Lise Øvreås

Microbial diversity and function in soil: from genes to ecosystems

Soils sustain an immense diversity of microbes, which, to a large extent, remains unexplored. A range of novel methods, most of which are based on rRNA and rDNA analyses, have uncovered part of the soil microbial diversity. The next step in the era of microbial ecology is to extract genomic, evolutionary and functional information from bacterial artificial chromosome libraries of the soil community genomes (the metagenome). Sophisticated analyses that apply molecular phylogenetics, DNA microarrays, functional genomics and in situ activity measurements will provide huge amounts of new data, potentially increasing our understanding of the structure and function of soil microbial ecosystems, and the interactions that occur within them. This review summarizes the recent progress in studies of soil microbial communities with focus on novel methods and approaches that provide new insight into the relationship between phylogenetic and functional diversity.

Novel techniques for analysing microbial diversity in natural and perturbed environments

Molecular techniques were applied for analysing the entire bacterial community, including both the cultivated and non-cultivated part of the community. DNA was extracted from samples of soils and sediments, and a combination of different molecular methods were used to investigate community structure and diversity in these environments. Reassociation of sheared and thermally denatured DNA in solution was used to measure the total genetical diversity. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of rRNA genes gave information about changes in the numerically dominating bacterial populations. Hybridisation with phylogenetic group specific probes, and sequencing provided information about the affiliation of the bacterial populations. Using DNA reassociation analysis we demonstrated that bacterial communities in pristine soil and sediments may contain more than 10,000 different bacterial types. The diversity of the total soil community was at least 200 times higher than the diversity of bacterial isolates from the same soil. This indicates that the culturing conditions select for a distinct subpopulation of the bacteria present in the environment. Molecular methods were applied to monitor the effects of perturbations due to antropogenic activities and pollution on microbial communities. Our investigations show that agricultural management, fish farming and pollution may lead to profound changes in the community structure and a reduction in the bacterial diversity.

Microbial Diversity and Community Structure in Two Different Agricultural Soil Communities

A bstractIn this study, two different agricultural soils were investigated: one organic soil and one sandy soil, from Stend (south of Bergen), Norway. The sandy soil was a field frequently tilled and subjected to crop rotations. The organic soil was permanent grazing land, infrequently tilled. Our objective was to compare the diversity of the cultivable bacteria with the diversity of the total bacterial population in soil. About 200 bacteria, randomly isolated by standard procedures, were investigated. The diversity of the cultivable bacteria was described at phenotypic, phylogenetic, and genetic levels by applying phenotypical testing (Biolog) and molecular methods, such as amplified rDNA restriction analysis (ARDRA); hybridization to oligonucleotide probes; and REP-PCR. The total bacterial diversity was determined by reassociation analysis of DNA isolated from the bacterial fraction of environmental samples, combined with ARDRA and DGGE analysis. The relationship between the diversity of cultivated bacteria and the total bacteria was elucidated. Organic soil exhibited a higher diversity for all analyses performed than the sandy soil. Analysis of cultivable bacteria resulted in different resolution levels and revealed a high biodiversity within the population of cultured isolates. The difference between the two agricultural soils was significantly higher when the total bacterial population was analyzed than when the cultivable population was. Thus, analysis of microbial diversity must ultimately embrace the entire microbial community DNA, rather than DNA from cultivable bacteria.

Correlating microbial community profiles with geochemical data in highly stratified sediments from the Arctic Mid-Ocean Ridge

Microbial communities and their associated metabolic activity in marine sediments have a profound impact on global biogeochemical cycles. Their composition and structure are attributed to geochemical and physical factors, but finding direct correlations has remained a challenge. Here we show a significant statistical relationship between variation in geochemical composition and prokaryotic community structure within deep-sea sediments. We obtained comprehensive geochemical data from two gravity cores near the hydrothermal vent field Loki’s Castle at the Arctic Mid-Ocean Ridge, in the Norwegian-Greenland Sea. Geochemical properties in the rift valley sediments exhibited strong centimeter-scale stratigraphic variability. Microbial populations were profiled by pyrosequencing from 15 sediment horizons (59,364 16S rRNA gene tags), quantitatively assessed by qPCR, and phylogenetically analyzed. Although the same taxa were generally present in all samples, their relative abundances varied substantially among horizons and fluctuated between Bacteria- and Archaea-dominated communities. By independently summarizing covariance structures of the relative abundance data and geochemical data, using principal components analysis, we found a significant correlation between changes in geochemical composition and changes in community structure. Differences in organic carbon and mineralogy shaped the relative abundance of microbial taxa. We used correlations to build hypotheses about energy metabolisms, particularly of the Deep Sea Archaeal Group, specific Deltaproteobacteria, and sediment lineages of potentially anaerobic Marine Group I Archaea. We demonstrate that total prokaryotic community structure can be directly correlated to geochemistry within these sediments, thus enhancing our understanding of biogeochemical cycling and our ability to predict metabolisms of uncultured microbes in deep-sea sediments.

Microbial community diversity in seafloor basalt from the Arctic spreading ridges

Microbial communities inhabiting recent (< or =1 million years old; Ma) seafloor basalts from the Arctic spreading ridges were analyzed using traditional enrichment culturing methods in combination with culture-independent molecular phylogenetic techniques. Fragments of 16S rDNA were amplified from the basalt samples by polymerase chain reaction, and fingerprints of the bacterial and archaeal communities were generated using denaturing gradient gel electrophoresis. This analysis indicates a substantial degree of complexity in the samples studied, showing 20-40 dominating bands per profile for the bacterial assemblages. For the archaeal assemblages, a much lower number of bands (6-12) were detected. The phylogenetic affiliations of the predominant electrophoretic bands were inferred by performing a comparative 16S rRNA gene sequence analysis. Sequences obtained from basalts affiliated with eight main phylogenetic groups of Bacteria, but were limited to only one group of the Archaea. The most frequently retrieved bacterial sequences affiliated with the gamma-proteobacteria, alpha-proteobacteria, Chloroflexi, Firmicutes, and Actinobacteria. The archaeal sequences were restricted to the marine Group 1: Crenarchaeota. Our results indicate that the basalt harbors a distinctive microbial community, as the majority of the sequences differed from those retrieved from the surrounding seawater as well as from sequences previously reported from seawater and deep-sea sediments. Most of the sequences did not match precisely any sequences in the database, indicating that the indigenous Arctic ridge basalt microbial community is yet uncharacterized. Results from enrichment cultures showed that autolithotrophic methanogens and iron reducing bacteria were present in the seafloor basalts. We suggest that microbial catalyzed cycling of iron may be important in low-temperature alteration of ocean crust basalt. The phylogenetic and physiological diversity of the seafloor basalt microorganisms differed from those previously reported from deep-sea hydrothermal systems.

Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy

A SYBR Green real-time quantitative PCR (Q-PCR) assay for the detection and quantification of Bacteria and Archaea present in the glassy rind of seafloor basalts of different ages and water depths is presented. Two sets of domain-specific primers were designed and validated for specific detection and quantification of bacterial and archaeal 16S rRNA genes in DNA extracted from basaltic glass. Total cell numbers were also estimated by fluorescence microscopy analysis of SYBR Gold-stained samples. The results from the two different approaches were concurrent, and Q-PCR results showed that the total number of cells present in basalts was in the range from 6 x 10(5) to 4 x 10(6) cells g(-1) basaltic glass. Further, it was demonstrated that these cells were almost exclusively from the domain Bacteria. When applying the same methods on samples of different ages (22 years-0.1 Ma) and water depths (139-3390 mbsl), no significant differences in cell concentrations or in the relative abundance of Archaea and Bacteria were detected.

CREST – Classification Resources for Environmental Sequence Tags

Sequencing of taxonomic or phylogenetic markers is becoming a fast and efficient method for studying environmental microbial communities. This has resulted in a steadily growing collection of marker sequences, most notably of the small-subunit (SSU) ribosomal RNA gene, and an increased understanding of microbial phylogeny, diversity and community composition patterns. However, to utilize these large datasets together with new sequencing technologies, a reliable and flexible system for taxonomic classification is critical. We developed CREST (Classification Resources for Environmental Sequence Tags), a set of resources and tools for generating and utilizing custom taxonomies and reference datasets for classification of environmental sequences. CREST uses an alignment-based classification method with the lowest common ancestor algorithm. It also uses explicit rank similarity criteria to reduce false positives and identify novel taxa. We implemented this method in a web server, a command line tool and the graphical user interfaced program MEGAN. Further, we provide the SSU rRNA reference database and taxonomy SilvaMod, derived from the publicly available SILVA SSURef, for classification of sequences from bacteria, archaea and eukaryotes. Using cross-validation and environmental datasets, we compared the performance of CREST and SilvaMod to the RDP Classifier. We also utilized Greengenes as a reference database, both with CREST and the RDP Classifier. These analyses indicate that CREST performs better than alignment-free methods with higher recall rate (sensitivity) as well as precision, and with the ability to accurately identify most sequences from novel taxa. Classification using SilvaMod performed better than with Greengenes, particularly when applied to environmental sequences. CREST is freely available under a GNU General Public License (v3) from http://apps.cbu.uib.no/crest and http://lcaclassifier.googlecode.com.

Planctomycetes dominate biofilms on surfaces of the kelp Laminaria hyperborea

BackgroundBacteria belonging to Planctomycetes display several unique morphological and genetic features and are found in a wide variety of habitats on earth. Their ecological roles in these habitats are still poorly understood. Planctomycetes have previously been detected throughout the year on surfaces of the kelp Laminaria hyperborea from southwestern Norway. We aimed to make a detailed investigation of the abundance and phylogenetic diversity of planctomycetes inhabiting these kelp surfaces.ResultsPlanctomycetes accounted for 51-53% of the bacterial biofilm cells in July and September and 24% in February according to fluorescence in situ hybridization (FISH) results. Several separate planctomycetes lineages within Pirellulae, Planctomyces and OM190 were represented in 16S rRNA gene clone libraries and the most abundant clones belonged to yet uncultured lineages. In contrast to the abundance, the diversity of the planctomycete populations increased from July to February and was probably influenced by the aging of the kelp tissue. One planctomycete strain that was closely related to Rhodopirellula baltica was isolated using selective cultivation techniques.ConclusionsBiofilms on surfaces of L. hyperborea display an even higher proportion of planctomycetes compared to other investigated planctomycete-rich habitats such as open water, sandy sediments and peat bogs. The findings agree well with the hypothesis of the role of planctomycetes as degraders of sulfated polymeric carbon in the marine environment as kelps produce such substances. In addition, the abundant planctomycete populations on kelp surfaces and in association with other eukaryotes suggest that coexistence with eukaryotes may be a key feature of many planctomycete lifestyles.

Quantifying temporal change in biodiversity: challenges and opportunities

Growing concern about biodiversity loss underscores the need to quantify and understand temporal change. Here, we review the opportunities presented by biodiversity time series, and address three related issues: (i) recognizing the characteristics of temporal data; (ii) selecting appropriate statistical procedures for analysing temporal data; and (iii) inferring and forecasting biodiversity change. With regard to the first issue, we draw attention to defining characteristics of biodiversity time series—lack of physical boundaries, uni-dimensionality, autocorrelation and directionality—that inform the choice of analytic methods. Second, we explore methods of quantifying change in biodiversity at different timescales, noting that autocorrelation can be viewed as a feature that sheds light on the underlying structure of temporal change. Finally, we address the transition from inferring to forecasting biodiversity change, highlighting potential pitfalls associated with phase-shifts and novel conditions.

Phylogenetic analysis of bacterial communities associated with larvae of the Atlantic halibut propose succession from a uniform normal flora.

Halibut, the largest of all flatfishes is a valuable species with a great potential for aquaculture. Bacteria play an important role in regulating the health of the early life stages. The present article is the first broad-range molecular analysis of bacterial communities in larvae of the Atlantic halibut (Hippoglossus hippoglossus). DNA was extracted from larvae, water and silo biofilm from hatcheries in Norway, Scotland, Iceland and Canada. Eubacterial 16S rRNA gene fragments were amplified by polymerase chain reaction (PCR) with broad-range primers. Sequences spanning the hyper variable V3 region representing individual bacterial species were separated into community profiles by denaturing gradient gel electrophoresis (DGGE). The profiles revealed simple communities after hatching and bacterial succession following growth. Sequencing and phylogenetic analysis of excised DGGE bands suggested aerobic heterotrophs related to groups of Pseudomonas, Janthinobacterium and possibly Marinomonas to be the primary colonisers of the larvae. After onset of feeding, fermentative species (Vibrio) were detected as well. Comparative analysis of bacterial communities from different geographical regions indicated that larvae of the Atlantic halibut possess a distinct and specific normal flora.