Metagenomic analysis of a freshwater toxic cyanobacteria bloom

High molecular weight (HMW) DNA prepared from a toxic freshwater cyanobacterial bloom sample was used to construct a PCR-generated 75-clone, 16S rRNA gene library and a 2850-clone bacterial artificial chromosome (BAC) library. Phylogenetic analysis of the 16S rRNA gene library demonstrated that members of eight phyla of domain Bacteria, which included Cyanobacteria, Actinobacteria, Verrucomicrobium, Bacteriodetes, Planctomycetes, Chloroflexi, Candidate Division OP10 and Alpha-, Beta- and Gammaproteobacteria, were present in the bloom community. Diversity estimates determined from 16S rRNA gene analysis and direct cell counts and morphological identification of phytoplanktons suggested that the bloom community was dominated by members of the genera Aphanizomenon and Cylindrospermopsis, phylum Cyanobacteria. BAC-end sequencing of 37 randomly selected clones and subsequent sequence analysis provided a snapshot of the total bloom community putative metabolic activities. The sequencing of the entire inserts of seven clones (clones designated 578, 67, 142, 543, 905, 1664 and 2089) selected from BAC-end sequence studies resulted in the generation of a total of 144-kb sequence data and in the identification of 130 genes for putative proteins representing at least four phyla, Proteobacteria, Actinobacteria, Bacteroidetes and Cyanobacteria. This is the first report on a snapshot analysis of a limited metagenome of a toxic cyanobacterial freshwater bloom.     

Molecular phylogenetic diversity of the microbial community associated with a high-temperature petroleum reservoir at an offshore oilfield

The microbial community and its diversity in production water from a high-temperature, water-flooded petroleum reservoir of an offshore oilfield in China were characterized by 16S rRNA gene sequence analysis. The bacterial and archaeal 16S rRNA gene clone libraries were constructed from the community DNA and, using sequence analysis, 388 bacterial and 220 archaeal randomly selected clones were clustered with 60 and 28 phylotypes, respectively. The results showed that the 16S rRNA genes of bacterial clones belonged to the divisions Firmicutes, Thermotogae, Nitrospirae and Proteobacteria, whereas the archaeal library was dominated by methanogen-like rRNA genes (Methanothermobacter, Methanobacter, Methanobrevibacter and Methanococcus), with a lower percentage of clones belonging to Thermoprotei. Thermophilic microorganisms were found in the production water, as well as mesophilic microorganisms such as Pseudomonas and Acinetobacter-like clones. The thermophilic microorganisms may be common inhabitants of geothermally heated specialized subsurface environments, which have been isolated previously from a number of high-temperature petroleum reservoirs worldwide. The mesophilic microorganisms were probably introduced into the reservoir as it was being exploited. The results of this work provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs at offshore oilfields.     

Biological and chemical factors driving the temporal distribution of cyanobacteria and heterotrophic bacteria in a eutrophic lake (West Lake,China)

Physico-chemical parameters, hydrological conditions, and microbial interactions can affect the growth and persistence of cyanobacteria, but the interacting effects among these bloom-forming factors are still poorly known. This hampers our capacity to predict the occurrence of cyanobacterial bloom accurately. Here, we studied the relationship between temperature, N and P cycles, and the microbial community abundance and diversity at 0.5 m under the surface of West Lake (China) from January 21 to November 20, 2015, in order to better understand the key factors regulating temporal changes in the cyanobacterial community. Using high throughput sequencing of the 16S rRNA gene V3-V4 region, we studied the diversity and abundance of bacteria. In parallel, we measured physico-chemical parameters and followed the abundance of key genes involved in N fixation, denitrification, and nutrient uptake. Multivariate analyses suggest that P concentration and water temperature are the key factors controlling the outbreak of summer cyanobacterial bloom. RT-qPCR analyses of the bacterial community and measurements of the copy number of denitrification-related gene (nirK, nosZ, nirS) show that denitrification potential and denitrifying bacteria relative abundance (Pseudomonas and Bacillus) increased in concert with diazotrophic cyanobacterial genera (Anabaena, Nostoc, Aphanizomenon flos-aquae) and the common bloom-forming non-diazotrophic cyanobacterium genus Microcystis. The present study brings new insights on the complex interplay between physico-chemical parameters, heterotrophic bacterial community composition, nitrogen cycle, and cyanobacteria dominance in a eutrophic lake.

     

Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat.

Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment cultures of aerobic chemoorganotrophic bacteria from the ca. 50 degrees C region of the mat community. By varying the degree of dilution of the inoculum, medium composition, and enrichment conditions and duration and by analyzing the cultures by DGGE, we detected 14 unique 16S rRNA sequence types. These corresponded to alpha-, beta-, gamma-, and delta-proteobacteria, Thermus relatives, and gram-positive bacteria with high G + C ratio and, at the highest inoculum dilutions, Chloroflexus aurantiacus relatives, which were estimated to still be approximately 300 times less abundant than cells of the mat primary producer, Synechococcus spp. Only three of these populations were previously cultivated on solidified medium after similar enrichment. Only two of these population have 16S rRNA sequences which were previously cloned directly from the mat. These results reveal a diversity of bacterial populations in enrichment culture which were not detected by either molecular retrieval or strain purification techniques.     

Species composition and cyanotoxin production in periphyton mats from three lakes of varying trophic status

In lakes, benthic micro-algae and cyanobacteria (periphyton) can contribute significantly to total primary productivity and provide important food sources for benthic invertebrates. Despite recognition of their importance, few studies have explored the diversity of the algal and cyanobacterial composition of periphyton mats in temperate lakes. In this study, we sampled periphyton from three New Zealand lakes: Tikitapu (oligotrophic), ōkāreka (mesotrophic) and Rotoiti (eutrophic). Statistical analysis of morphological data showed a clear delineation in community structure among lakes and highlighted the importance of cyanobacteria. Automated rRNA intergenic spacer analysis (ARISA) and 16S rRNA gene clone libraries were used to investigate cyanobacterial diversity. Despite the close geographic proximity of the lakes, cyanobacterial species differed markedly. The 16S rRNA gene sequence analysis identified eight cyanobacterial OTUs. A comparison with other known cyanobacterial sequences in GenBank showed relatively low similarities (91-97%). Cyanotoxin analysis identified nodularin in all mats from Lake Tikitapu. ndaF gene sequences from these samples had very low (≤ 89%) homology to sequences in other known nodularin producers. To our knowledge, this is the first detection of nodularin in a freshwater environment in the absence of Nodularia. Six cyanobacteria species were isolated from Lake Tikitapu mats. None were found to produce nodularin. Five of the species shared low (< 97%) 16S rRNA gene sequence similarities with other cultured cyanobacteria.     

不同轮作茬口土壤细菌群落及后茬小麦产量

评估不同轮作模式的生态可持续性和作物生产力,可为调整优化种植结构提供理论依据。设置7个不同轮作作物和周期茬口处理,采用实时荧光定量PCR技术测定不同轮作茬口的土壤细菌群落丰度,采用16S rRNA基因扩增子高通量测序技术分析土壤细菌群落多样性与物种组成,并测定土壤速效养分状况和后茬小麦产量。结果表明: 与夏玉米茬口相比,不同轮作周期夏花生或夏大豆茬口处理降低了土壤有机碳、无机氮和速效钾含量,显著增加了土壤有效磷含量。不同轮作周期夏花生或夏大豆茬口处理的土壤细菌16S rRNA基因拷贝数显著降低,而群落丰富度和多样性有所增加。不同轮作作物显著改变了土壤细菌群落结构和物种组成。与夏玉米茬口相比,不同轮作周期夏大豆茬口显著增加了后茬冬小麦籽粒千粒重和产量。综上,不同轮作周期夏花生或夏大豆茬口有利于增加土壤有效磷含量和细菌群落多样性,显著改变土壤细菌群落结构,其中,夏大豆茬口对后茬冬小麦产量形成具有积极作用。     

Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes

The diversity of freshwater bacterioplankton communities has not been extensively studied despite their key role in foodwebs and the cycling of carbon and associated major elements. In order to explore and characterize the composition of bacterioplankton associated with cyanobacterial blooms, large 16S rRNA clone libraries from four lakes experiencing such blooms were analysed. The four libraries contained 1461 clones, of which 559 were prokaryotic sequences of non-cyanobacterial origin. These clones were classified into 158 operational taxonomic units affiliated mainly with bacterial divisions commonly found in freshwater systems, e.g. Proteobacteria, Bacteriodetes, Actinobacteria, Verrucomicrobia and Planctomycetes. Richness and evenness of non-cyanobacterial clones were similar to other clone libraries obtained for freshwater bacterioplankton, suggesting that bacterial communities accompanying cyanobacterial blooms are as diverse as non-bloom communities. Many of the identified operational taxonomic units grouped with known freshwater clusters but the libraries also contained novel clusters of bacterial sequences that may be characteristic for cyanobacterial blooms. About 25% of the operational taxonomic units were detected in more than one lake. Even so, 16S rRNA heterogeneity analysis demonstrated large differences in community composition between lakes regardless of their similar characteristics and close proximity. Hence even the similar environmental conditions created by different cyanobacterial blooms may foster very dissimilar bacterial communities, which could indicate that the genetic diversity in lake bacteria have been underestimated in the past.     

Influence of depth and time on diversity of free-living microbial community in the variably saturated zone of a granitic aquifer

We investigated the temporal and vertical changes in the microbial communities related to hydrological variations an aquifer (Brittany, France). Five water samplings were carried out, spanning three hydrological cycles in the variably and the permanently saturated zones. Seasonal variations in the major anion concentrations (NO3 -, SO4 2- and Cl(-) ) indicated that different physical processes occurred during the recharge process in the two zones. The variably saturated zone is mainly dominated by diffusion and advection processes from the soil, whereas the permanently saturated zone is controlled by moderate advective transfer from the variably saturated zone. Bacterial diversity was investigated by flow cytometry, 16S rRNA and narG genes analyses. Part of this diversity was new in that 6 of the 27 16S rRNA gene sequence phylotypes were unknown even at the class or phylum level. The narG gene analysis did not reveal any clear variation in time or depth within the nitrate reducers' community. In contrast, 16S rRNA gene analyses showed modifications of community composition that could be related to the hydrologic and chemical contrast between the two zones. It was concluded that the physical processes of water transfer could influence bacterial diversity at the soil-aquifer interface.     

Seasonal distributions of dominant 16S rRNA-defined populations in a hot spring microbial mat examined by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments was used to examine the distributions of bacterial populations within a hot spring microbial mat (Octopus Spring, Yellowstone National Park). Populations at sites along the thermal gradient of the spring's effluent channel were surveyed at seasonal intervals. No shift in the thermal gradient was detected, and populations at spatially or temperature-defined sites exhibited only slight changes over the annual sampling period. A new cyanobacterial 16S rRNA sequence type was detected at temperatures from 63 to 75 degrees C. A new green nonsulfur bacterium-like sequence type was also detected at temperatures from 53 to 62 degrees C. Genetically unique though closely related cyanobacterial and green nonsulfur bacterium-like populations were successively distributed along the thermal gradient of the Octopus Spring effluent channel. At least two cyanobacterial populations were detected at each site; however, a limited ability to detect some cyanobacterial populations suggests that only dominant populations were observed.     

Substitution by Inosine at the 3��-Ultimate and Penultimate Positions of 16S rRNA Gene Universal Primers

Universal 16S rRNA gene primers (8F and 518R) bearing inosine substitutions at either the 3??-ultimate or the 3??-ultimate and penultimate base positions were exploited for the first time to study the bacterial community associated with coral polymicrobial Black Band Disease (BBD). Inosine-modified universal primer pairs display some shifting in the composition of 16S rRNA gene libraries, as well as expanding the observed diversity of a BBD bacterial community at the family/class level. Possible explanations for the observed shifts are discussed. These results thus point to the need for adopting multiple approaches in designing 16S rRNA universal primers for PCR amplification and subsequent construction of 16S rRNA gene libraries or pyrosequencing in the exploration of complex microbial communities.     

Monitoring changing toxigenicity of a cyanobacterial bloom by molecular methods

Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001. Malpas Dam has been shown in the past to have toxic blooms of Microcystis aeruginosa that have caused liver damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the genes for the beta and alpha subunits. The potential for microcystin production was determined by PCR amplification of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by PCR amplification of a region of the 16S rRNA gene of Anabaena circinalis strains. Toxicity of samples was established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy and mouse bioassay. We also show that toxigenic strains of Anabaena and Microcystis spp. occur at this site and that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR detection of potential toxicity can enhance the management of a significant public health hazard.     

Comparative microbial diversity analyses of modern marine thrombolitic mats by barcoded pyrosequencing

Thrombolites are unlaminated carbonate structures that form as a result of the metabolic interactions of complex microbial mat communities. Thrombolites have a long geological history; however, little is known regarding the microbes associated with modern structures. In this study, we use a barcoded 16S rRNA gene-pyrosequencing approach coupled with morphological analysis to assess the bacterial, cyanobacterial and archaeal diversity associated with actively forming thrombolites found in Highborne Cay, Bahamas. Analyses revealed four distinct microbial mat communities referred to as black, beige, pink and button mats on the surfaces of the thrombolites. At a coarse phylogenetic resolution, the domain bacterial sequence libraries from the four mats were similar, with Proteobacteria and Cyanobacteria being the most abundant. At the finer resolution of the rRNA gene sequences, significant differences in community structure were observed, with dramatically different cyanobacterial communities. Of the four mat types, the button mats contained the highest diversity of Cyanobacteria, and were dominated by two sequence clusters with high similarity to the genus Dichothrix, an organism associated with the deposition of carbonate. Archaeal diversity was low, but varied in all mat types, and the archaeal community was predominately composed of members of the Thaumarchaeota and Euryarchaeota. The morphological and genetic data support the hypothesis that the four mat types are distinctive thrombolitic mat communities.     

Uncultivated cyanobacteria, Chloroflexus-like inhabitants, and spirochete-like inhabitants of a hot spring microbial mat.

Analysis of 16S rRNA sequences retrieved as cDNA (16S rcDNA) from the Octopus Spring cyanobacterial mat has permitted phylogenetic characterization of some uncultivated community members, expanding our knowledge or diversity within this microbial community. Two new cyanobacterial 16S rRNA sequences were discovered, raising to four the number of cyanobacterial sequence types known to occur in the mat. None of the sequences found is that of the cultivated thermophilic cyanobacterium Synechococcus lividus. A new 16S rRNA sequence characteristic of green nonsulfur bacteria and their relatives was discovered, raising to two the number of such sequences known to exist in the mat. Both are unique among the 16S rRNA sequences of cultivated members of this group, including an Octopus Spring isolate of Chloroflexus aurantiacus and Heliothrix oregonensis, whose sequences we report herein. Two spirochete-like 16S rRNA sequences were discovered. One can be placed in the leptospira subdivision of the spirochete group, but the other has such a loose affiliation with the spirochete group that it might actually belong to an as yet unrecognized subdivision or even to a new eubacterial line of descent.     

Uncultivated cyanobacteria, Chloroflexus-like inhabitants, and spirochete-like inhabitants of a hot spring microbial mat.

Analysis of 16S rRNA sequences retrieved as cDNA (16S rcDNA) from the Octopus Spring cyanobacterial mat has permitted phylogenetic characterization of some uncultivated community members, expanding our knowledge or diversity within this microbial community. Two new cyanobacterial 16S rRNA sequences were discovered, raising to four the number of cyanobacterial sequence types known to occur in the mat. None of the sequences found is that of the cultivated thermophilic cyanobacterium Synechococcus lividus. A new 16S rRNA sequence characteristic of green nonsulfur bacteria and their relatives was discovered, raising to two the number of such sequences known to exist in the mat. Both are unique among the 16S rRNA sequences of cultivated members of this group, including an Octopus Spring isolate of Chloroflexus aurantiacus and Heliothrix oregonensis, whose sequences we report herein. Two spirochete-like 16S rRNA sequences were discovered. One can be placed in the leptospira subdivision of the spirochete group, but the other has such a loose affiliation with the spirochete group that it might actually belong to an as yet unrecognized subdivision or even to a new eubacterial line of descent.     

Bacterial diversity based on 16S rRNA and gyrB genes at Yinshan mine, China

The diversity of bacterial communities at three sites impacted by acid mine drainage (AMD) from the Yinshan Mine in China was studied using comparative sequence analysis of two molecular markers, the 16S rRNA and gyrB genes. The phylogenetic analyses retrieved sequences from six classes of bacteria, Nitrospira, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Acidobacteria, and Actinobacteria, as well as sequences related to the plastid of the cyanobacterium Cyanidium acidocaldarium and also some unknown bacteria. The results of phylogenetic analyses based on gyrB and 16S rRNA were compared. This confirmed that gyrB gene analysis may be a useful tool, in addition to the comparative sequence analysis of the 16S rRNA gene, for the analysis of microbial community compositions. Moreover, the Mantel test showed that the geochemical characteristics, especially the pH value and the concentration of iron, strongly influenced the composition of the microbial communities.     

Soil Bacterial Diversity Screening Using Single 16S rRNA Gene V Regions Coupled with Multi-Million Read Generating Sequencing Technologies

The novel multi-million read generating sequencing technologies are very promising for resolving the immense soil 16S rRNA gene bacterial diversity. Yet they have a limited maximum sequence length screening ability, restricting studies in screening DNA stretches of single 16S rRNA gene hypervariable (V) regions. The aim of the present study was to assess the effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. Using an in silico approach, the performance of each V region was compared with the complete 16S rRNA gene stretch. We assessed related properties of the soil derived bacterial sequence collection of the Ribosomal Database Project (RDP) database and concomitantly performed simulations based on published datasets. Results indicate that overall the most prominent V region for soil bacterial diversity studies was V3, even though it was outperformed in some of the tests. Despite its high performance during most tests, V4 was less conserved along flanking sites, thus reducing its ability for bacterial diversity coverage. V5 performed well in the non-redundant RDP database based analysis. However V5 did not resemble the full-length 16S rRNA gene sequence results as well as V3 and V4 did when the natural sequence frequency and occurrence approximation was considered in the virtual experiment. Although, the highly conserved flanking sequence regions of V6 provide the ability to amplify partial 16S rRNA gene sequences from very diverse owners, it was demonstrated that V6 was the least informative compared to the rest examined V regions. Our results indicate that environment specific database exploration and theoretical assessment of the experimental approach are strongly suggested in 16S rRNA gene based bacterial diversity studies.     

Phylogenetic diversity of bacteria in an earth-cave in Guizhou province, southwest of China

The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.     

Cultivation of aerobic chemoorganotrophic proteobacteria and gram-positive bacteria from a hot spring microbial mat.

The diversity of aerobic chemoorganotrophic bacteria inhabiting the Octopus Spring cyanobacterial mat community (Yellowstone National Park) was examined by using serial-dilution enrichment culture and a variety of enrichment conditions to cultivate the numerically significant microbial populations. The most abundant bacterial populations cultivated from dilutions to extinction were obtained from enrichment flasks which contained 9.0 x 10(2) primary producer (Synechococcus spp.) cells in the inoculum. Two isolates exhibited 16S rRNA nucleotide sequences typical of beta-proteobacteria. One of these isolates contained a 16S rRNA sequence identical to a sequence type previously observed in the mat by molecular retrieval techniques. Both are distantly related to a new sequence directly retrieved from the mat and contributed by a beta-proteobacterial community member. Phenotypically diverse gram-positive isolates genetically similar to Bacillus flavothermus were obtained from a variety of dilutions and enrichment types. These isolates exhibited identical 16S rRNA nucleotide sequences through a variable region of the molecule. Of the three unique sequences observed, only one had been previously retrieved from the mat, illustrating both the inability of the cultivation methods to describe the composition of a microbial community and the limitations of the ability of molecular retrieval techniques to describe populations which may be less abundant in microbial communities.