一株兼性氧化亚氮还原菌的还原N2O能力

【目的】从水稻土中分离筛选出一株兼性氧化亚氮还原菌,并探索其在不同条件下还原N_2O的能力,为减少温室气体N_2O的排放提供重要依据。【方法】通过微生物富集培养分离技术从水稻土中分离得到纯菌;利用nosZ基因和16S rRNA的测序分析鉴定菌株;通过测定菌株在不同条件下N_2O的还原量,分析该菌株还原N_2O的能力及调控因子。【结果】经鉴定,该菌株含有nos Z基因,属于假单胞菌属,在温度30°C、厌氧条件下还原N_2O速率高达0.0219μmol/min以上,改变不同温度和氧气浓度后其能力相对减弱,但仍具备较强的还原N_2O作用。【结论】从水稻土中分离筛选得到的兼性氧化亚氮还原菌为假单胞菌,它在不同环境条件下都具备较强的还原N_2O能力,该菌株可能为减少土壤N_2O排放提供新途径,对保障生态环境安全具有重要的应用价值。     

微生物胞外长距离电子传递网络研究进展

[目的] 解析一株从黄河三角洲湿地甲烷氧化富集物中分离获得的甲烷氧化菌伴生菌的生理学及电化学特性,并探究该菌株对甲烷氧化过程的影响。[方法] 使用高通量测序技术解析甲烷氧化富集物的菌群结构,采用稀释涂布法、平板划线法分离甲烷氧化菌的伴生菌,通过16S rRNA基因测序技术进行菌株初步鉴定。利用扫描电子显微镜观察菌株形态,并通过气相色谱（gas chromatography,GC）检测伴生菌利用甲烷情况及对甲烷氧化菌氧化甲烷效率的影响。采用双室微生物燃料电池（microbial fuel cells,MFCs）及差分脉冲伏安法（differential pulse voltammetry,DPV）检测菌株的电化学活性。[结果] 黄河三角洲湿地土壤甲烷氧化富集物主要的好氧甲烷氧化菌为甲基杆菌属Methylobacter,同时还发现一些伴生菌。分离得到一株甲醇利用菌P7,其16S rRNA基因序列与恶臭假单胞菌Pseudomonasputida的相似性达99.79%。扫描电镜结果显示该菌株为杆状,长约1.5-2.5μm,宽度约为0.5μm。GC检测结果显示,该菌株不能利用甲烷,但与甲烷氧化菌共培养时,可以促进甲烷氧化（P<0.05）。双室MFCs检测结果显示该菌株具有电活性,最大电流输出密度为28 mA/m²,DPV检测结果显示该菌株主要的氧化峰和还原峰分别位于-0.17 V和-0.25 V。[结论] 本研究从黄河三角洲湿地甲烷氧化富集物中获得一株具有电活性的甲烷氧化菌的伴生菌恶臭假单胞菌Pseudomonas putida P7,该菌株可以促进甲烷氧化。本研究加深了对甲烷氧化过程中伴生菌的生理学特性及功能的认识。     

中国北部湾光裸方格星虫可培养微生物的分离鉴定及其活性代谢物产生菌筛选

【目的】本研究从北部湾海域光裸方格星虫(Sipunculus nudus)肠道中分离鉴定可培养微生物,并对筛选菌株的代谢物活性进行研究,为后续开发和利用光裸方格星虫肠道微生物代谢产物提供理论支持。【方法】通过微生物培养、菌株分离纯化和16S rRNA基因序列分析,分析鉴定湛江、北海、防城港三地光裸方格星虫肠道可培养微生物;采用透明圈法、可见分光光度法、平板打孔法等对产胞外活性代谢物的菌株进行筛选和活性分析。【结果】中国北部湾不同海域光裸方格星虫肠道可培养微生物包括弧菌属(Vibrio)、希瓦氏菌属(Shewanella)、假交替单胞菌属(Pseudoalteromonas)、发光杆菌属(Photobacterium)和芽孢杆菌属(Bacillus)等12个细菌属。弧菌属(Vibrio)是3个地区样本共有的优势菌群。具有产胞外水解蛋白酶、壳聚糖酶、多糖以及抑菌活性等能力的菌株主要来自假交替单胞菌属(Pseudoalteromonas)、发光杆菌属(Photobacterium)和芽孢杆菌属(Bacillus)。【结论】中国北部湾不同海域光裸方格星虫肠道可培养微生物在属的种类上存在显著性差异,且光裸方格星虫肠道菌株具有产生多种胞外活性代谢物的能力,是一种良好的海洋活性代谢物来源。     

硇洲岛褐蓝子鱼肠道抗菌活性菌株的分离及多样性

【目的】了解广东湛江硇洲岛褐蓝子鱼(Siganus fuscescen)肠道抗菌物质产生菌的多样性。【方法】采用传统的分离方法与牛津杯扩散法对褐蓝子鱼肠道菌进行分离纯化与抗菌活性测定,运用16S rRNA基因序列的系统发育分析对抗菌活性菌株进行多样性分析。【结果】根据形态观察和部分生理生化实验去冗余,从褐蓝子鱼肠道样品中分离到68株细菌,其中抗菌活性菌株19株,占分离株的27.9%。19株不同种类的活性菌株分属于细菌域的放线杆菌门(Actinobacteria)、变形杆菌门(Proteobacteria)与厚壁菌门(Firmicutes)的11个科、12个属。多数菌株属于厚壁菌门(8株,42.1%),其次是变形杆菌门(7株,36.8%)和放线杆菌门(4株,21.1%)。大多数抗菌活性菌株与其系统发育关系最密切的已知典型菌株之间存在一定的遗传差异(16S rRNA基因序列相似性为96.2%-99.9%),其中有4株代表潜在的新种。【结论】广东湛江硇洲岛褐蓝子鱼肠道中存在较为丰富的细菌抗菌物质产生菌,并蕴藏着较多的微生物新类群。     

微小卡罗藻(Karlodinium micrum)共附生微生物抗菌与细胞毒活性

【目的】本文对微小卡罗藻共附生微生物进行分离并对其抗菌和细胞毒活性进行初步研究,以期望获得既具有抗菌又具有细胞毒的高活性菌株,为从共附生微生物的角度去研究微小卡罗藻毒素的合成途径以及真正来源提供研究材料。【方法】利用琼脂扩散法和MTT法对细菌培养液的乙酸乙酯提取物进行抗菌和细胞毒活性筛选,并对具有细胞毒活性的细菌菌株进行了16S rRNA系统发生学分析。【结果】在分离到的38株海洋细菌中,25株细菌具有抗菌活性,5株细菌(W-14-2、W-2-2、W-12、E-8-2和W-4)具有细胞毒活性。对这5株具有细胞毒活性的细菌菌株进行16S rRNA系统发生学分析显示它们分别与Alteromonas alvinellae、Stappia aggregata、Pelagibaca bermudensis、Marinobacter kribbensis和Maribacter dokdonensis的16S rRNA基因序列具有较高的相似性。【结论】在分离到的微小卡罗藻共附生微生物中含有较为丰富的活性菌株,且获得5株具有抗菌活性又具有细胞毒的高活性菌株。     

异化铁还原梭菌Clostridium bifermentans EZ-1产氢与电化学特性

【目的】从黄河三角洲滨海湿地土壤中获得同时具备产氢、产电以及异化铁还原能力的多功能菌株。【方法】通过厌氧分离技术从黄河三角洲土壤中分离得到纯菌株,16S r RNA基因测序后与数据库已有序列进行比对。利用革兰氏染色及扫描电镜观察菌株形态,并用气相色谱(gas chromatography,GC)和液相色谱(high performance liquid chromatography,HPLC)检测其生理代谢底物和产物。通过添加不同形态铁氧化物检测该菌株Fe(Ⅲ)的还原能力。构建微生物燃料电池(Microbial fuel cells,MFCs)检测该菌株的电化学活性。【结果】16S r RNA基因序列比对发现其与双酶梭菌Clostridium bifermentans的相似性达97.99%。革兰氏染色结果显示为阳性菌。能够利用葡萄糖为底物发酵产生氢气、二氧化碳、乙酸和丁酸。Fe(Ⅲ)还原能力检测发现,其不仅可以还原柠檬酸铁(FeC_6H_5O_7)中可溶性的Fe(Ⅲ),还可以还原无定形铁水铁矿(FeOOH)和晶型纳米磁铁矿(Fe_3O_4)中的Fe(Ⅲ)。此外,经MFCs检测发现,该菌具有电化学活性,最大电流输出密度可达6.5 m A/m~2,且在0.15 V位置存在氧化峰。【结论】本研究从土壤中成功分离得到了一株同时具有产氢、产电以及异化铁还原能力的多功能梭菌菌株,保藏并命名为Clostridium bifermentans EZ-1。     

ε-聚赖氨酸发酵过程污染微生物的分离与鉴定

【目的】研究ε-聚赖氨酸发酵过程中污染微生物的种类。【方法】采用稀释涂布法、划线法、环境胁迫法和液体营养富集法等对污染样本进行微生物的分离与纯化,通过菌落形态和显微观察,再结合16S rRNA基因序列分析,确定分离菌株的系统发育地位,并对分离菌株的ε-聚赖氨酸耐受性进行考察。【结果】液体营养富集法实现了污染微生物的分离,通过16S rRNA基因序列分析鉴定其为一株Acinetobacter bereziniae,并证实该菌能在高浓度ε-聚赖氨酸条件下生长。【结论】Acinetobacter bereziniae是ε-聚赖氨酸发酵过程中的主要污染微生物,这为后期发酵污染防治提供了一定的指导作用。     

基于活性和基因从海洋微生物中筛选烯二炔类抗生素

【目的】高质量的海洋微生物菌种库及其天然产物库是新药开发的重要来源。在本研究中我们通过筛选新的烯二炔类抗生素来对已经构建的海洋微生物天然产物库进行质量评价。【方法】首先我们根据烯二炔类抗生素能引起DNA断裂的活性构建了活性筛选模型,并对我们的天然产物库进行筛选;其次根据合成烯二炔核心结构的独特且保守的重复I型聚酮合成酶设计了引物,通过PCR扩增的方法对海洋微生物库进行序列筛选。【结果】通过活性筛选从我们的海洋微生物天然产物库中获得一个阳性的发酵产物。对该阳性菌(LS481)的系统发育学分析表明该菌属于能产生烯二炔类化合物—Dynemicin的Micromonospora chersina,对其发酵产物TLC分析证明该菌确实产生Dynemicin类化合物。通过基因筛选得到了2个具备合成烯二炔核心结构聚酮合成酶的菌株,16S rRNA基因分析显示其中一个很可能为灰色链霉菌(MS098),另外一株菌则同Streptomyces vinaceus NBRC 13425T和Streptomyces cirratus NRRLB-3250T最相近。【结论】我们的活性筛选模型能够有效获得烯二炔类物质,结合基因筛选能够进一步获得可能产生烯二炔物质的菌株。初筛结果也再一次验证了我们海洋微生物天然产物库的质量较好。     

奥奈达希瓦氏菌CctA介导周质甲基橙还原的电子传递机理

电活性微生物奥奈达希瓦氏菌的胞外电子传递（extracellular electron transfer,EET）在污染物降解、环境修复、生物电化学传感、能源利用等方面具有广泛的应用潜力;四血红素细胞色素CctA （small tetraheme cytochrome）是希瓦氏菌周质空间中最丰富的蛋白质之一,能够参与多种氧化还原过程,但目前对CctA在EET中的行为和机理认识仍然有限。【目的】研究阐明CctA蛋白在希瓦氏菌模式菌株MR-1周质空间以偶氮染料作为电子受体的EET中的作用,补充和拓展希瓦氏菌的厌氧呼吸产能机制。【方法】以周质还原型偶氮染料甲基橙（methyl orange,MO）作为电子受体,在mteal reduction （Mtr）蛋白缺失菌株Δmtr中研究MO的周质还原特点,并通过基因敲除和回补表达研究CctA蛋白在周质电子传递中的作用。【结果】在缺失Mtr通道的情况下,细胞色素CctA可以介导周质空间的电子传递而还原MO。重组表达CctA在低水平时,MO在周质空间中的还原速率与其表达水平呈正相关,更高水平的CctA表达无助于进一步提高MO的还原速率。蛋白膜伏安结果展示了CctA与周质空间内其他高电位氧化还原蛋白的显著区别,可能参与构成一条低电位的MO还原通道。【结论】从分子动力学层面揭示了CctA在周质MO还原中的独特电子传递行为,为进一步推进对细菌周质电子传递机制的理解,以及通过合成生物学设计或改造胞外氧化还原系统、强化生物电化学在污染物降解中的应用提供了重要信息。     

一株盐湖芽孢杆菌LG的鉴定及其抗金黄色葡萄球菌活性研究

【目的】从运城盐湖中分离获得一株耐盐细菌LG,对其进行分类鉴定及抗菌特性研究。【方法】利用16S rRNA基因序列分析对菌株进行分类鉴定。以金黄色葡萄球菌为指示菌,采用杯碟法对菌株LG发酵上清液进行抗菌活性检测,利用扫描电镜和透射电镜观察其抗菌效果。研究不同因素对上清液抗菌活性的影响,并采用PCR技术对菌株基因组进行功能基因筛查。【结果】系统发育分析表明该菌为Bacillus属成员,在0–25%的NaCl浓度范围内生长良好,为耐盐细菌。电镜观察发现,菌株LG发酵上清液处理金黄色葡萄球菌可导致细胞结构明显出现异常,细胞质泄漏。抗菌稳定性研究表明,菌株LG发酵上清液活性稳定,表现出了良好的对紫外光、温度、pH和NaCl的耐受性。采用特异性引物,通过PCR筛查发现菌株LG基因组中含有聚酮合酶(PKS I)基因和非核糖体肽合成酶(NRPS)基因,表明该菌具有产多种代谢产物的潜力。【结论】极端环境中的微生物资源可作为抗菌活性物质的潜在新来源。     

Coccystes undoxylophus

Ohne Zusammenfassung     

UeberAquila pennata undminuta

Ohne Zusammenfassung     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Aquila pennata undminuta

Ohne Zusammenfassung     

Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.