玛珥湖好氧不产氧光合细菌pufM基因DNA和mRNA的定量及多样性分析

【目的】湖光岩玛珥湖是一类特殊的火山口湖,它完全封闭,地质年代久远,尚未受人类活动的剧烈影响,孕育着丰富而特殊的微生物种群。好氧不产氧光合细菌(AAPB)是以其在有氧情况下能行使光合功能而定义的一类专性异养细菌,其生理生态特征独特,进化年代久远,在水生生态系统的上层水体中广泛分布。目前,AAPB在玛珥湖水体中是否有分布仍是未知。【方法】构建和比较夏季湖水1 m、5 m、12 m三个水层的总DNA和总RNA的AAPB光合中心合成的关键基因pufM的6个克隆文库,并结合定量PCR技术,分析了不同水层AAPB的分布、系统发育多样性及其在总细菌中的比重。【结果】6个文库覆盖率和稀释曲线显示样本初步揭示了各水层优势AAPB类群的多样性。BLAST核苷酸同源性介于80%93%;多样性指数表明,湖光岩表层和底层多样性相当,中间层最低,总RNA的多样性高于总DNA。系统发育分析结果表明,OTU21 24所含的序列(占总序列的49.43%)与β-变形细菌的进化距离最接近,是湖光岩玛珥湖的优势AAPB菌群。定量PCR结果显示1 m水层中AAPB在总细菌中的比重最高,可达38.06%;而5 m水层中AAPB所占的比重最低,仅为0.85%;12 m为9.54%。【结论】湖光岩玛珥湖孕育着丰富而多样的AAPB类群。     

基于pufM基因的乌梁素海富营养化湖区好氧不产氧光合细菌系统发育多样性分析

好氧不产氧光合细菌(AAPB)的多样性在海洋中已经广泛研究,但在富营养化湖泊中却研究甚少。通过构建和分析AAPB光合中心合成中的关键基因pufM克隆文库,以期揭示乌梁素海富营养化湖区AAPB分布及其系统发育多样性,探讨其在富营养化湖泊中的功能和作用。对乌梁素海红圪卜湖区水体文库中的52个克隆子进行分析,产生了28个OTU,文库覆盖度达到71.4%,反映出文库有较好的代表性。同源性和系统发育分析结果表明,乌梁素海红圪卜湖区AAPB有较高的多样性,与我们之前所发现的同一湖区总细菌多样性较低形成鲜明对比。所获得的序列分属7个亚群,即γ-Proteobacteria(44.2%,含Group-1,-2和-3共3个亚群)、β-Proteobacteria(21.2%)、Rhodobacter-like(7.69%)及2个未知亚群unknown Group-1(21.2%)和Group-2(5.77%)。其中γ-Proteobacteria占到总克隆的44.2%,在低盐的乌梁素海环境中出现高比例的γ-Proteobacteria之前并未见类似报道,并且乌梁素海中存在一些可能是富营养化湖泊特有的AAPB。这表明AAPB可能在富营养化湖泊生态系统的维持和稳定中有重要作用。     

含好氧不产氧光合基因簇和Xanthorhodopsin-like基因的Sphingomonas sp. MIM37：基因组及光促生长分析

【目的】菌株MIM37为具有两种光能利用途径的光合异养细菌,分析其基因组和光照对生长的影响,为理解光能利用途径、光营养生物多样性以及光合作用的进化和功能等提供线索。【方法】采用平板涂布划线法分离菌株,结合形态观察及16S rRNA基因和光合基因序列同源性与系统发育分析进行初步分类鉴定;以分光光度法和荧光显微观察法测定光照和黑暗培养下培养液细胞浓度和单细胞体积;构建片段长度为300?500 bp的Illumina PE文库,以Illumina Hiseq2000进行基因组测序,以SOAPdenovo和GapCloser组装序列,以RAST在线软件注释基因组。【结果】从内蒙古腾格里沙漠天鹅湖表层水中分离获得一株细菌MIM37,经16S rRNA基因、pufM和视紫质基因同源性和系统发育分析均显示其与Sphingomonas属亲缘关系最为密切;相对黑暗培养,光照刺激下的最大细胞浓度和单细胞体积大小分别提高了1.2和5.6倍;基因组注释显示MIM37代谢途径多样,含典型好氧菌的呼吸电子传递链,具有完整的好氧不产氧细菌的光合基因簇及xanthorhodopsin-like视紫质蛋白基因,合成铁载体,还原重金属,降解微囊藻毒素和多环芳烃类等。【结论】MIM37属于Sphingomonas属,具有两种光能利用途径,光照可明显促进其生长,多样的代谢模式可能使其在自然环境中极具竞争力、分布广泛并具有应用于修复环境污染的潜力。     

Source environment feature related phylogenetic distribution pattern of anoxygenic photosynthetic bacteria as revealed by pufM analysis

Anoxygenic photosynthesis, performed primarily by anoxygenic photosynthetic bacteria (APB), has been supposed to arise on Earth more than 3 billion years ago. The long established APB are distributed in almost every corner where light can reach. However, the relationship between APB phylogeny and source environments has been largely unexplored. Here we retrieved the pufM sequences and related source information of 89 pufM containing species from the public database. Phylogenetic analysis revealed that horizontal gene transfer (HGT) most likely occurred within 11 out of a total 21 pufM subgroups, not only among species within the same class but also among species of different phyla or subphyla. A clear source environment feature related phylogenetic distribution pattern was observed, with all species from oxic habitats and those from anoxic habitats clustering into independent subgroups, respectively. HGT among ancient APB and subsequent long term evolution and adaptation to separated niches may have contributed to the coupling of environment and pufM phylogeny.     

锡林河流域潜在不产氧光合细菌陆向分异特征及影响因素

【目的】为探明锡林河流域潜在不产氧光合细菌（anoxygenic photosynthetic bacteria,AnPB）的陆向分异特征及影响因素。【方法】本研究沿着陆向梯度依次采集水生湍流带、缓流带、滞流带、水偏湿生样带、湿偏旱生样带、旱生样带土壤样品。基于文献建立AnPB在科水平的数据库,运用16S rRNA基因高通量测序筛选科水平潜在AnPB类群及其组成丰度的陆向分异,运用皮尔逊相关性及冗余分析等研究土壤理化因子对潜在AnPB陆向分异的影响。【结果】紫色硫细菌（外硫红螺菌科）和紫色非硫细菌（红杆菌科、红环菌科、醋酸杆菌科、丛毛单胞菌科、全噬菌科）主要分布在水生及水偏湿生生境,其相对丰度与湿度呈显著（P<0.05）或极显著（P<0.01）正相关关系;紫色非硫细菌（红螺菌科、慢生根瘤菌科、生丝微菌科、红菌科）、芽单胞菌科、酸杆菌科、绿色非硫细菌（蔷薇菌科）等主要分布在湿偏旱生和旱生环境中,其相对丰度与盐度和全氮含量呈显著（P<0.05）或极显著（P<0.01）正相关关系;多元回归树分析显示,盐度、湿度、全氮对潜在AnPB陆向分异的总解释度分别为62.39%、...     

Memoir of a 1949 railway journey with photosynthetic bacteria

A serendipic observation at the Hopkins Marine Station of Stanford University in 1948 led to the discovery that anoxygenic photosynthetic bacteria can fix molecular nitrogen. To confirm the discovery, an unusual collaborative event was arranged between laboratories at Washington University (St. Louis) and the University of Wisconsin (Madison).     

土生空团菌的遗传多样性分析

为了研究土生空团菌的遗传多样性,对来自中国、美国、瑞士和法国等菌株的rDNA ITS区进行序列分析,利用Popgene32和phylip软件进行数据计算和聚类分析。序列分析表明土生空团菌rDNA ITS区的序列长度为422–447bp,遗传距离在0.000–0.051之间。居群结构和聚类分析结果表明:(1)土生空团菌有一定的遗传多样性,且遗传差异主要来自于居群内;(2)基因流Nm>1,遗传漂变不是导致土生空团菌居群遗传分化的主要因素;(3)土生空团菌的遗传分化受到地理环境的影响,而与宿主来源没有明显相关性。     

Lasers-an effective artificial source of radiation for the cultivation of anoxygenic photosynthetic bacteria

The laser diode (LD) is a unique light source that can efficiently produce all radiant energy within the narrow wavelength range used most effectively by a photosynthetic microorganism. We have investigated the use of a single type of LD for the cultivation of the well-studied anoxygenic photosynthetic bacterium, Rhodobacter capsulatus (Rb. capsulatus). An array of vertical-cavity surface-emitting lasers (VCSELs) was driven with a current of 25 mA, and delivered radiation at 860 nm with 0.4 nm linewidth. The emitted light was found to be a suitable source of radiant energy for the cultivation of Rb. capsulatus. The dependence of growth rate on incident irradiance was quantified. Despite the unusual nearly monochromatic light source used in these experiments, no significant changes in the pigment composition and in the distribution of bacteriochlorophyll between LHII and LHI-RC were detected in bacterial cells transferred from incandescent light to laser light. We were also able to show that to achieve a given growth rate in a light-limited culture, the VCSEL required only 30% of the electricity needed by an incandescent bulb, which is of great significance for the potential use of laser-devices in biotechnological applications and photobioreactor construction.     

中国黄瓜主栽品种SSR遗传多样性分析及指纹图谱构建

应用简单重复序列(SSR)标记对从国内主要黄瓜育种单位征集到的116份生产上主栽品种进行遗传多样性分析。结果表明35对引物共扩增出86个等位基因,每对引物平均扩增出2.46个等位基因,其中有效等位基因占70.99%,平均Shannon’s信息指数为0.639,平均PIC为0.382。116个品种的遗传相似系数(GS)分布在0.5029~0.9797之间。聚类分析结果表明在遗传距离0.25处可将供试材料分为2大类群。第1类共106个品种,可分为5个亚族,主要包括华北密刺型、华南型、日本少刺型这3大类型;第2类包含10个欧洲温室型品种。     

南京小龙山钾矿区植物根际可培养细菌的遗传多样性分析

摘要:【目的】矿区优势植物可培养细菌生物多样性研究将有助于了解植物根际细菌与矿物,植物根系相互作用及对矿物风化和土壤形成的重要影响。【方法】采用纯培养法分离南京小龙山废钾矿区野生植物野塘蒿,千金子和栽培植物甘薯根内与根周围土壤的可培养细菌, 通过16S rDNA限制性酶切多态性分析(amplified rDNA restriction analysis, ARDRA)和16S rDNA序列分析研究了可培养细菌的多样性。【结果】分离纯化到60株具不同菌落形态的可培养细菌, 在60%相似水平上可分为18个OTU. 19株代表菌株分别属于3个门, 10个科, 11个属。多数菌株属于变形菌门(α-proteobacteria, 4株, 21.1%; β-proteobacteria, 2株, 10.5%; γ-proteobacteria, 6株, 31.6%)。假单胞菌属(Pseudomonas), 泛菌属(Pantoea)和根瘤菌属(Rhizobium)为优势种群。【结论】小龙山废矿区优势植物根围具有丰富的微生物种群多样性。     

光合菌生物制氢技术

简要分析了光合细菌产氢的主要影响因素,介绍了国内外光合细菌生物制氢技术的研究和应用现状,并对光合制氢技术的发展趋势和应用前景进行了评述。     

中国7个地方绵羊品遗传多样性的微卫星分析

利用26个微卫星标记分析了中国7个地方绵羊（Ovis aries）品种的遗传多样性。通过计算基因频率,平均杂合度（H）、多态信息含量（PIC）及有效等位基因数（Ne）,并根据Nei氏标准遗传距离,利用UPGMA法进行了聚类分析,评估其种内遗传变异和品种间遗传关系。结果表明：26个微卫星位点共检测到278个等位基因,Ne在2.1288-13.3924之间;26个位点均为高度多态位点,PIC在0.6628-0.8712之间,聚类分析表明哈萨克羊、阿勒泰羊和巴音布鲁克羊遗传关系最近;然后与白藏羊,黑藏羊聚为一类,湖羊和晋中羊聚为一类,各绵羊各种的聚类关系与其来源,育成史及地理分布基本一致。     

Genetic diversity of carbofuran-degrading soil bacteria

The genetic diversity of 128 carbofuran-degrading bacteria was determined by ARDRA (amplified ribosomal DNA restriction analysis) of 16S rDNA and restriction fragment length polymorphism analysis of the 16S-23S rDNA spacer region (IGS) using five endonucleases. The isolates were distributed in 26 distinct ARDRA groups and 45 IGS types revealing a high level of microbial diversity confirmed by ARDRA clustering and sequencing of 16S rDNA. The occurrence of a methylcarbamate-degrading gene (mcd) was monitored by polymerase chain reaction amplification using specific primers. The mcd gene was detected only in 58 bacteria and there was no clear relationship between the presence of this gene and the phylogenetic position of the strain.     

Abundance and diversity of aerobic anoxygenic phototrophic bacteria in saline lakes on the Tibetan plateau

Aerobic anoxygenic phototrophic (AAP) bacteria are heterotrophic prokaryotes that are capable of utilizing light as an energy source but are not capable of producing molecular oxygen. Recently, multiple studies have found that AAP bacteria are widely distributed in oceans and estuaries and may play an important role in carbon cycling. However, AAP bacteria in inland lake ecosystems have not been investigated in depth. In this study, the abundance and diversity of the pufL-M genes, encoding photosynthetic reaction centers of AAP bacteria, were determined in the oxic water column and anoxic sediments of saline lakes (Qinghai, Erhai, and Gahai Lakes) on the Tibetan Plateau, China. Our results indicated that AAP bacteria were abundant in inland lakes, with the proportion of AAP bacteria (in total bacteria) comparable to those in the oceans, but with a lower diversity. Salinity and pH were found to be potential factors controlling the AAP bacterial diversity and community composition. Our data have implications for a better understanding of the potential role of AAP bacteria in carbon cycling in inland lake ecosystems.     

Real-time PCR for quantification of aerobic anoxygenic phototrophic bacteria based on pufM gene in marine environment

The abundance of aerobic anoxygenic phototrophic bacteria (AAPB), a new functional group that plays important roles in marine carbon cycling, is determined frequently by infrared epifluorescence microscopic analysis (IREM) or high-performance liquid chromatography (HPLC) based on detecting BChl a (bacteriochlorophyll a) fluorescence signal at 880 nm. Unfortunately, the fluorescence signal is often influenced by environmental variables and physiological state of cell. Here we developed a real-time quantitative PCR (qPCR) assay based on pufM gene to specifically quantify AAPB in marine environments. High specificity and sensitivity for estimation of AAPB abundance were revealed by analysis of amplification products, melting curves and target sequences. The phylogenetic tree indicated that this primer set is suitable for a wide genetic diversity of AAPB, including α-3, α-4 Proteobacteria and clones of unclear taxonomic position. In contrast, no amplicon was obtained from green non-sulphur bacteria and oxygenic phototrophic bacteria such as Cyanobacterial genomic DNA. The melting behavior could indicate predominant phenotypes in AAPB community in addition to validating the products of qPCR. The AAPB was estimated to range from 1.3 × 10⁴ cell/ml to 3.4 × 10⁵ cell/ml in our 10 tested water samples by this qPCR assay. Further investigations on the abundance distribution of AAPB in marine environments using the qPCR assay may provide new insight into their ecological functions.     

Phylogenetic analysis of photosynthetic genes of Rhodocyclus gelatinosus: Possibility of horizontal gene transfer in purple bacteria

Nucleotide sequences of the genes coding for the M and cytochrome subunits of the photosynthetic reaction center of Rhodocyclus gelatinosus, a purple bacterium in the subdivision, were determined. The deduced amino acid sequences of these proteins were compared with those of other photosynthetic bacteria. Based on the homology of these two photosynthetic proteins, Rc. gelatinosus was placed in the subdivision of purple bacteria, which disagrees with the phylogenetic trees based on 16S rRNA and soluble cytochrome c ₂. Horizontal transfer of the genes which code for the photosynthetic apparatus in purple bacteria can be postulated if the phylogenetic trees based on 16S rRNA and soluble cytochrome c ₂ reflect the real history of purple bacteria.Abbreviations LH I light harvesting complex I - RC reaction center     

罗汉松遗传多样性的SCoT分析

采用SCoT分子标记技术对8份罗汉松种质材料进行遗传多样性研究。结果表明,从80条引物中筛选出10条重复性好、条带清晰的引物进行PCR扩增,共产生136条带,其中多态性带122条(占88.97%),8个罗汉松种质间的遗传相似系数范围在0.39～0.80说明罗汉松的遗传多样性丰富。利用UPGMA进行系统的聚类分析显示,将8份罗汉松材料分为2大类;主成分分析结果与聚类分析结果相一致。可见,利用SCoT分子标记可有效的分析罗汉松种质资源的遗传多样性,为罗汉松种质亲缘关系的鉴别和分类提供理论依据。     

Stepwise reduction of the culture redox potential allows the analysis of microaerobic metabolism and photosynthetic membrane synthesis in Rhodospirillum rubrum

Bacterial growth under oxygen‐limited (microaerobic) conditions is often accompanied by phenomena of great interest for fundamental research and industrial application. The microaerobic lifestyle of anoxygenic photosynthetic bacteria like Rhodospirillum rubrum harbors such a phenomenon, as it allows the formation of photosynthetic membranes and related interesting products without light. However, due to the technical difficulties in process control of microaerobic cultivations and the limited sensitivity of available oxygen sensors, the analysis of microaerobic growth and physiology is still underrepresented in current research. The main focus of the present study was to establish an experimental set‐up for the systematic study of physiological processes, associated with the growth of R. rubrum under microaerobic conditions in the dark. For this purpose, we introduce a robust and reliable microaerobic process control strategy, which applies the culture redox potential (CRP) for assessing different degrees of oxygen limitation in bioreactor cultivations. To describe the microaerobic growth behavior of R. rubrum cultures for each of these defined CRP reduction steps, basic growth parameters were experimentally determined. Flux variability analysis provided an insight into the metabolic activity of the TCA cycle and implied its connection to the respiratory capacity of the cells. In this context, our results suggest that microaerobic growth of R. rubrum can be described as an oxygen‐activated cooperative mechanism. The present study thus contributes to the investigation of metabolic and regulatory events responsible for the redox‐sensitive formation of photosynthetic membranes in facultative photosynthetic bacteria. Furthermore, the introduced microaerobic cultivation setup should be generally applicable for any microbial system of interest which can be cultivated in common stirred‐tank bioreactors. Biotechnol. Bioeng. 2013; 110: 573–585. © 2012 Wiley Periodicals, Inc.     

共代谢条件下光合细菌对2-氯苯酚的生物降解

光合细菌PSB-1D不能利用2-氯苯酚(2-CP)作为唯一的碳源和能源.选用苹果酸、丙酸钠、乙酸钠、柠檬酸钠、苯酚、葡萄糖和可溶性淀粉等7种不同碳源作为光合细菌PSB-1D降解2-CP的共代谢基质,考察了在黑暗好氧培养条件下,不同共代谢基质对PSB-1D生长及降解2-CP效果的影响.结果表明:葡萄糖能够很好地促进PSB-1D的大量繁殖,提高降解效果,缩短降解周期,为最佳共代谢基质.对葡萄糖的投加浓度进行了优化,当葡萄糖的投加浓度为3 g·L-1时,菌株PSB-1D培养168 h后的菌体生长浓度△D560为1.749,2-CP的半衰期为3.9 d,降解速率常数为0.00864 h-1.采用SDS-PAGE对微生物全细胞蛋白质进行分析发现,在共代谢过程中当菌株PSB-1D利用葡萄糖作为底物提供能源和碳源时,可诱导产生2-CP特异性降解酶.