Christian Quast

SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB

Sequencing ribosomal RNA (rRNA) genes is currently the method of choice for phylogenetic reconstruction, nucleic acid based detection and quantification of microbial diversity. The ARB software suite with its corresponding rRNA datasets has been accepted by researchers worldwide as a standard tool for large scale rRNA analysis. However, the rapid increase of publicly available rRNA sequence data has recently hampered the maintenance of comprehensive and curated rRNA knowledge databases. A new system, SILVA (from Latin silva, forest), was implemented to provide a central comprehensive web resource for up to date, quality controlled databases of aligned rRNA sequences from the Bacteria, Archaea and Eukarya domains. All sequences are checked for anomalies, carry a rich set of sequence associated contextual information, have multiple taxonomic classifications, and the latest validly described nomenclature. Furthermore, two precompiled sequence datasets compatible with ARB are offered for download on the SILVA website: (i) the reference (Ref) datasets, comprising only high quality, nearly full length sequences suitable for in-depth phylogenetic analysis and probe design and (ii) the comprehensive Parc datasets with all publicly available rRNA sequences longer than 300 nucleotides suitable for biodiversity analyses. The latest publicly available database release 91 (August 2007) hosts 547 521 sequences split into 461 823 small subunit and 85 689 large subunit rRNAs.

The SILVA ribosomal RNA gene database project: improved data processing and web-based tools

SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches.

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.

The SILVA and “All-species Living Tree Project (LTP)” taxonomic frameworks

SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive resource for up-to-date quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. SILVA provides a manually curated taxonomy for all three domains of life, based on representative phylogenetic trees for the small- and large-subunit rRNA genes. This article describes the improvements the SILVA taxonomy has undergone in the last 3 years. Specifically we are focusing on the curation process, the various resources used for curation and the comparison of the SILVA taxonomy with Greengenes and RDP-II taxonomies. Our comparisons not only revealed a reasonable overlap between the taxa names, but also points to significant differences in both names and numbers of taxa between the three resources.

Characterization of a marine gammaproteobacterium capable of aerobic anoxygenic photosynthesis

Members of the gammaproteobacterial clade NOR5/OM60 regularly form an abundant part, up to 11%, of the bacterioplankton community in coastal systems during the summer months. Here, we report the nearly complete genome sequence of one cultured representative, Congregibacter litoralis strain KT71, isolated from North Sea surface water. Unexpectedly, a complete photosynthesis superoperon, including genes for accessory pigments, was discovered. It has a high sequence similarity to BAC clones from Monterey Bay [Beja O, Suzuki MT, Heidelberg JF, Nelson WC, Preston CM, et al. (2002) Nature 415:630–633], which also share a nearly identical gene arrangement. Although cultures of KT71 show no obvious pigmentation, bacteriochlorophyll a and spirilloxanthin-like carotenoids could be detected by HPLC analysis in cell extracts. The presence of two potential BLUF (blue light using flavin adenine dinucleotide sensors), one of which was found adjacent to the photosynthesis operon in the genome, indicates a light- and redox-dependent regulation of gene expression. Like other aerobic anoxygenic phototrophs (AAnPs), KT71 is able to grow neither anaerobically nor photoautotrophically. Cultivation experiments and genomic evidence show that KT71 needs organic substrates like carboxylic acids, oligopeptides, or fatty acids for growth. The strain grows optimally under microaerobic conditions and actively places itself in a zone of ≈10% oxygen saturation. The genome analysis of C. litoralis strain KT71 identifies the gammaproteobacterial marine AAnPs, postulated based on BAC sequences, as members of the NOR5/OM60 clade. KT71 enables future experiments investigating the importance of this group of gammaproteobacterial AAnPs in coastal environments.

Microbial and Chemical Characterization of Underwater Fresh Water Springs in the Dead Sea

Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water’s chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea−Dead Sea water conduit.

Recurring patterns in bacterioplankton dynamics during coastal spring algae blooms

A process of global importance in carbon cycling is the remineralization of algae biomass by heterotrophic bacteria, most notably during massive marine algae blooms. Such blooms can trigger secondary blooms of planktonic bacteria that consist of swift successions of distinct bacterial clades, most prominently members of the Flavobacteriia, Gammaproteobacteria and the alphaproteobacterial Roseobacter clade. We investigated such successions during spring phytoplankton blooms in the southern North Sea (German Bight) for four consecutive years. Dense sampling and high-resolution taxonomic analyses allowed the detection of recurring patterns down to the genus level. Metagenome analyses also revealed recurrent patterns at the functional level, in particular with respect to algal polysaccharide degradation genes. We, therefore, hypothesize that even though there is substantial inter-annual variation between spring phytoplankton blooms, the accompanying succession of bacterial clades is largely governed by deterministic principles such as substrate-induced forcing. DOI: http://dx.doi.org/10.7554/eLife.11888.001

Transcriptional response of the model planctomycete Rhodopirellula baltica SH1T to changing environmental conditions

BackgroundThe marine model organism Rhodopirellula baltica SH1T was the first Planctomycete to have its genome completely sequenced. The genome analysis predicted a complex lifestyle and a variety of genetic opportunities to adapt to the marine environment. Its adaptation to environmental stressors was studied by transcriptional profiling using a whole genome microarray.ResultsStress responses to salinity and temperature shifts were monitored in time series experiments. Chemostat cultures grown in mineral medium at 28°C were compared to cultures that were shifted to either elevated (37°C) or reduced (6°C) temperatures as well as high salinity (59.5‰) and observed over 300 min. Heat shock showed the induction of several known chaperone genes. Cold shock altered the expression of genes in lipid metabolism and stress proteins. High salinity resulted in the modulation of genes coding for compatible solutes, ion transporters and morphology. In summary, over 3000 of the 7325 genes were affected by temperature and/or salinity changes.ConclusionTranscriptional profiling confirmed that R. baltica is highly responsive to its environment. The distinct responses identified here have provided new insights into the complex adaptation machinery of this environmentally relevant marine bacterium. Our transcriptome study and previous proteome data suggest a set of genes of unknown functions that are most probably involved in the global stress response. This work lays the foundation for further bioinformatic and genetic studies which will lead to a comprehensive understanding of the biology of a marine Planctomycete.

Megx.net—database resources for marine ecological genomics

Marine microbial genomics and metagenomics is an emerging field in environmental research. Since the completion of the first marine bacterial genome in 2003, the number of fully sequenced marine bacteria has grown rapidly. Concurrently, marine metagenomics studies are performed on a regular basis, and the resulting number of sequences is growing exponentially. To address environmentally relevant questions like organismal adaptations to oceanic provinces and regional differences in the microbial cycling of nutrients, it is necessary to couple sequence data with geographical information and supplement them with contextual information like physical, chemical and biological data. Therefore, new specialized databases are needed to organize and standardize data storage as well as centralize data access and interpretation. We introduce Megx.net, a set of databases and tools that handle genomic and metagenomic sequences in their environmental contexts. Megx.net includes (i) a geographic information system to systematically store and analyse marine genomic and metagenomic data in conjunction with contextual information; (ii) an environmental genome browser with fast search functionalities; (iii) a database with precomputed analyses for selected complete genomes; and (iv) a database and tool to classify metagenomic fragments based on oligonucleotide signatures. These integrative databases and webserver will help researchers to generate a better understanding of the functioning of marine ecosystems. All resources are freely accessible at .

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.