了不同细菌类群的数量及其与沉积物中5项理化指标间的相关性.结果显示:水中大多数细菌类群的周年变化呈夏季高、冬季低的趋势,与温度的变化趋势基本一致,弧菌的变化幅度较异养菌大;硝化细菌较硫化细菌高一个数量级,但变化趋势一致;水体的pH与溶解氧均在夏季高温出现最低值,与温度变化趋势相反;沉积物中异养菌数变化不大,弧菌数量在夏季和冬季出现两个高值.氮循环相关细菌的数量高于硫循环相关细菌.相关性分析结果显示,刺参养殖池塘沉积物的pH与氧化还原电位呈显著正相关(P0.05),二者与硫化物含量均呈极显著负相关(P0.01).硫化物含量与总异养菌、硫酸盐还原菌和硝化细菌均呈极显著正相关(P0.01),与硫化菌和硝酸盐还原菌呈显著负相关(P0.05),总氮含量与硝化细菌正相关性显著(P0.05).各类细菌数量之间也存在一定的相关性:总异养菌与硝化细菌、硫酸盐还原菌之间、硝化细菌与硫酸盐还原菌之间均存在极显著正相关性(P0.01),弧菌与总异养菌、硝化细菌之间存在正相关性,硝酸盐还原菌与硝化细菌之间、硫酸盐还原菌与硫化细菌之间存在负相关性.本研究表明刺参养殖池塘环境中多项理化参数和细菌数量之间相关性显著,其关系错综复杂,可为防治刺参病害提供理论依据.     

含铜多金属复杂矿区的一株浸矿细菌的分离与鉴定

为寻找新的浸矿细菌,从江西城门山矿区硫化矿矿坑水中使用平板分离法得到了一株能够浸矿的细菌,命名为CMS.经电镜和生理生化试验,鉴定该菌株为革兰氏阴性细菌,短杆状,菌体大小为(0.4±0.1)靘祝±0.5)靘, 最适生长温度为25～30 ℃,最适pH值为2.0,化能自养型,能利用亚铁、单质硫和葡萄糖生长,不能利用硫代硫酸钠、蛋白胨生长.16S rRNA系统发育树的结果表明,CMS菌株与嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans,简称A.f)AFY菌株位于系统发育树一个同的分支中,相似度99.72%.克隆其代谢系统关键基因亚铁氧化酶(Iro protein)基因并测定其序列,与其他相关菌株比对,结果显示编码区的核酸序列与报道序列完全一致.另外,其铁闪锌矿(ZnFe)S摇瓶浸出试验显示,在浸出28 d后,含菌株的摇瓶的锌离子质量浓度即达到615.50 mg·L-1,而无菌浸出仅有392.25 mg·L-1.     

固定CO2氢细菌的筛选及其培养条件优化

从土壤中分离筛选到一株高效固定CO2 的氢氧化细菌—专性化能自养菌BHA 15 ,在最佳培养条件下(培养基 ρ/gL-1:NH4 Cl 4.0 ,Na2 HPO4 ·12H2 O 0 .75 ,KH2 PO4 0 .5 ,MgSO4 ·7H2 O 0 .3;V(H2 )∶V(O2 )∶V(CO2 ) =3 .5∶1∶1;pH =7;最适生长温度θ =30℃ ) ,密闭摇瓶中恒温 (30℃ )培养 40h后 ,菌体浓度 ρ(DW)达 1.6mg/mL ,菌体蛋白含量 71.8% .图 7表 2参 12     

污水深度处理生物滤层中菌群的时空分布特征

以生物滤柱为反应器,对污水进行深度处理。试验表明,生物滤层中的菌群由于生化特性的不同,启动阶段异养菌要比自养菌的生长提前一周左右。生物滤柱异养菌、自养菌的形成对稳定运行非常关键,启动阶段较高的COD容积负荷不利于亚硝化、硝化细菌的产生。异养菌在竞争中占有优势,主要生长在滤柱的进水端和悬浮生物膜中。进水有机物浓度较低,造成亚硝化细菌的分布同异养菌分布基本一致。硝化细菌由于较弱的竞争能力,主要生长在滤柱的出水端和吸附生物膜中。底物浓度的改变使生物膜中的菌群对有机物、溶解氧以及生存空间的竞争也发生变化,最终导致其分布也随之改变。     

土壤氢氧化细菌促进作物生长机理研究进展

综述了近十年来国内外关于豆科作物根际土壤促生菌中氢氧化细菌的研究进展,讨论了根瘤释放H2促进作物生长的可能机制.与豆科作物进行轮作、间作是提高土壤肥力、增加作物产量的一项传统的农业耕作方式.对于这种耕作方式优势的机制研究,过去大多数主要集中在土壤氮(N)元素含量的提高.而近期研究表明土壤氢氧化细菌以豆科作物根瘤菌在固氮过程中释放的H2为能量来源进行化能自养改变土壤微生物种群结构.一些土壤氢氧化细菌通过产生1-氨基环丙烷-1-羧酸脱氨酶和根瘤菌毒素抑制植物体内乙烯的合成,促进作物生长.此外,本文进一步讨论了利用现代分子生物学方法研究氢氧化细菌促进作物生长的途径.     

磁性壳聚糖凝胶微球固定反硝化菌去除地下水中硝酸盐氮

本研究从活性污泥中分离出氢自养反硝化细菌,在厌氧条件下利用氢气作为电子受体,将硝酸盐氮污染物彻底还原为氮气.通过原位共沉淀/柠檬酸钠交联法制备了一种磁性壳聚糖微球,将氢自养反硝化菌固定于磁性壳聚糖微球上组成固定化微生物反硝化体系.利用16SrDNA菌种鉴定、扫描电镜(SEM)、傅里叶红外光谱(FTIR)对固定化前后的材料进行了表征,并与游离的氢自养反硝化菌进行对比,同时进行静态批实验考察了在不同影响因素下硝酸盐去除效果.结果表明,分离出的氢自养反硝化菌属于陶厄氏菌属(MK928401),且被成功固定在磁性壳聚糖微球上;相同时间内,固定化氢自养反硝化菌对硝酸盐氮去除率高出游离细菌59%,说明固定化菌克服了由于游离菌易团聚而限制反硝化速率的缺点;磁性壳聚糖微球的加入,在一定程度上拓宽了氢自养反硝化菌对硝酸盐氮浓度的适应范围,同时拓宽了氢自养反硝化菌对pH的耐受范围;固定化氢自养反硝化菌经5次重复利用后,仍能高效还原硝酸盐氮,相比于游离细菌具有可回收和循环利用性.以上结果得出,以磁性壳聚糖微球固定氢自养反硝化菌,为高效去除地下水中的硝酸盐氮提供了一种更有效的途径.     

Localization of RubisCO and sulfur in endosymbiotic bacteria of the gutless marine oligochaete Inanidrilus leukodermatus (Annelida)

 The chemoautotrophic potential of the two co-occurring larger and smaller bacterial endosymbionts of the gutless marine oligochaete Inanidrilus leukodermatus was determined using immunocytochemistry. An antibody directed against the Form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key CO₂-fixing enzyme of the Calvin–Benson cycle, consistently labeled the larger symbionts. Electron microscopic spectroscopy showed that the larger symbionts contained sulfur in intracellular globules and to a lesser degree in the cytoplasm. The presence of RubisCO and sulfur indicates that the larger endosymbionts of I. leukodermatus are chemoautotrophic sulfur-oxidizing bacteria. In contrast, no RubisCO or sulfur was detected in the smaller endosymbionts of this host. Received: 28 September 1999 / Accepted: 29 May 2000     

Ralstonia eutropha菌株镉抗性调节基因CzcR的克隆与序列测定

G-细菌镉抗性决定子是一个编码4个蛋白的CzcCBAD操纵子,参照GenBank已登录Czc基因序列进行引物设计,利用PCR技术,从可在含350mg/LCdCl2的培养基上生长的抗镉菌株Ralstoniaeutropha质粒中,扩增出长度约为1200bp的革兰氏阴性细菌镉抗性系统中的抗性调节基因CzcR,然后将其亚克隆到pGEM-T-easy载体上,并转化至受体菌中,构建重组质粒,采用碱性裂解法提取质粒DNA后,经EcoRI酶切分析和核苷酸序列分析,其与GenBank中登录的CzcR基因序列相似性高达98%,显示其具有正确的CzcR基因核苷酸序列。镉抗性基因的获得将大大推动对微生物抗重金属机理的研究,并为进一步构建耐镉基因工程菌,高效净化重金属污染环境打下坚实的基础。     

Chemoautotrophic symbionts and translocation of fixed carbon from bacteria to host tissues in the littoral bivalve Loripes lucinalis (Lucinidae)

Specimens of Loripes lucinalis (Lucinidae) living in reducing sediments were collected near a sewage outfall at low tide on the Moulin Blanc beach, Brest, France, from January to March 1987. Electron microscope studies revealed numerous Gram-negative-type bacteria in the gill cells. Ribulosebiphosphate carboxylase, a diagnostic enzyme of the Calvin-Benson cycle of CO₂-fixation was measured only in the gill extracts. Various tissues of L. lucinalis were examined for activity of APS reductase, (EC 1.8.99.2), ATP sulphurylase (EC 2.7.7.4) and rhodanese (EC 2.8.1.1), enzymes involved in sulphide oxidation. APS reductase was only found in symbiont-containing tissues, i.e., gills. These enzymatic studies characterise the symbionts as chemoautotrophic sulphide-oxidizing bacteria. Histoautoradiography demonstrated that part of the carbon dioxide fixed by symbiotic bacteria in the gills is translocated to symbiont-free tissues of the bivalve. The ultrastructure of the gill is detailed and a nomenclature based on established and new terminology is proposed to describe the various cellular types comprising the gill filament.     

Activities of sulfur-oxidizing bacteria at the 21°N East Pacific Rise vent site

The incorporation of inorganic and organic carbon into cell material, as well as the activities of carboxylating enzymes (ribulose bisphosphate carboxylase and phosphoenol pyruvate carboxylase), were measured in waters emitted from warm vents at the 21°N ocean spreading site (depth 2 600 m) of the East Pacific Rise. Both obligately and facultatively chemoautotrophic bacteria were present and comprised a significant but variable portion of the total microbial population as indicated by comparisons of microscopic cell counts with liquid enrichments and colony counts on media containing reduced sulfur compounds. The proportion of chemoautotrophic, sulfur-oxidizing bacteria maximally reached 79% of the total microbial population based on ribulose bisphosphate carboxylase activity. Variability of chemoautotrophic activity occurred between vents at different locations, but was also observed in one individual vent. Maximum rates of CO₂ incorporation in warm vent water were similar to levels measured previously in the O₂/H₂S interface of the Black Sea. In shipboard experiments, these rates were virtually unaffected by in-situ pressures (ca 260 atm). Rate measurements at various temperatures, as well as the observed mesophilic characteristics of all isolates obtained, suggest that the microbial, chemoautotrophic activity decreases rapidly as freshly emitted vent water is diluted with cold, ambient, deep-sea water.Contribution No. 6071 of the Woods Hole Oceanographic InstitutionContribution No. 1708 of the Center for Environmental and Estuarine Studies of the University of Maryland     

Cytoenzymatic investigation of intracellular digestion in the symbiont-bearing hydrothermal bivalve <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis>

Invertebrates harbouring endosymbiotic chemoautotrophic bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. Bathymodiolids are dominants of the biomass at geochemically distinct vent sites of the Mid Atlantic Ridge (MAR) and thus are good candidates to study biological processes in response to site-specific conditions. To satisfy their nutritional requirements, these organisms depend to varying extent on two types of chemoautotrophic symbionts and on filterfeeding. The quantitative relationships of the nutritional modes are poorly understood. Using enzyme cytochemistry, electron microscopy and X-ray microanalysis, the structural and functional aspects of the cellular equipment necessary for lysosomal digestion was studied. We provide evidence for the following: (1) the basis of intracellular digestion of symbionts in Bathymodiolus azoricus from two geochemically distinct vent sites was not mainly in the large lysosomal bodies as previously thought (based on the membranous content resembling bacteria); (2) senescent bacteria are autolysed, possibly by bacterial acid phosphatase, that is more likely a cell cycling of the symbionts rather than an active lysosomal digestion by the host; (3) the consistent absence of hydrolases may indicate the improper use of the name “lysosome” for large vesicles at the base of the gill bacteriocytes (4) nutrient transfer in B. azoricus, therefore, may more likely be accomplished through leaking of metabolites from the symbiont to the host, not excluding lysosomal resorption of dead bacteria as an auxiliary strategy for organic molecule transfer; (5) evidence is provided for microvillar transfer of substances from the seawater that may indicate filter-feeding, in non-symbiotic ciliated gill cells of mussels from Lucky Strike; (6) two types of lysosomal vesicles can be distinguished in digestive cells based on their enzymatic content and their elemental composition.     

苏云金芽胞杆菌YBT-1520中SigL及其增强子结合蛋白的结构与功能分析

为揭示SigL及其增强子结合蛋白(EBPs)在苏云金芽胞杆菌(Bt)中的调控功能,在全基因组测序的基础上,采用生物信息学方法,对YBT-1520菌株的SigL及其EBPs的结构和功能进行深入分析.结果表明,YBT-1520基因组中存在1个SigL和6个EBPs,而且EBPs在结构上具有丰富的多样性,包含了EBPs的所有可能的结构域组织类型.SigL所调控的基因涉及11个假定的COG代谢途径,其中包括能量代谢、氨基酸代谢、翻译与细胞周期等.根据EBPs在基因组的位置推测,YBT-1520的EBPs参与γ-氨基丁酸代谢途径、精氨酸代谢途径、支链脂肪酸降解途径、多糖分解代谢等代谢途径的调控.本研究将为揭示Bt杀虫晶体蛋白大量表达的调控机制提供新的思路.     

Microbial activities in a permanently stratified estuary. II. Microbial activities at the oxic-anoxic interface

Bacterial populations and activities at the oxic-anoxic interface of Saelenvann, a permanently stratified estuary in western Norway, have been studied. Concentrations of particulate material and direct counts of bacteria were higher at the interface than in the surrounding water. Dark assimilation of CO₂ was maximal at the interface, indicating high rates of chemoautotrophic productivity. Nitrification could possibly account for 10 to 50% of the measured dark CO₂ fixation. Nitrate is rapidly consumed by dentrifying organisms in the anoxic water and by photosynthetic organisms in the oxic water. A characteristic population of photosynthetic bacteria similar to Chlorobium phaeovibrioides flourishes at the interface, imparting a reddishbrown discoloration to the water.     

群体感应现象及其在生物膜法水处理中的应用

群体感应是指细菌通过感知分泌到环境中自诱导物浓度的变化来调整基因表达,增强其在复杂环境中的生存能力,是细菌行为社会化的体现.研究者对群体感应的微生态凋控机制进行了深入的探讨,包括自诱导物的合成、运输和对目的基因的表达调控,以及各信号通路之间的竞争关系对细菌适应环境的影响.本文分析了当前相关研究中的热点问题,提出了群体感应机制在生物法水处理领域中的潜在应用方式.图2参32     

A new bathymodioline mussel symbiosis at the Juan de Fuca hydrothermal vents

Until recently, the only major hydrothermal vent biogeographic province not known to include bathymodioline mussels was the spreading centers of the northeast Pacific, but deep-sea dives using DSV Alvin on the Endeavor segment of the Juan de Fuca Ridge (47°56N 129°06W; ∼2,200 m depth) in August 1999 yielded the only recorded bathymodioline mytilids from these northeastern Pacific vents. One specimen in good condition was evaluated for its relatedness to other deep-sea bathymodioline mussels and for the occurrence of chemoautotrophic and/or methanotrophic symbionts in the gills. Phylogenetic analyses of the host cytochrome oxidase I gene show this mussel shares evolutionary alliances with hydrothermal vent and cold seep mussels from the genus Bathymodiolus, and is distinct from other known species of deep-sea bathymodiolines, suggesting this mussel is a newly discovered species. Ultrastructural analyses of gill tissue revealed the presence of coccoid bacteria that lacked the intracellular membranes observed in methanotrophic symbionts. The bacteria may be extracellular but poor condition of the fixed tissue complicated conclusions regarding symbiont location. A single gamma-proteobacterial 16S rRNA sequence was amplified from gill tissue and directly sequenced from gill tissue. This sequence clusters with other mussel chemoautotrophic symbiont 16S rRNA sequences, which suggests a chemoautotrophic, rather than methanotrophic, symbiosis in this mussel. Stable carbon (δ¹³C = −26.6%) and nitrogen (δ¹⁵N = +5.19%) isotope ratios were also consistent with those reported for other chemoautotroph-mussel symbioses. Despite the apparent rarity of these mussels at the Juan de Fuca vent sites, this finding extends the range of the bathymodioline mussels to all hydrothermal vent biogeographic provinces studied to date.     

POM in macro-/meiofaunal food webs associated with three flow regimes at deep-sea hydrothermal vents on Axial Volcano,Juan de Fuca Ridge

Deep-sea hydrothermal vent ecosystems host both symbiotic and non-symbiotic invertebrates. The non-symbiotic vent fauna is generally assumed to rely on free-living chemoautotrophic bacteria as their main food source but other sources such as detritus have recently been suggested to be a part of the invertebrate diets. Little is known about how food availability influences the distribution of vent organisms on a small scale. In addition, the feeding ecology and role of small, often numerically dominant invertebrates, the meiofauna is poorly understood at vents. In this study, we used stable carbon and nitrogen isotopic analysis to investigate the role of particulate detritus in the diets of macro- and meiobenthic invertebrates within three vent assemblages at Axial Volcano, Juan de Fuca Ridge, and Northeast Pacific. Particulate organic matter of a detrital origin became more important in the diet of invertebrates in assemblages typically associated with low-hydrothermal flow intensities. Meiobenthic species occupied several different feeding guilds and trophic levels in the assemblages investigated. We conclude that small-scale spatial variability in food sources is an important feature of vent food webs and that spatial patterns observed here and elsewhere are shaped by variations in hydrothermal discharge.