Anna Klindworth

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies.

16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.

Substrate-controlled succession of marine bacterioplankton populations induced by a phytoplankton bloom.

Blooming Succession Algal blooms in the ocean will trigger a succession of microbial predators and scavengers. Teeling et al. (p. 608) used a combination of microscopy, metagenomics, and metaproteomics to analyze samples from a North Sea diatom bloom over time. Distinct steps of polysaccharide degradation and carbohydrate uptake could be assigned to clades of Flavobacteria and Gammaproteobacteria, which differ profoundly in their transporter profiles and their uptake systems for phosphorus. The phytoplankton/bacterioplankton coupling in coastal marine systems is of crucial importance for global carbon cycling. Bacterioplankton clade succession following phytoplankton blooms may be predictable enough that it can be included in models of global carbon cycling. Seasonal diatom growth in the North Sea results in a temporal succession of metabolically specialized bacteria. Phytoplankton blooms characterize temperate ocean margin zones in spring. We investigated the bacterioplankton response to a diatom bloom in the North Sea and observed a dynamic succession of populations at genus-level resolution. Taxonomically distinct expressions of carbohydrate-active enzymes (transporters; in particular, TonB-dependent transporters) and phosphate acquisition strategies were found, indicating that distinct populations of Bacteroidetes, Gammaproteobacteria, and Alphaproteobacteria are specialized for successive decomposition of algal-derived organic matter. Our results suggest that algal substrate availability provided a series of ecological niches in which specialized populations could bloom. This reveals how planktonic species, despite their seemingly homogeneous habitat, can evade extinction by direct competition.

Expression of sulfatases in Rhodopirellula baltica and the diversity of sulfatases in the genus Rhodopirellula.

The whole genome sequence of Rhodopirellula baltica SH1(T), published nearly 10years ago, already revealed a high amount of sulfatase genes. So far, little is known about the diversity and potential functions mediated by sulfatases in Planctomycetes. We combined in vivo and in silico techniques to gain insights into the ecophysiology of planktomycetal sulfatases. Comparative genomics of nine recently sequenced Rhodopirellula strains detected 1120 open reading frames annotated as sulfatases (Enzyme Commission number (EC) 3.1.6.*). These were clustered into 173 groups of orthologous and paralogous genes. To analyze the functional aspects, 708 sulfatase protein sequences from these strains were aligned with 67 sulfatase reference sequences of reviewed functionality. Our analysis yielded 22 major similarity clusters, but only five of these clusters contained Rhodopirellula sequences homologous to reference sequences, indicating a surprisingly high diversity. Exemplarily, R. baltica SH1(T) was grown on different sulfated polysaccharides, chondroitin sulfate, λ-carrageenan and fucoidan. Subsequent gene expression analyses using whole genome microarrays revealed distinct sulfatase expression profiles based on substrates tested. This might be indicative for a high structural diversity of sulfated polysaccharides as potential substrates. The pattern of sulfatases in individual planctomycete species may reflect ecological niche adaptation.

Practical application of self-organizing maps to interrelate biodiversity and functional data in NGS-based metagenomics

Next-generation sequencing (NGS) technologies have enabled the application of broad-scale sequencing in microbial biodiversity and metagenome studies. Biodiversity is usually targeted by classifying 16S ribosomal RNA genes, while metagenomic approaches target metabolic genes. However, both approaches remain isolated, as long as the taxonomic and functional information cannot be interrelated. Techniques like self-organizing maps (SOMs) have been applied to cluster metagenomes into taxon-specific bins in order to link biodiversity with functions, but have not been applied to broad-scale NGS-based metagenomics yet. Here, we provide a novel implementation, demonstrate its potential and practicability, and provide a web-based service for public usage. Evaluation with published data sets mimicking varyingly complex habitats resulted into classification specificities and sensitivities of close to 100% to above 90% from phylum to genus level for assemblies exceeding 8 kb for low and medium complexity data. When applied to five real-world metagenomes of medium complexity from direct pyrosequencing of marine subsurface waters, classifications of assemblies above 2.5 kb were in good agreement with fluorescence in situ hybridizations, indicating that biodiversity was mostly retained within the metagenomes, and confirming high classification specificities. This was validated by two protein-based classifications (PBCs) methods. SOMs were able to retrieve the relevant taxa down to the genus level, while surpassing PBCs in resolution. In order to make the approach accessible to a broad audience, we implemented a feature-rich web-based SOM application named TaxSOM, which is freely available at http://www.megx.net/toolbox/taxsom. TaxSOM can classify reads or assemblies exceeding 2.5 kb with high accuracy and thus assists in linking biodiversity and functions in metagenome studies, which is a precondition to study microbial ecology in a holistic fashion.

Diversity and activity of marine bacterioplankton during a diatom bloom in the North Sea assessed by total RNA and pyrotag sequencing.

A recent investigation of bacterioplankton communities in the German Bight towards the end of a diatom-dominated spring phytoplankton bloom revealed pronounced successions of distinct bacterial clades. A combination of metagenomics and metaproteomics indicated that these clades had distinct substrate spectra and consumed different algal substrates. In this study we re-analyzed samples from the initial study by total community RNA (metatranscriptomics) and 16S rRNA gene amplicon sequencing. This complementary approach provided new insights into the community composition and expressed genes as well as the assessment of metabolic activity levels of distinct clades. Flavobacteria (genera Ulvibacter, Formosa, and Polaribacter), Alphaproteobacteria (SAR11 clade and Rhodobacteraceae) and Gammaproteobacteria (genus Reinekea and SAR92 clade) were the most abundant taxa. Mapping of the metatranscriptome data on assembled and taxonomically classified metagenome data of the same samples substantiated that Formosa and Polaribacter acted as major algal polymer degraders, whereas Rhodobacteraceae and Reinekea spp. exhibited less specialized substrate spectra. In addition, we found that members of the Rhodobacteraceae and SAR92 clade showed high metabolic activity levels, which suggests that these clades played a more important role during the bloom event as indicated by their in situ abundances.

The founding charter of the Genomic Observatories Network

The co-authors of this paper hereby state their intention to work together to launch the Genomic Observatories Network (GOs Network) for which this document will serve as its Founding Charter. We define a Genomic Observatory as an ecosystem and/or site subject to long-term scientific research, including (but not limited to) the sustained study of genomic biodiversity from single-celled microbes to multicellular organisms.An international group of 64 scientists first published the call for a global network of Genomic Observatories in January 2012. The vision for such a network was expanded in a subsequent paper and developed over a series of meetings in Bremen (Germany), Shenzhen (China), Moorea (French Polynesia), Oxford (UK), Pacific Grove (California, USA), Washington (DC, USA), and London (UK). While this community-building process continues, here we express our mutual intent to establish the GOs Network formally, and to describe our shared vision for its future. The views expressed here are ours alone as individual scientists, and do not necessarily represent those of the institutions with which we are affiliated.

Permanent draft genomes of the two Rhodopirellula europaea strains 6C and SH398

The genomes of two Rhodopirellula europaea strains were sequenced as permanent drafts to study the genomic diversity within this genus, especially in comparison with the closed genome of the type strain Rhodopirellula baltica SH1(T). The isolates are part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the second of a series of five publications describing a total of eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genome of Rhodopirellula sallentina SM41

The genome of Rhodopirellula sallentina SM41 was sequenced as a permanent draft to supplement the full genome sequence of the type strain Rhodopirellula baltica SH1(T). This isolate is part of a larger study to gain insights into the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the third of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genomes of the Rhodopirellula maiorica strain SM1.

The genome of Rhodopirellula maiorica strain SM1 was sequenced as a permanent draft to complement the full genome sequence of the type strain Rhodopirellula baltica SH1(T). This isolate is part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the fifth of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.

Permanent draft genomes of the three Rhodopirellula baltica strains SH28, SWK14 and WH47.

The genomes of three Rhodopirellula baltica strains were sequenced as permanent drafts to complement the full genome sequence of the type strain R. baltica SH1(T). The isolates are part of a larger study to infer the biogeography of Rhodopirellula species in European marine waters, as well as to amend the genus description of R. baltica. This genomics resource article is the first of a series of five publications reporting in total eight new permanent daft genomes of Rhodopirellula species.