Actinomycetal Community Structures in Seawater and Freshwater Examined by DGGE Analysis of 16S rRNA Gene Fragments

The actinomycetal community structures in marine and freshwater environments (the Pacific Ocean, East China Sea, Tokyo Bay, and Arakawa River) were investigated by a culture-independent molecular method to clarify spatial and seasonal distributions. Deoxyribonucleic acid (DNA) was extracted from environmental water samples, and a community analysis was carried out on polymerase chain reaction-amplified 16S ribosomal DNA. The amplified DNA fragments were analyzed by denaturing gradient gel electrophoresis (DGGE) and nonmetric multidimensional scaling analysis, followed by sequencing analysis. The actinomycetal community structures were different at each station in the Pacific Ocean, the East China Sea, Tokyo Bay, and Arakawa River, and different populations predominated in each area. There were vertical variations in actinomycetal communities in the Pacific Ocean and East China Sea between the surface and 100-m depth, but communities were similar from 200- to 1,000-m depths. There were also distinct seasonal variations in communities in Tokyo Bay. Phylogenetic analysis of DNA fragments recovered from DGGE bands revealed that most of the predominant actinomycetal strains were uncultured and were quite different from well known culturable strains, such as the Streptomyces, Micromonospora, Microbispora, Salinispora, and Actinoplanes groups. These results suggest that the marine environment is an attractive target for discovering new actinomycetal populations producing bioactive compounds and that sampling depth and season are important considerations for isolating various populations effectively.     

Phylogenetic Diversity and Ecological Pattern of Ammonia-oxidizing Archaea in the Surface Sediments of the Western Pacific

The phylogenetic diversity of ammonia-oxidizing archaea (AOA) was surveyed in the surface sediments from the northern part of the South China Sea (SCS). The distribution pattern of AOA in the western Pacific was discussed through comparing the SCS with other areas in the western Pacific including Changjiang Estuary and the adjacent East China Sea where high input of anthropogenic nitrogen was evident, the tropical West Pacific Continental Margins close to the Philippines, the deep-sea methane seep sediments in the Okhotsk Sea, the cold deep sea of Northeastern Japan Sea, and the hydrothermal field in the Southern Okinawa Trough. These various environments provide a wide spectrum of physical and chemical conditions for a better understanding of the distribution pattern and diversities of AOA in the western Pacific. Under these different conditions, the distinct community composition between shallow and deep-sea sediments was clearly delineated based on the UniFrac PCoA and Jackknife Environmental Cluster analyses. Phylogenetic analyses showed that a few ammonia-oxidizing archaeal subclades in the marine water column/sediment clade and endemic lineages were indicative phylotypes for some environments. Higher phylogenetic diversity was observed in the Philippines while lower diversity in the hydrothermal vent habitat. Water depth and possibly with other environmental factors could be the main driving forces to shape the phylogenetic diversity of AOA observed, not only in the SCS but also in the whole western Pacific. The multivariate regression tree analysis also supported this observation consistently. Moreover, the functions of current and other climate factors were also discussed in comparison of phylogenetic diversity. The information collectively provides important insights into the ecophysiological requirements of uncultured ammonia-oxidizing archaeal lineages in the western Pacific Ocean.     

长江口及东海春季底栖硅藻、原生动物和小型底栖生物的生态特点

利用Ludox-QPS方法并结合沉积环境因子的综合分析,研究了2011年4月采自长江口及东海10个站位以底栖硅藻、纤毛虫和异养小鞭毛虫为代表的微型底栖生物及小型底栖生物的组成、丰度和生物量、分布及生态特点。结果表明,底栖硅藻的丰度 (5.92 ? 104 ind/10 cm2) 和生物量 (83.29 ?g C/10 cm2) 远高于纤毛虫 (丰度为1036 ind/10 cm2,生物量为3.33 ?g C/10 cm2)、异养小鞭毛虫 (丰度为4451 ind/10 cm2,生物量为2.51 ?g C/10 cm2) 和小型底栖生物 (丰度为1947 ? 849 ind/10 cm2,生物量为49.01? 22.05 ?g C/10 cm2)。在鉴定出的11个小型底栖生物类群中,线虫占小型底栖生物总丰度的90%和总生物量的37%。底栖硅藻生物量在长江口及东海海域呈由近岸向外海逐渐降低的分布特点,而底栖纤毛虫、异养小鞭毛虫及小型底栖生物的分布则正相反。在垂直分布上,76%的硅藻和80%的线虫分布在0–2 cm沉积物表层,仅1%的硅藻和6%的线虫分布在5–8 cm分层。统计分析表明,底栖硅藻的现存量与沉积物中叶绿素a含量呈极显著的正相关,与底层水温度呈弱的正相关;该海域底栖原生动物和小型底栖生物的分布受多个因子而非单一环境因子的共同作用。对比分析表明,长江口及东海单位体积沉积物中的硅藻丰度较水体中的硅藻丰度高2个数量级,沉积物中相当部分的叶绿素a含量可能系底栖硅藻所贡献;表层8 cm沉积物中纤毛虫的丰度约是上层30 m水柱中纤毛虫丰度的30倍,生物量约是后者的40倍。尽管纤毛虫在生物量上远小于小型底栖生物,但其估算的生产力约是后者的3倍;而异养小鞭毛虫由于个体更小,其周转率可能较纤毛虫更高。长江口及东海陆架区原生动物和小型底栖生物的高现存量及生产力预示着其在该海域生态系统中的重要作用。     

Diversity,Abundance, and Spatial Distribution of Ammonia-Oxidizing β-Proteobacteria in Sediments from Changjiang Estuary and Its Adjacent Area in East China Sea

Changjiang Estuary, the largest estuary in China, encompasses a wide range of nutrient loading and trophic levels from the rivers to the sea, providing an ideal natural environment to explore relationships between functional diversity, physical/chemical complexity, and ecosystem function. In this study, molecular biological techniques were used to analyze the community structure and diversity of ammonia-oxidizing bacteria (AOB) in the sediments of Changjiang Estuary and its adjacent waters in East China Sea. The amoA gene (encoding ammonia monooxygenase subunit A) libraries analysis revealed extensive diversity within the β-Proteobacteria group of AOB, which were grouped into Nitrosospira-like and Nitrosomonas-like lineages. The majority of amoA gene sequences fell within Nitrosospira-like clade, and only a few sequences were clustered with the Nitrosomonas-like clade, indicating that Nitrosospira-like lineage may be more adaptable than Nitrosomonas-like lineage in this area. Multivariate statistical analysis indicated that the spatial distribution of the sedimentary β-Proteobacterial amoA genotype assemblages correlated significantly with nitrate, nitrite, and salinity. The vertical profile of amoA gene copies in gravity cores showed that intense sediment resuspension led to a deeper mixing layer. The horizontal distribution pattern of amoA gene copies was nearly correlated with the clayey mud belt in Changjiang Estuary and its adjacent area in East China Sea, where higher β-Proteobacteria phylogenetic diversity was observed. Meanwhile, those areas with high amoA copies in the surface sediments nearly matched those with low concentrations of dissolved oxygen and ammonium in the bottom water.     

应用DGGE法对青海相邻两盐湖中细菌多样性的快速检测

分别对青海相邻两盐湖柯柯盐湖、茶卡盐湖土样、泥样进行富集培养后,从中提取的DNA用两套不同的细菌通用引物进行扩增,分别得到包含V8和V9高变区的16SrDNA片断。经变性梯度凝胶电泳(DGGE)分析,结果显示这两个盐湖富集样品中细菌多样性具有较大的差异,而且同一盐湖不同性质样品中的细菌多样性差异也较大,两湖的泥样富集样品中均表现出了稍丰富的多样性。     

呼伦贝尔草原不同退化梯度土壤细菌多样性季节变化

为了研究草地退化程度与土壤微生物多样性的关系,在呼伦贝尔草地上选取羊草草甸草原和贝加尔针茅草甸草原两个典型放牧点,按照轻度、中度和重度划分取样点,分别于6、8月份和10月份3个不同季节采集土壤样品。应用变性梯度凝胶电泳技术(PCR-DGGE)研究两个放牧地点不同退化程度、不同季节草地的细菌群落结构变化。结果表明,呼伦贝尔草地不同退化梯度的草地土壤中细菌种类较为丰富。从丰富度和Shannon-Winner指数的变化看,两个放牧点8月份丰富度和Shannon-Winner指数最高,8月份的丰富度平均为32.4,比6月和10月份分别高11%和7.4%;8月份Shannon-Winner指数平均为4.15,比6月和10月份分别高7.7%和5.4%。DGGE图谱聚类分析结果显示,随着季节变化和草地退化程度由轻至重的变化,土壤中的细菌优势种群没有受到明显的影响。回收DGGE图谱中10个条带进行测序分析,结果显示,所有序列与GenBank数据库中的相似度在87%100%之间。基于98%的相似度,可将其中的7个鉴定为Proteobacteria(变形菌门),将其中的1个鉴定为Actinobacteria(放线菌门)。另外2个同已知序列相似性较低,可能是未知的细菌。结果表明,Proteobacteria(变形菌门)为呼伦贝尔草原土壤中的优势细菌类群,尽管所选取样点草地植被有不同程度的退化,但土壤微生物优势种群并没有发生变化。     

Vertical distribution of bacterial community structure in the sediments of two eutrophic lakes revealed by denaturing gradient gel electrophoresis (DGGE) and multivariate analysis techniques

Vertical distribution of bacterial community structure was investigated in the sediments of two eutrophic lakes of China, Lake Taihu and Lake Xuanwu. Profiles of bacterial communities were generated using a molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequence analysis, and the results were interpreted with multivariate statistical analysis. To assess changes in the genetic diversity of bacterial communities with changing depth, DGGE banding patterns were analysed by cluster analysis. Distinct clusters were recognized in different sampling stations of Lake Taihu. Canonical correspondence analysis (CCA) was carried out to infer the relationship between environmental variables and bacterial community structure. DGGE samples collected at the same sampling site clustered together in both lakes. Total phosphorus, organic matter and pH were considered to be the key factors driving the changes in bacterial community composition.     

长江口及其陆架春季小型底栖生物丰度和生物量

2007年4—5月对长江口及其陆架海域小型底栖生物进行了调查。结果表明:调查海域有18个类群小型底栖生物,平均丰度为(1117.19±820.36)个/10 cm2,平均生物量为(754.87±546.37)(μg.干重)/10 cm2,其中线虫是绝对优势类群,占小型底栖生物总丰度的95.42%,占总生物量的55.35%。其他优势类群分别是桡足类、多毛类和动吻类。垂直分布研究表明:小型底栖生物丰度随着深度的增加而减少,其数量的65%分布于0—2 cm层,其中线虫分布于0—2 cm层的数量约占64%;桡足类集中分布于0—2 cm层,占该类总量的88%以上;多毛类分布于0—2 cm层的数量约占61%。各区块小型底栖生物总丰度从高到低依次为东海陆架深水区长江口近岸杭州湾。与环境因子相关分析表明:盐度与小型底栖生物丰度,线虫丰度成显著的正相关,与多毛类,桡足类丰度以及小型底栖生物生物量呈极显著的正相关。近底溶解氧与小型底栖生物丰度,多毛类,线虫丰度以及小型底栖生物生物量呈显著的负相关,与桡足类丰度呈极显著的负相关。调查海域沉积物的中值粒径,粘土粉砂含量与小型底栖生物丰度,多毛类,线虫丰度以及小型底栖生物生物量的相关性不显著。位沉积物叶绿素a和脱镁叶绿素含量与小型底栖生物丰度,线虫丰度、多毛类丰度,桡足类丰度相关不显著;沉积物中异养细菌含量与多毛类丰度呈显著负相关。     

Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H'), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from beta-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.     

Picophytoplankton, nanophytoplankton, heterotrohpic bacteria and viruses in the Changjiang Estuary and adjacent coastal waters

Flow cytometry (FCM) was used to examine the abundances anddistributions of different picophytoplankton groups (i.e. Synechococcus,Prochlorococcus and picoeukaryotes), nanophytoplankton, heterotrophicbacteria and viruses were examined in the Changjiang Estuary,China and adjacent coastal waters during autumn 2004. Watertemperature and light availability were found to be criticalfactors for picophytoplankton growth. Positive correlationswere found between picophytoplankton, heterotrophic bacteriaand viruses, and a seaward-increasing trend in the V-I (thegroup yielding high green fluorescence according to FCM) populationwithin viruses was detected. The importance of nanophytoplanktonis progressively usurped by picophytoplankton with increasingdistance offshore. Picoeukaryotes are the most successful groupamong picophytoplankton in near-shore eutrophic waters, whereasProchlorococcus surpasses other groups within the pico- andnanophytoplankton community in offshore oligotrophic regionsof the East China Sea Shelf.     

长江口及邻近海域浮游动物群落结构及季节变化

根据2006—2007年长江口及其邻近海域(29°30'N—32°30'N,120°00'E—127°30'E)150个站位4个季节的调查资料,对长江口海域浮游动物群落结构、种类组成、优势种及其季节变化进行研究。结果表明,长江口及其邻近海域浮游动物群落物种多样性丰富,四季共鉴定浮游动物460种,隶属7个门,246属,此外,另有54类浮游幼体。其中,桡足类是最优势类群,有193种,占41.96%;端足类为第二优势类群,有51种,占11.09%;水螅水母为第三优势类群,有34种,占7.39%。长江口及其邻近海域浮游动物的物种多样性呈现明显季节变化,其特征为:夏季(317种)秋季(309种)春季(230种)冬季(138种)。中华哲水蚤和百陶带箭虫为长江口及其邻近海域的四季优势种。长江口及其邻近海域浮游动物大体可划分为5种生态类群:近岸低盐类群、广温广盐类群、低温高盐类群、高温广盐类群和高温高盐类群。结合同步调查的水文和水化学数据,进行浮游动物群落丰度与环境因子的相关分析表明:盐度是影响长江口及其邻近海域的浮游动物群落丰度的主要环境因子。     

Differential response of archaeal and bacterial communities to nitrogen inputs and pH changes in upland pasture rhizosphere soil

Grassland management regimens influence the structure of archaeal communities in upland pasture soils, which appear to be dominated by as yet uncultivated non-thermophilic Crenarchaeota. In an attempt to determine which grassland management factors select for particular crenarchaeal community structures, soil microcosm experiments were performed examining the effect of increased pH, application of inorganic fertilizer (ammonium nitrate) and sheep urine deposition on both archaeal and bacterial communities in unmanaged grassland soil. As grassland management typically increases pH, a further experiment examined the effect of a reduction in pH, to that typical of unimproved grassland soils, on archaeal and bacterial communities. The RT-PCR amplification of 16S rRNA followed by denaturing gradient gel electrophoresis analysis demonstrated a distinct and reproducible effect on bacterial communities after incubation for 28 or 30 days. In contrast, none of the treatments had a significant effect on the structure of the crenarchaeal community, indicating that these factors are not major drivers of crenarchaeal community structures in grassland soils.     

黄海和东海营养盐分布及其对浮游植物的限制

根据1997～1999年黄海和东海4个季节的现场调查资料,分析探讨了黄海、东海的营养盐分布特征及其对浮游植物生长的限制状况．结果表明,在长江口以东及其东北部海域终年存在一个范围很大的营养盐高值区．分析表明,这些营养盐主要来自长江冲淡水的扩展及苏北沿岸流的输送．此外,还获得了1998年长江流域特大洪水期间,迄今被观测到的长江冲淡水中营养盐的最大扩展范围,计算并研究了黄海、东海上层水中Si／N／P比值,结果表明,黄东海上层水中Si／N比值较高,Si不会成为黄东海浮游植物生长的限制因子;但在南黄海南部尤其是西南部、东海近岸及长江口以东海域,N／P比值很高(>30),说明与一般海洋环境的情况不同,黄海、东海有很大一部分海域浮游植物的生长受磷限制,而不是受氮限制．     

Links between archaeal community structure, vegetation type and methanogenic pathway in Alaskan peatlands

Although northern peatlands contribute significantly to natural methane emissions, recent studies of the importance and type of methanogenesis in these systems have provided conflicting results. Mechanisms controlling methanogenesis in northern peatlands remain poorly understood, despite the importance of methane as a greenhouse gas. We used 16S rRNA gene retrieval and denaturing gradient gel electrophoresis (DGGE) to analyse archaeal communities in 15 high-latitude peatland sites in Alaska and three mid-latitude peatland sites in Massachusetts. Archaeal community composition was analysed in the context of environmental, vegetation and biogeochemical factors characterized in a parallel study. Phylogenetic analysis revealed that Alaskan sites were dominated by a cluster of uncultivated crenarchaeotes and members of the families Methanomicrobiaceae and Methanobacteriaceae, which are not acetoclastic. Members of the acetoclastic family Methanosarcinaceae were not detected, whereas those of the family Methanosaetaceae were either not detected or were minor. These results are consistent with biogeochemical evidence that acetoclastic methanogenesis is not a predominant terminal decomposition pathway in most of the sites analysed. Ordination analyses indicated a link between vegetation type and archaeal community composition, suggesting that plants (and/or the environmental conditions that control their distribution) influence both archaeal community activity and dynamics.     

Stratified Communities of Active Archaea in Shallow Sediments of the Pearl River Estuary, Southern China

Marine subsurface sediments represent a novel archaeal biosphere with unknown physiology. To get to know the composition and ecological roles of the archaeal communities within the sediments of the Pearl River Estuary, Southern China, the diversity and vertical distribution of active archaea in a sediment core were characterized by 16S rRNA phylogenetic analysis of clone libraries derived from RNA. In this study, the archaeal diversity above, within, and beneath the sulfate-methane transition zone (SMTZ) in the Pearl River Estuary sediment core was described. The majority of the clones obtained from the metabolically active fraction of the archaeal community were most closely related to miscellaneous crenarchaeotal group and terrestrial miscellaneous euryarchaeotal group. Notably, although the Pearl River Estuary sediment belong to high methane and high organic carbon environment, sequences affiliated with methanotrophic and methanogenic archaea were detected as minor group in 16S rRNA clone libraries. No obvious evidence suggested that these unknown archaeal phylotypes related directly to anaerobic oxidation of methane in SMTZ. This is the first phylogenetic analysis of the metabolically active fraction of the archaeal community in the coastal sediment environments.     

Succession of Bacterial Community Structure along the Changjiang River Determined by Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H′), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from β-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.     

Effects of water regime on archaeal community composition in Arctic soils

Effects of water regime on archaeal communities in Arctic soils from Spitsbergen were studied using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA genes, with subsequent sequencing of amplicons and ordination analysis of binary DGGE data. Samples with major differences in soil water regime showed significant differences in their archaeal community profiles. Methanomicrobiales, Methanobacteriaceae and Methanosaeta were detectable only in environments that were wet during most of the growth season, while a novel euryarchaeotal cluster was detected only in less reduced solifluction material. Group 1.3b of Crenarchaeota had a high relative abundance within the archaeal community in a wide range of wet soils. Along a natural soil moisture gradient, changes in archaeal community composition were observed only in upper soil layers. The results indicated that members of Methanomicrobiales were relatively tolerant to soil aeration. Differences in archaeal community composition associated with soil water regime were predominant over regional and seasonal variation, and over differences between individual wetlands. The results suggest that the observed 'on-off switch' mechanism of soil hydrology for large-scale variations in methane emissions from northern wetlands is at least partly caused by differences in the community structure of organisms involved in methane production.     

Direct comparison of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) to characterize a microbial community on the basis of 16S rRNA gene fragments

Characterization of microbial communities using single-strand conformation polymorphism (SSCP) was compared with that using denaturing gradient gel electrophoresis (DGGE). This comparison was based on the V3-4 region (Escherichia coli positions: 341-806) of 16S rRNA gene of bacterial or archaeal communities obtained from a methanogenic bioreactor. Significant differences in the bacterial banding profiles were observed while attempting to detect the diversity of the community and its succession during the reactor operation. The SSCP produced a number of sharp bands and differentiated the bacterial community structures to which the DGGE gave an identical pattern. On the other hand, the SSCP and DGGE provided similar succession patterns for archaeal community.     

Different Effects of Transgenic Maize and Nontransgenic Maize on Nitrogen-Transforming Archaea and Bacteria in Tropical Soils

The composition of the rhizosphere microbiome is a result of interactions between plant roots, soil, and environmental conditions. The impact of genetic variation in plant species on the composition of the root-associated microbiota remains poorly understood. This study assessed the abundances and structures of nitrogen-transforming (ammonia-oxidizing) archaea and bacteria as well as nitrogen-fixing bacteria driven by genetic modification of their maize host plants. The data show that significant changes in the abundances (revealed by quantitative PCR) of ammonia-oxidizing bacterial and archaeal communities occurred as a result of the maize host being genetically modified. In contrast, the structures of the total communities (determined by PCR-denaturing gradient gel electrophoresis) were mainly driven by factors such as soil type and season and not by plant genotype. Thus, the abundances of ammonia-oxidizing bacterial and archaeal communities but not structures of those communities were revealed to be responsive to changes in maize genotype, allowing the suggestion that community abundances should be explored as candidate bioindicators for monitoring the possible impacts of cultivation of genetically modified plants.     

Distribution of Cren- and Euryarchaeota in Scots Pine Mycorrhizospheres and Boreal Forest Humus

Archaeal 16S rRNA gene sequences have been found in a variety of moderate-temperature habitats including soil and rhizospheres. In this study, the differences of archaeal communities associated with Scots pine (Pinus sylvestris L.) short roots, different types of mycorrhizospheric compartments, and uncolonized boreal forest humus were tested by direct DNA extraction, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE), and sequencing. The results indicated that mycorrhizal colonization of Scots pine roots substantially influence the archaeal community of pine rhizospheres. Colonization of short roots by most mycorrhizal fungi tested increased both archaeal frequency and diversity. Most of the archaeal sequences encountered in mycorrhizas belonged to the phylum Euryarchaeota, order of Halobacteriales. The difference in archaeal diversity between the mycorrhizospheric compartments and humus was profound. Most compartments with fungal components contained euryarchaeotal 16S rRNA gene sequences, whereas a high diversity of crenarchaeotal sequences and no euryarchaeotal sequences were found in forest humus outside mycorrhizospheres.