Metagenomic and geochemical characterization of pockmarked sediments overlaying the Troll petroleum reservoir in the North Sea

ABSTRACT: BACKGROUND: Pockmarks (depressions in the seabed) have been discovered throughout the world's oceans and are often related to hydrocarbon seepage. Although high concentrations of pockmarks are present in the seabed overlaying the Troll oil and gas reservoir in the northern North Sea, geological surveys have not detected hydrocarbon seepage in this area at the present time. In this study we have used metagenomics to characterize the prokaryotic communities inhabiting the surface sediments in the Troll area in relation to geochemical parameters, particularly related to hydrocarbon presence. We also investigated the possibility of increased potential for methane oxidation related to the pockmarks. Five metagenomes from pockmarks and plain seabed sediments were sequenced by pyrosequencing (Roche/454) technology. In addition, two metagenomes from seabed sediments geologically unlikely to be influenced by hydrocarbon seepage (the Oslofjord) were included. The taxonomic distribution and metabolic potential of the metagenomes were analyzed by multivariate analysis and statistical comparisons to reveal variation within and between the two sampling areas. RESULTS: The main difference identified between the two sampling areas was an overabundance of predominantly autotrophic nitrifiers, especially Nitrosopumilus, and oligotrophic marine Gammaproteobacteria in the Troll metagenomes compared to the Oslofjord. Increased potential for degradation of hydrocarbons, especially aromatic hydrocarbons, was detected in two of the Troll samples: one pockmark sample and one from the plain seabed. Although presence of methanotrophic organisms was indicated in all samples, no overabundance in pockmark samples compared to the Oslofjord samples supports no, or only low level, methane seepage in the Troll pockmarks at the present time. CONCLUSIONS: Given the relatively low content of total organic carbon and great depths of hydrocarbon containing sediments in the Troll area, it is possible that at least part of the carbon source available for the predominantly autotrophic nitrifiers thriving in this area originates from sequential prokaryotic degradation and oxidation of hydrocarbons to CO2. By turning CO2 back into organic carbon this subcommunity could play an important environmental role in these dark oligotrophic sediments. The oxidation of ammonia to nitrite and nitrate in this process could further increase the supply of terminal electron acceptors for hydrocarbon degradation.     

SUBPOPULATION DIFFERENTIATION ASSOCIATED WITH NONRIBOSOMAL PEPTIDE SYNTHETASE GENE CLUSTER DYNAMICS IN THE CYANOBACTERIUM PLANKTOTHRIX SPP.1

The Plankthotrix Anagn. et Komárek population in the mesotrophic Lake Steinsfjorden has been intensively studied over several decades. This Planktothrix population produces a number of different classes of oligopetides. However, over the study period, only four main oligopeptide profiles (chemotypes) have been associated with the strains isolated from the lake. The chemotypes show distinct interactions with the environment, demonstrated by shifts in abundance along time series and vertical profiles. Here, we present genetic analysis of nonribosomal peptide synthetase (NRPS) gene regions in strains representing the four Planktothrix chemotypes in Lake Steinsfjorden. On the basis of phylogenetic analyses, we show that the NRPS genes for microcystin (mcy) and cyanopeptolin (oci) display the same clustering as do the chemotypes. Nucleotide diversity in mcy and oci was significantly higher between strains of different chemotypes than between strains of the same chemotype. K_a/K_s (nonsynonymous vs. synonymous mutations) values indicated positive selection in several polymorphic regions of the mcy and oci genes. Notably, incongruence between the phylogenetic trees for different gene segments and split decomposition analyses for segments of oci suggested horizontal gene transfer (HGT) events between strains showing different oligopeptide profiles. The oci HGT region encodes a module responsible for incorporating a variable amino acid in cyanopeptolin and is one of the regions suggested to be under positive selection. Taken together, our data suggest that there are four genetically distinct sympatric subpopulations—displayed as distinct chemotypes—in Lake Steinsfjorden. The diversification process of the chemotypes, and consequently the subpopulations, is driven by HGT and reinforced by positive selection of the corresponding NRPS gene regions.     

Efficient protocols for CAPS-based mapping inArabidopsis

Positional cloning continues to be an essential method for gene identification and characterisation. The introduction of PCR-based techniques such as Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Length Polymorphisms (SSLP) and Cleaved Amplified Polymorphic Sequences (CAPS) has greatly increased the efficiency of gene mapping in arabidopsis. To develop the CAPS marker approach further, we have altered several critical mapping parameters. Efficiency was improved by using a small volume of dry seed for DNA extraction instead of the commonly used vegetative tissue. Reproducibility of PCR reactions was enhanced by faster and reduced protocols for PCR and restriction enzyme digestion and optimisation of PCR conditions for over 50 CAPS primer pairs. Finally, the density of genetic markers was increased by providing polymorphic information for all CAPS markers in arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col) and Cape Verde Islands (Cvi).     

Microbial community analysis by FISH for mathematical modelling of selective enrichment of gel-entrapped nitrifiers obtained from domestic wastewater

Nitrifying activated sludge from natural domestic sewage was entrapped in hydrogel beads, which were subsequently enriched for nitrifiers in a continuous stirred tank reactor (CSTR). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidisers were used in combination with DAPI staining to monitor the selectivity of the enrichment process. The growth of both nitrifying and heterotrophic bacteria was more pronounced in the periphery of the beads, leading to a biofilm-like stratification of the biomass during the enrichment. Quantitatively, the relative number of nitrifiers increased from 20% immediately after immobilisation up to 64% after 30 days, but decreased again due to extensive heterotrophic growth. These changes were accompanied by an increase in nitrifying activity for about 30 days, whereupon it reached a stable level. This selective enrichment was mathematically modelled by applying finite difference techniques to the diffusion-reaction mass balances of all soluble substrates relevant in the nitrification process. To model biomass growth and spreading, balanced by both decay and detachment at the surface of the beads, the differential methods were combined with a descrete cellular automaton approach. The spatially two-dimensional model was used to calculate radial concentration profiles within a gel bead, as well as to estimate the corresponding total activity of the reactor. Qualitatively, this model could simulate all essential aspects observed experimentally. However, more and better population data as well as independent estimates of decay and hydrolysis rates are needed to refine and verify the quantitative model. In conclusion, even in the absence of an external carbon source and with excess ammonium, it was only possible to obtain a moderate enrichment of nitrifying cells compared to heterotrophs. Under long-term cultivation, the biofilm-like structure developed in the outer gel layers led to a vigorous competition between auto- and heterotrophs for space, and thereby, access to oxygen. FISH analysis in combination with mathematical modelling constitute a suitable toolbox for analysing the population dynamics and biocatalytic performance of such an ecosystem based on lithoautotrophic primary production.     

Differentiation and levels of genetic variation in northern European lynx (Lynx lynx) populations revealed by microsatellites and mitochondrial DNA analysis

The European lynx (Lynx lynx) hasexperienced significant decline in populationnumbers over large parts of its formerdistribution area in central and northernEurope. In Scandinavia (Sweden and Norway), thespecies has been subject to intense hunting and inthe early 20th century the population size mayhave been as low as about 100 animals. Duringthe rest of the century there have beenalternating periods of restricted hunting andtotal protection. Future management of theScandinavian lynx population will requireinsight into what effects demographicbottlenecks may have had on genetic variabilityand structure. For this purpose, 276 lynxesfrom Sweden, Norway, Finland, Estonia andLatvia were analysed for polymorphism at 11feline microsatellite loci and at themitochondrial DNA (mtDNA) control region.Scandinavian lynxes were found to be fixed fora single mtDNA haplotype, while this and threeadditional haplotypes were seen in Finland andthe Baltic States (Estonia and Latvia); thehaplotypes were all very similar, onlydiffering at 1–4 sites within a 700 bp regionsequenced. Microsatellite variability wasmoderate (H_e = 0.51–0.62) with lowerheterozygosity and fewer alleles in Scandinaviathan in Finland and the Baltic States together,though significant so only for the latter.Heterozygosity data in Scandinavia wereconsistent with a recent population bottleneck.Various analyses (e.g. F_st, individual-basedtree, assignment test) revealed distinctgenetic differentiation between Scandinavianlynxes and animals from Finland and the BalticStates. Some structure was evident withinScandinavia as well, suggesting an isolation bydistance. The observed partition of geneticvariability between Scandinavia and the easterncountries thereof indicates that lynxpopulations from the two regions may need to beseparately managed. We discuss what factors canhave contributed to the population geneticstructure seen in northern European lynxpopulations of today.     

Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

Background  

Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy). mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer.     

Multiple-locus variable-number tandem repeat analysis of Legionella pneumophila using multi-colored capillary electrophoresis

Several methods for typing of Legionella pneumophila exist, one of which is an 8-locus variable-number of tandem repeats analysis (MLVA). This method is based on separating and sizing amplified VNTR PCR products by agarose gel electrophoresis. In the present work, the existing L. pneumophila MLVA-8 assay is adapted to capillary electrophoresis. The assay was multiplexed by using multiple fluorescent labeling dyes and tested on a panel of L. pneumophila strains with known genotypes. The results from the capillary electrophoresis-based assay are shown to be equivalent to, and in a few cases more sensitive than, the gel-based genotyping assay. The assay presented here allows for a swift, automated and precise typing of L. pneumophila from patient or environmental samples and represents an improvement over the current gel-based method.