Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: a polyphasic study

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N(2)-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.     

Growth dynamics of a laminated microbial mat in response to variable irradiance in an Antarctic lake

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广西水牛瘤胃中的细菌多样性

[目的]了解广西水牛瘤胃中细菌的组成及其可能的降解纤维素细菌的主要类群。[方法]提取水牛瘤胃内容物和高效降解滤纸的水牛瘤胃内容物的富集培养物的宏基因组DNA,以宏基因组DNA为模板,扩增16S rRNA基因序列,构建该两种样品的细菌的16S rRNA基因文库。通过对16S rRNA基因序列的分析,了解这两种样品的细菌群体种类及数量。 [结果] 水牛瘤胃内容物与其富集培养物中均主要含有LGCGPB (low G+C Gram-Positive Bacteria)、CFB (Cytophaga-Flexibacter-Bacteroides)两大类菌群和少数的螺旋体菌（Spirochaetes）,且LGCGPB所占的比例都是最高的,LGCGPB在水牛瘤胃内容物细菌中的比例为56.66%,而在富集培养物细菌中的比例升高为73.33%。在水牛瘤胃内容物中,丝状杆菌（Fibrobacteres）占3.33%,但在富集培养物中未被检测到。而在富集培养物中占13.33%的变形杆菌（Proteobacteria）,在水牛瘤胃内容物中未被检测到。本研究还发现了分类地位尚未明确的一菌群（R46）。[结论]细菌类群LGCGPB、Proteobacteria可能在水牛瘤胃中的纤维素降解过程中起重要作用。此外,水牛瘤胃中的细菌组成和牦牛、牛、羊瘤胃中的细菌组成较相似但比例有所不同。     

Bacterial diversity in organically-enriched fish farm sediments

The bacterial diversity and community structure within both organically enriched and adjacent, unimpacted, near-shore marine sediments at two fish farms in southern Tasmania, Australia, was examined using 16S rRNA gene clone library construction and analysis. Sediments at both caged and reference sites at both farms showed a very high level of microbial diversity. Over 900 clones were analysed and grouped into 631 unique phylotypes. Reference sites were dominated by Delta- and Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cage site sediments were also dominated by these phylotypes, as well as members of the Alpha- and Epsilonproteobacteria. Diversity and coverage indices indicated that the actual diversity of the sediments was much greater than that detected, despite a large sampling effort. All libraries were shown to be statistically different from one another (P < 0.05). Many phylotypes did not group with cultured bacteria, but grouped with other environmental clones from a wide array of marine benthic environments. Diversity and evenness indices suggested that although both parameters changed after farming, diverse communities were present in all sediments. The response of the microbial community to organic load suggested that random, rather than predictable, succession events determine community composition and diversity, and that sediment type may influence bacterial community and sediment response to organic perturbation.     

Ultrastructural and genetic characteristics of endolithic cyanobacterial biofilms colonizing Antarctic granite rocks

The precise identification of the cyanobacteria that comprise an endolithic biofilm is hindered by difficulties in culturing the organisms found in these biofilms and a lack of previous molecular and ultrastructural data. This study characterizes, both at the ultrastructural and molecular level, two different cyanobacterial biofilms found in fissures of granite from continental Antarctica. Electron microscopy revealed structural differences between the two biofilms. One was only loosely adhered to the substrate, while the other biofilm showed a closer association between cells and rock minerals and was tightly attached to the substrate. Cells from both biofilms where ultrastructurally distinct, displaying, for instance, clear differences in their sheaths. The amounts of EPS and their organization associated with the cyanobacteria may determine the differences in adhesion and effects on the lithic substrate observed in the biofilms. By sequencing part of the 16S rRNA gene, the two cyanobacteria were also genetically characterized. The gene sequence of the cells comprising the biofilm that was tightly attached to the lithic substrate showed most homology with that of an endolithic cyanobacterium from Switzerland (AY153458), and the cyanobacterial type loosely adhered to the rock, clustered with Acaryochloris marina, the only organism unequivocally known to contain chlorophyll d. This study reveals the presence of at least two different types of endolithic biofilm, dominated each by a single type of cyanobacterium, able to withstand the harsh conditions of the Antarctic climate.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Phylogenetic and statistical analyses of 16S rRNA gene libraries were used for the investigation of actinobacterial communities present in two tropical estuarine sediments (Santos-São Vicente estuary, Brazil). The libraries were constructed from samples collected at the brackish end of the estuary, highly hydrocarbon-contaminated, and at the marine end, uncontaminated. Clones from the marine end of the estuary were all related to sequences from non-cultured Actinobacteria and unidentified bacteria recovered from a wide range of environmental samples, whereas clones from the brackish end were mainly related to sequences from cultured Actinobacteria. Statistical analyses showed that the community recovered from the hydrocarbon-contaminated sediment sample, at the brackish end, was less diverse than the uncontaminated one, at the marine end, and that the communities from the two libraries were differently structured, suggesting that these may have not originated from the same community. The recognition of the spatial pattern of actinobacterial distribution in a natural environment is a first step towards understanding the way these communities are organized, providing valuable data for further investigations of their taxonomic and functional diversity.     

Common evolutionary origin of planktonic and benthic nitrogen-fixing oscillatoriacean cyanobacteria from tropical oceans

The filamentous cyanobacteria belonging to the genus Hydrocoleum (Blennothrix) are among the most common mat-forming cyanobacteria in tropical oceans. We present here the evidence that these benthic cyanobacteria are morphologically and phylogenetically very close to the planktonic species of Trichodesmium. Genetic relationship was established independently with regard to sequences of the 16S rRNA gene, nifH gene, and phycocyanin and phycoerythrin intergenic spacers. The species of both genera formed a common distinct branch in phylogenetically reconstructed cyanobacterial trees, suggesting that the main constituents of cyanobacterial benthos and plankton have an early common origin and both represent major contributors to nitrogen budget of tropical oceans today as in the distant geological past.     

Seasonal rather than spatial variability drives planktonic and benthic bacterial diversity in a microtidal lagoon and the adjacent open sea

Coastal lagoons are highly productive ecosystems, which are experiencing a variety of human disturbances at increasing frequency. Bacteria are key ecological players within lagoons, yet little is known about the magnitude, patterns and drivers of diversity in these transitional environments. We carried out a seasonal study in the Venice Lagoon (Italy) and the adjacent sea, to simultaneously explore diversity patterns in different domains (pelagic, benthic) and their spatio‐temporal variability, and test the role of environmental gradients in structuring assemblages. Community composition differed between lagoon and open sea, and between domains. The dominant phyla varied temporally, with varying trends for the two domains, suggesting different environmental constraints on the assemblages. The percentage of freshwater taxa within the lagoon increased during higher river run‐off, pointing at the lagoon as a dynamic mosaic of microbial taxa that generate the metacommunity across the whole hydrological continuum. Seasonality was more important than spatial variability in shaping assemblages. Network analyses indicated more interactions between several genera and environmental variables in the open sea than the lagoon. Our study provides evidences for a temporally dynamic nature of bacterial assemblages in lagoons and suggests that an interplay of seasonally influenced environmental drivers shape assemblages in these vulnerable ecosystems.     

青岛太平湾潮间带趋磁细菌多样性

在青岛太平湾潮间带沉积物中发现了一定量的海洋趋磁细菌,最大丰度可达350个/cm~3。透射电镜观察发现该区域趋磁细菌均为趋磁球菌。磁小体个体形状单一,皆是立方体状;磁小体排列方式多样,以链状排列为主,包括单链、双链与多链,也有少数成簇排列。EDS结果表明,磁小体成分为四氧化三铁。据估算,趋磁细菌的铁元素含量(干重)范围在0.40%—6.91%之间,平均为2.19%。通过16S rRNA基因文库的构建与测序得到了47个趋磁细菌序列,分属13个OTU。系统发育分析结果表明,它们都属于α-变形菌纲,其中9个OTU与已知最相似序列的相似性低于97%,有5个OTU与已知最相似序列的相似性低于93%,可能代表了趋磁细菌的9个新种、5个新属,说明该区域潜在的微生物新种质资源十分可观。     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.     

A function of cyanobacterial mats in phosphorus-limited tropical wetlands

Cyanobacterial mats are important components of oligotrophic wetland ecosystems in the limestone-based regions of the Caribbean. Our goals were to: (1) Estimate the biomass and primary production of cyanobacterial mats, quantify the extent of nitrogen fixation and measure the activity of alkaline phosphatase (APA) in representative marshes of northern Belize; (2) Record changes in these variables following nutrient additions. The mat biomass ranged from 200 to 700 g m^–2 AFDM, with the epipelon contributing up to 87% of the total. Tissue nitrogen was similar in all marshes (1.1–1.5%), while tissue phosphorus was extremely low (0.0055–0.0129%) and well correlated with the N:P ratio in water. Nitrogen fixation expressed as nitrogenase activity was high in some marshes (17.5 nmol C₂H₄ cm^–2 h^–1) and low (< 5 nmol C₂H₄ cm^–2 h^–1) in others depending mainly on the proportion of heterocyst-forming cyanobacteria (Nostocales, Stigonematales) in the mat. Alkaline phosphatase activity was positively correlated with the N:P ratio of the mat. Experimental addition of phosphorus resulted in significant increase in primary production and nitrogen fixation while it suppressed the APA activity. The presented data clearly showed that oligotrophic marshes of northern Belize are strongly P limited. Increased input of phosphorus would profoundly change their structure and functions.     

新疆艾比湖和伊吾湖可培养嗜盐古菌多样性

新疆地区盐湖密布,蕴藏着丰富的微生物资源。为保护和利用微生物物种与基因资源,作者从新疆准噶尔盆地的艾比湖和天山山间盆地的伊吾湖分离纯化嗜盐微生物。采用PCR方法扩增其中65株嗜盐古菌16SrRNA基因序列。序列分析表明,分离的嗜盐古菌分属6个属,艾比湖以Haloterrigena和Natrinema属的菌株为主,伊吾湖由Haloarcula和Halorubrum两个属的菌株构成。通过多样性指数、丰富度指数、均匀度指数和物种相对多度模型对分离的菌株进行多样性分析和比较,结果表明,盐湖嗜盐古菌的多样性指数、丰富度指数和均匀度指数具有一定相关性,艾比湖可培养嗜盐古菌的多样性高于伊吾湖。研究发现了一些新的物种资源,表明新疆盐湖中孕育的特色微生物资源亟待保护与利用。     

On the use of high‐throughput sequencing for the study of cyanobacterial diversity in Antarctic aquatic mats

The study of Antarctic cyanobacterial diversity has been mostly limited to morphological identification and traditional molecular techniques. High‐throughput sequencing (HTS) allows a much better understanding of microbial distribution in the environment, but its application is hampered by several methodological and analytical challenges. In this work, we explored the use of HTS as a tool for the study of cyanobacterial diversity in Antarctic aquatic mats. Our results highlight the importance of using artificial communities to validate the parameters of the bioinformatics procedure used to analyze natural communities, since pipeline‐dependent biases had a strong effect on the observed community structures. Analysis of microbial mats from five Antarctic lakes and an aquatic biofilm from the Sub‐Antarctic showed that HTS is a valuable tool for the assessment of cyanobacterial diversity. The majority of the operational taxonomic units retrieved were related to filamentous taxa such as Leptolyngbya and Phormidium, which are common genera in Antarctic lacustrine microbial mats. However, other phylotypes related to different taxa such as Geitlerinema, Pseudanabaena, Synechococcus, Chamaesiphon, Calothrix, and Coleodesmium were also found. Results revealed a much higher diversity than what had been reported using traditional methods and also highlighted remarkable differences between the cyanobacterial communities of the studied lakes. The aquatic biofilm from the Sub‐Antarctic had a distinct cyanobacterial community from the Antarctic lakes, which in turn displayed a salinity‐dependent community structure at the phylotype level.     

Covariation between zooplankton community composition and cyanobacterial community dynamics in Lake Blaarmeersen (Belgium)

The cyanobacterial community composition in the mesotrophic Lake Blaarmeersen was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments during two consecutive years to assess the importance of different classes of explanatory variables (bottom-up and top-down factors, physical variables and phytoplankton) in cyanobacterial community dynamics. The most dominant cyanobacteria in Lake Blaarmeersen were Synechococcus (three genotypes), Limnothrix redekei and Anabaena/Aphanizomenon. Analyses of Similarity revealed that the cyanobacterial community in Lake Blaarmeersen differed significantly between the growing season and the winter season as well as between the epilimnion and hypolimnion during the stratified periods. Mantel tests revealed significant correlations between the DGGE data and bottom-up factors, physical variables, the phytoplankton community composition and, interestingly, the zooplankton community composition. In general, the zooplankton community composition (especially the cladoceran community) was more important in structuring the cyanobacterial community than the total zooplankton biomass. This study shows that grazing zooplankton communities can have a relatively strong impact on the cyanobacterial community dynamics and that this impact can be equally important as bottom-up processes regulated by nutrient concentrations and/or physical variables.     

普通和稀释培养基研究太湖沉积物可培养细菌的多样性

采用普通牛肉汁培养基和 10倍稀释的普通牛肉汁培养基 (以下简称稀释培养基 )研究太湖沉积物中细菌多样性 ,发现在稀释培养基上生长的细菌数量普遍是在普通牛肉汁琼脂培养基上生长的细菌数量的 3～ 5倍。分离得到纯培养物的 16SrDNA部分序列 (5′端约 5 0 0bp)分析表明 ,不同培养基上生长的优势细菌类群存在差别 :普通培养基生长的细菌主要为γ_Proteobacteria(35. 1% ) ,其次为Actinobacteria(2 4 5 % )和Firmicutes(2 2 . 3% )等类群 ,其中大部分细菌与假单胞菌属 (Pseudomoas)、芽孢杆菌属 (Bacillus)和节杆菌属 (Archrobacter)细菌的系统关系密切 ;稀释培养基生长的细菌则主要为Actinobacteria(2 7. 1% )、Firmicutes(2 5 . 7% )、α_Proteobacteria(2 1. 4 % )和γ_Proteobacteria(15. 7% )等类群 ,与芽孢杆菌属 (Bacillus) (2 5. 7% )发育系统关系密切的细菌为优势属。研究结果表明同时采用两种培养基有助于从太湖沉积物中分离到更多种微生物。     

Effect of sample handling on estimation of bacterial diversity in marine sediments by 16S rRNA gene sequence analysis

Abstract The diversity of bacterial communities in deep marine sediments, up to 503 metres below the sea floor of the Japan Sea, was investigated by sequence analysis of amplified 16S rRNA genes. The use of different sample handling procedures greatly affected the types and diversity of sequences obtained. DNA from sediment samples stored aerobically for up to 24 h before freezing was dominated by sequences belonging to the β- and γ-proteobacteria, many of which appeared to originate from aerobic bacteria. Sub-samples equilibrated anaerobically at 16°C, were then injected with a radiotracer and immediately frozen, to simulate the conditions of a typical control sample from a radiotracer based activity assay, contained mostly α-proteobacterial sequences. Pristine sediment samples taken anaerobically and frozen within 2 h contained the widest diversity of sequences from α-, γ-, δ-proteobacteria and Gram-positive bacteria, which appeared to have originated from predominantly anaerobic or facultative bacteria. It was clear that both samples that were not frozen immediately (within 2 h) showed signs of enrichment of specific bacterial groups. Our results strongly suggest that immediate freezing should always be employed when sediment samples are to be used to assess bacterial diversity by molecular methods.     

Feedbacks of plant identity and diversity on the diversity and community composition of rhizosphere microbiomes from a long‐term biodiversity experiment

Soil microbes are known to be key drivers of several essential ecosystem processes such as nutrient cycling, plant productivity and the maintenance of plant species diversity. However, how plant species diversity and identity affect soil microbial diversity and community composition in the rhizosphere is largely unknown. We tested whether, over the course of 11 years, distinct soil bacterial communities developed under plant monocultures and mixtures, and if over this time frame plants with a monoculture or mixture history changed in the bacterial communities they associated with. For eight species, we grew offspring of plants that had been grown for 11 years in the same field monocultures or mixtures (plant history in monoculture vs. mixture) in pots inoculated with microbes extracted from the field monoculture and mixture soils attached to the roots of the host plants (soil legacy). After 5 months of growth in the glasshouse, we collected rhizosphere soil from each plant and used 16S rRNA gene sequencing to determine the community composition and diversity of the bacterial communities. Bacterial community structure in the plant rhizosphere was primarily determined by soil legacy and by plant species identity, but not by plant history. In seven of the eight plant species the number of individual operational taxonomic units with increased abundance was larger when inoculated with microbes from mixture soil. We conclude that plant species richness can affect below‐ground community composition and diversity, feeding back to the assemblage of rhizosphere bacterial communities in newly establishing plants via the legacy in soil.