Jörg Peplies

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.

SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes

MOTIVATION In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. RESULTS In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. AVAILABILITY Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license.

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.

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.

Isolation of Novel Pelagic Bacteria from the German Bight and Their Seasonal Contributions to Surface Picoplankton

ABSTRACT We tested new strategies for the isolation of abundant bacteria from coastal North Sea surface waters, which included reducing by several orders of magnitude the concentrations of inorganic N and P compounds in a synthetic seawater medium. Agar plates were resampled over 37 days, and slowly growing colonies were allowed to develop by repeatedly removing all newly formed colonies. A fivefold increase of colonies was observed on plates with reduced nutrient levels, and the phylogenetic composition of the culture collection changed over time, towards members of the Roseobacter lineage and other alpha-proteobacteria. Novel gamma-proteobacteria from a previously uncultured but cosmopolitan lineage (NOR5) formed colonies only after 12 days of plate incubation. A time series of German Bight surface waters (January to December 1998) was screened by fluorescence in situ hybridization (FISH) with isolate-specific and general probes. During spring and early summer, a prominent fraction of FISH-detectable bacteria (mean, 51%) were affiliated with theCytophaga-Flavobacterium group (CF) of theBacteroidetes. One Cytophaga sp. lineage with cultured representatives formed almost 20% of the CF group. Members of the Roseobacter cluster constituted approximately 50% of alpha-proteobacteria, but none of the Roseobacter-related isolates formed populations of >1% in the environment. Thus, the readily culturable members of this clade are probably not representative of Roseobacter species that are common in the water column. In contrast, members of NOR5 were found at high abundances (>105 cells ml−1) in the summer plankton. Some abundant pelagic bacteria are apparently able to form colonies on solid media, but appropriate isolation techniques for different species need to be developed.

JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison.

Abstract Summary: JSpecies Web Server (JSpeciesWS) is a user-friendly online service for in silico calculating the extent of identity between two genomes, a parameter routinely used in the process of polyphasic microbial species circumscription. The service measures the average nucleotide identity (ANI) based on BLAST+ (ANIb) and MUMmer (ANIm), as well as correlation indexes of tetra-nucleotide signatures (Tetra). In addition, it provides a Tetra Correlation Search function, which allows to rapidly compare selected genomes against a continuously updated reference database with currently about 32 000 published whole and draft genome sequences. For comparison, own genomes can be uploaded and references can be selected from the JSpeciesWS reference database. The service indicates whether two genomes share genomic identities above or below the species embracing thresholds, and serves as a fast way to allocate unknown genomes in the frame of the hitherto sequenced species. Availability and implementation: JSpeciesWS is available at http://jspecies.ribohost.com/jspeciesws. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: mrichter@ribocon.com

Optimization strategies for DNA microarray-based detection of bacteria with 16S rRNA-targeting oligonucleotide probes.

ABSTRACT The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 oligonucleotide probes. Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization. With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation. Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41). Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules. False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture oligonucleotides. The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested. Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of oligonucleotide probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort.

Combined Approach for Characterization of Uncultivated Magnetotactic Bacteria from Various Aquatic Environments

ABSTRACT Both magnetic collection and “race track” purification techniques were highly effective for selective enrichment of magnetotactic bacteria (MTB) from complex communities, as suggested by amplified ribosomal DNA restriction analysis and denaturing gradient gel electrophoresis combined with sequence analysis of 16S rRNA genes. Using these purification methods, the occurrence and diversity of MTB in microcosms from various marine and freshwater environments were assayed by using a combined microscopic, molecular, and cultivation approach. Most microcosms were dominated by magnetotactic cocci. Consistently, the majority of retrieved 16S RNA sequences were affiliated with a distinct cluster in the Alphaproteobacteria. Within this lineage the levels of sequence divergence were <1 to 11%, indicating genus-level diversity between magnetotactic cocci from various microcosms, as well as between MTB from different stages of succession of the same microcosms. The community composition in microscosms underwent drastic succession during incubation, and significant heterogeneities were observed between microcosms from the same environmental sources. A novel magnetotactic rod (MHB-1) was detected in a sediment sample from a lake in northern Germany by fluorescence in situ hybridization. MHB-1 falls into the Nitrospira phylum, displaying 91% 16S rRNA sequence similarity to “Magnetobacterium bavaricum.” In extensive cultivation attempts, we failed to isolate MHB-1, as well as most other MTB present in our samples. However, although magnetotactic spirilla were not frequently observed in the enrichments, 10 novel isolates of the genus Magnetospirillum which had not routinely been isolated in pure culture before were obtained.