25 years of serving the community with ribosomal RNA gene reference databases and tools.

SILVA (lat. forest) is a comprehensive web resource, providing services around up to date, high-quality datasets of aligned ribosomal RNA gene (rDNA) sequences from the Bacteria, Archaea, and Eukaryota domains. SILVA dates back to the year 1991 when Dr. Wolfgang Ludwig from the Technical University Munich started the integrated software workbench ARB (lat. tree) to support high-quality phylogenetic inference and taxonomy based on the SSU and LSU rDNA marker genes. At that time, the ARB project maintained both, the sequence reference datasets and the software package for data analysis. In 2005, with the massive increase of DNA sequence data, the maintenance of the software system ARB and the corresponding rRNA databases SILVA was split between Munich and the Microbial Genomics and Bioinformatics Research Group in Bremen. ARB has been continuously developed to include new features and improve the usability of the workbench. Thousands of users worldwide appreciate the seamless integration of common analysis tools under a central graphical user interface, in combination with its versatility. The first SILVA release was deployed in February 2007 based on the EMBL-EBI/ENA release 89. Since then, full SILVA releases offering the database content in various flavours are published at least annually, complemented by intermediate web-releases where only the SILVA web dataset is updated. SILVA is the only rDNA database project worldwide where special emphasis is given to the consistent naming of clades of uncultivated (environmental) sequences, where no validly described cultivated representatives are available. Also exclusive for SILVA is the maintenance of both comprehensive aligned 16S/18S rDNA and 23S/28S rDNA sequence datasets. Furthermore, the SILVA alignments and trees were designed to include Eukaryota, another unique feature among rDNA databases. With the termination of the European Ribosomal RNA Database Project in 2007, the SILVA database has become the authoritative rDNA database project for Europe. The application spectrum of ARB and SILVA ranges from biodiversity analysis, medical diagnostics, to biotechnology and quality control for academia and industry.

A phylogenetic framework for the kingdom Fungi based on 18S rRNA gene sequences.

The usage of molecular phylogenetic approaches is critical to advance the understanding of systematics and community processes in the kingdom Fungi. Among the possible phylogenetic markers (or combinations of them), the 18S rRNA gene appears currently as the most prominent candidate due to its large availability in public databases and informative content. The purpose of this work was the creation of a reference phylogenetic framework that can serve as ready-to-use package for its application on fungal classification and community analysis. The current database contains 9329 representative 18S rRNA gene sequences covering the whole fungal kingdom, a manually curated alignment, an annotated and revised phylogenetic tree with all the sequence entries, updated information on current taxonomy, and recommendations of use. Out of 201 total fungal taxa with more than two sequences in the dataset, 179 were monophyletic. From another perspective, 66% of the entries had a tree-derived classification identical to that obtained from the NCBI taxonomy, whereas 34% differed in one or the other rank. Most of the differences were associated to missing taxonomic assignments in NCBI taxonomy, or the unexpected position of sequences that positioned out of their theoretically corresponding clades. The strong correlation observed with current fungal taxonomy evidences that 18S rRNA gene sequence-based phylogenies are adequate to reflect genealogy of Fungi at the levels of order and above, and justify their further usage and exploration.

Expanding the World of Marine Bacterial and Archaeal Clades.

Determining which microbial taxa are out there, where they live, and what they are doing is a driving approach in marine microbial ecology. The importance of these questions is underlined by concerted, large-scale, and global ocean sampling initiatives, for example the International Census of Marine Microbes, Ocean Sampling Day, or Tara Oceans. Given decades of effort, we know that the large majority of marine Bacteria and Archaea belong to about a dozen phyla. In addition to the classically culturable Bacteria and Archaea, at least 50 "clades," at different taxonomic depths, exist. These account for the majority of marine microbial diversity, but there is still an underexplored and less abundant portion remaining. We refer to these hitherto unrecognized clades as unknown, as their boundaries, names, and classifications are not available. In this work, we were able to characterize up to 92 of these unknown clades found within the bacterial and archaeal phylogenetic diversity currently reported for marine water column environments. We mined the SILVA 16S rRNA gene datasets for sequences originating from the marine water column. Instead of the usual subjective taxa delineation and nomenclature methods, we applied the candidate taxonomic unit (CTU) circumscription system, along with a standardized nomenclature to the sequences in newly constructed phylogenetic trees. With this new phylogenetic and taxonomic framework, we performed an analysis of ICoMM rRNA gene amplicon datasets to gain insights into the global distribution of the new marine clades, their ecology, biogeography, and interaction with oceanographic variables. Most of the new clades we identified were interspersed by known taxa with cultivated members, whose genome sequences are available. This result encouraged us to perform metabolic predictions for the novel marine clades using the PICRUSt approach. Our work also provides an update on the taxonomy of several phyla and widely known marine clades as our CTU approach breaks down these randomly lumped clades into smaller objectively calculated subgroups. Finally, all taxa were classified and named following standards compatible with the Bacteriological Code rules, enhancing their digitization, and comparability with future microbial ecological and taxonomy studies.

Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences.

Publicly available sequence databases of the small subunit ribosomal RNA gene, also known as 16S rRNA in bacteria and archaea, are growing rapidly, and the number of entries currently exceeds 4 million. However, a unified classification and nomenclature framework for all bacteria and archaea does not yet exist. In this Analysis article, we propose rational taxonomic boundaries for high taxa of bacteria and archaea on the basis of 16S rRNA gene sequence identities and suggest a rationale for the circumscription of uncultured taxa that is compatible with the taxonomy of cultured bacteria and archaea. Our analyses show that only nearly complete 16S rRNA sequences give accurate measures of taxonomic diversity. In addition, our analyses suggest that most of the 16S rRNA sequences of the high taxa will be discovered in environmental surveys by the end of the current decade.

New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities.

Bleaching of Oculina patagonica has been extensively studied in the Eastern Mediterranean Sea, although no studies have been carried out in the Western basin. In 1996 Vibrio mediterranei was reported as the causative agent of bleaching in O. patagonica but it has not been related to bleached or healthy corals since 2003, suggesting that it was no longer involved in bleaching of O. patagonica. In an attempt to clarify the relationship between Vibrio spp., seawater temperature and coral diseases, as well as to investigate the putative differences between Eastern and Western Mediterranean basins, we have analysed the seasonal patterns of the culturable Vibrio spp. assemblages associated with healthy and diseased O. patagonica colonies. Two sampling points located in the Spanish Mediterranean coast were chosen for this study: Alicante Harbour and the Marine Reserve of Tabarca. A complex and dynamic assemblage of Vibrio spp. was present in O. patagonica along the whole year and under different environmental conditions and coral health status. While some Vibrio spp. were detected all year around in corals, the known pathogens V. mediteranei and V. coralliilyticus were only present in diseased specimens. The pathogenic potential of these bacteria was studied by experimental infection under laboratory conditions. Both vibrios caused diseased signs from 24 °C, being higher and faster at 28 °C. Unexpectedly, the co-inoculation of these two Vibrio species seemed to have a synergistic pathogenic effect over O. patagonica, as disease signs were readily observed at temperatures at which bleaching is not normally observed.

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.

Sequencing orphan species initiative (SOS): Filling the gaps in the 16S rRNA gene sequence database for all species with validly published names.

High quality 16S ribosomal RNA (rRNA) gene sequences from the type strains of all species with validly published names, as defined by the International Code of Nomenclature of Bacteria, are a prerequisite for their accurate affiliations within the global genealogical classification and for the recognition of potential new taxa. During the last few years, the Living Tree Project (LTP) has taken care to create a high quality, aligned 16S and 23S rRNA gene sequence database of all type strains. However, the manual curation of the sequence dataset and type strain information revealed that a total of 552 "orphan" species (about 5.7% of the currently classified species) had to be excluded from the reference trees. Among them, 322 type strains were not represented by an SSU entry in the public sequence repositories. The remaining 230 type strains had to be discarded due to bad sequence quality. Since 2010, the LTP team has coordinated a network of researchers and culture collections in order to improve the situation by (re)-sequencing the type strains of these "orphan" species. As a result, we can now report 351 16S rRNA gene sequences of type strains. Nevertheless, 201 species could not be sequenced because cultivable type strains were not available (121), the cultures had either been lost or were never deposited in the first place (66), or it was not possible due to other constraints (14). The International Code of Nomenclature of Bacteria provides a number of mechanisms to deal with the problem of missing type strains and we recommend that due consideration be given to the appropriate mechanisms in order to help solve some of these issues.

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.

Virioplankton community structure in Tunisian solar salterns.

The microbial community inhabiting Sfax solar salterns on the east coast of Tunisia has been studied by means of different molecular and culture-dependent tools that have unveiled the presence of novel microbial groups as well as a community structure different from that of other coastal hypersaline environments. We have focused on the study of the viral assemblages of these salterns and their changes along the salinity gradient and over time. Viruses from three ponds (C4, M1, and TS) encompassing salinities from moderately hypersaline to saturated (around 14, 19, and 35%, respectively) were sampled in May and October 2009 and analyzed by transmission electron microscopy (TEM) and pulsed-field gel electrophoresis (PFGE). Additionally, for all three October samples and the May TS sample, viral metagenomic DNA was cloned in fosmids, end sequenced, and analyzed. Viral concentration, as well as virus-to-cell ratios, increased along the salinity gradient, with around 10(10) virus-like particles (VLPs)/ml in close-to-saturation ponds, which represents the highest viral concentration reported so far for aquatic systems. Four distinct morphologies could be observed with TEM (spherical, tailed, spindled, and filamentous) but with various proportions in the different samples. Metagenomic analyses indicated that every pond harbored a distinct viral assemblage whose G+C content could be roughly correlated with that of the active part of the microbial community that may have constituted the putative hosts. As previously reported for hypersaline metaviromes, most sequences did not have matches in the databases, although some were conserved among the Sfax metaviromes. BLASTx, BLASTp, and dinucleotide frequency analyses indicated that (i) factors additional to salinity could be structuring viral communities and (ii) every metavirome had unique gene contents and dinucleotide frequencies. Comparison with hypersaline metaviromes available in the databases indicated that the viral assemblages present in close-to-saturation environments located thousands of kilometers apart presented some common traits among them in spite of their differences regarding the putative hosts. A small core metavirome for close-to-saturation systems was found that contained 7 sequences of around 100 nucleotides (nt) whose function was not hinted at by in silico search results, although it most likely represents properties essential for hyperhalophilic viruses.

Culture-independent approaches for studying viruses from hypersaline environments.

Hypersaline close-to-saturation environments harbor an extremely high concentration of virus-like particles, but the number of haloviruses isolated so far is still very low. Haloviruses can be directly studied from natural samples by using different culture-independent techniques that include transmission electron microscopy, pulsed-field gel electrophoresis, and different metagenomic approaches. Here, we review the findings of these studies, with a main focus on the metagenomic approaches. The analysis of bulk viral nucleic acids directly retrieved from the environment allows estimations of viral diversity, activity, and dynamics and tentative host assignment. Results point to a diverse and active viral community in constant interplay with its hosts and to a "hypersalineness" quality common to viral assemblages present in hypersaline environments that are thousands of kilometers away from each other.

Taxonomic and functional metagenomic profiling of the microbial community in the anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, Central Spain).

The phylogenetic and functional structure of the microbial community residing in a Ca(2+)-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO(4) × 2 H(2)O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1 Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca(2+) and SO (4) (2-) being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physical-chemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.

Response of sulfate-reducing bacteria to an artificial oil-spill in a coastal marine sediment.

In situ mesocosm experiments using a calcareous sand flat from a coastal area of the island of Mallorca in the Mediterranean Sea were performed in order to study the response of sulfate-reducing bacteria (SRB) to controlled crude oil contamination, or heavy contamination with naphthalene. Changes in the microbial community caused by the contamination were monitored by a combination of comparative sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, cultivation approaches and metabolic activity rates. Our results showed that crude oil and naphthalene negatively influenced the total microbial community as the natural increase in cell numbers due to the seasonal dynamics was attenuated. However, both contaminants enhanced the sulfate reduction rates, as well as the culturability of SRB. Our results suggested the presence of autochthonous deltaproteobacterial SRBs that were able to degrade crude oil or polycyclic aromatic hydrocarbons such as naphthalene in anaerobic sediment layers.

Update of the All-Species Living Tree Project based on 16S and 23S rRNA sequence analyses.

The "All-Species Living Tree Project" (LTP) provides the scientific community with a useful taxonomic tool consisting of a curated database of type strain sequences, a universal and optimized alignment and a single phylogenetic tree harboring all the type strains of the hitherto classified species. On the website http://www.arb-silva.de/projects/living-tree an update has been regularly maintained by including the 1301 new descriptions that have appeared in the validation and notification lists of the IJSEM journal. The topology of the 16S rRNA-based tree was validated with a detailed comparison against a collection of taxa-specific and broad-range trees made using different approaches, subsets of sequences and alignments. Seven percent of the classified species is still missing, as their type strains do not have a good quality SSU sequence. In addition, a new database of type strains for which adequate 23S rRNA entries existed in public repositories was built. Among the 8602 species with validly published names until February 2010, we were able to find good quality LSU representatives for 792 type strains, whereas around 91% of the complete catalogue still remains unsequenced. Despite the scarce representation of some groups in LSU databases, we have devised a highly optimized alignment and a reliable LSU tree in order to set up a stable phylogenetic starting point for taxonomic purposes. The current release corresponds to the fourth update of the project (LTPs102), and contains additional features which increase usability and compatibility. Use the contact address living-tree@arb-silva.de to provide additional input for the development of this taxonomic tool.

The metavirome of a hypersaline environment.

Hypersaline environments harbour the highest number of virus-like particles reported for planktonic systems. However, very little is known about the genomic diversity of these virus assemblages since most of the knowledge on halophages is based on the analysis of a few isolates infecting strains of hyperhalophilic Archaea that may not be representatives of the natural microbiota. Here, we report the characterization, through a metagenomic approach, of the viral assemblage inhabiting a crystallizer pond (CR30) from a multi-pond solar saltern in Santa Pola (SE Spain). A total of 1.35 Mbp were cloned that yielded a total of 620 kb sequenced viral DNA. The metavirome was highly diverse and different from virus communities of marine and other aquatic environments although it showed some similarities with metaviromes from high-salt ponds in solar salterns in San Diego (SW USA), indicating some common traits between high-salt viromes. A high degree of diversity was found in the halophages as revealed by the presence of 2479 polymorphic nucleotides. Dinucleotide frequency analysis of the CR30 metavirome showed a good correlation with GC content and enabled the establishment of different groups, and even the assignment of their putative hosts: the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber.

Pseudomonas arsenicoxydans sp nov., an arsenite-oxidizing strain isolated from the Atacama desert.

A Gram-negative, arsenite-oxidizing bacterial strain, designated VC-1, was isolated from sediment samples from the Camarones Valley in the Atacama Desert, Chile. Strain VC-1 was strictly aerobic, oxidase and catalase positive, rod shaped, of about 5.5 microm in length and 0.5-1.0 microm in diameter. It was motile by means of multiple polar flagella. The phylogenetic reconstruction of the 16S rRNA gene sequence, an MLSA study by concatenating six genes, and DDH studies indicated that the strain differed genotypically from its closest relatives and was therefore recognized as a new species within the genus Pseudomonas. Phenotypic analysis combining metabolic tests, fatty acid profiles and MALDI-TOF profiles of total cell extracts supported the classification of the new species for which we propose the designation Pseudomonas arsenicoxydans sp. nov. The type strain is accessible under the culture collection numbers CCUG 58201(T) and CECT 7543(T).

Fine-scale evolution: genomic, phenotypic and ecological differentiation in two coexisting Salinibacter ruber strains.

Genomic and metagenomic data indicate a high degree of genomic variation within microbial populations, although the ecological and evolutive meaning of this microdiversity remains unknown. Microevolution analyses, including genomic and experimental approaches, are so far very scarce for non-pathogenic bacteria. In this study, we compare the genomes, metabolomes and selected ecological traits of the strains M8 and M31 of the hyperhalophilic bacterium Salinibacter ruber that contain ribosomal RNA (rRNA) gene and intergenic regions that are identical in sequence and were simultaneously isolated from a Mediterranean solar saltern. Comparative analyses indicate that S. ruber genomes present a mosaic structure with conserved and hypervariable regions (HVRs). The HVRs or genomic islands, are enriched in transposases, genes related to surface properties, strain-specific genes and highly divergent orthologous. However, the many indels outside the HVRs indicate that genome plasticity extends beyond them. Overall, 10% of the genes encoded in the M8 genome are absent from M31 and could stem from recent acquisitions. S. ruber genomes also harbor 34 genes located outside HVRs that are transcribed during standard growth and probably derive from lateral gene transfers with Archaea preceding the M8/M31 divergence. Metabolomic analyses, phage susceptibility and competition experiments indicate that these genomic differences cannot be considered neutral from an ecological perspective. The results point to the avoidance of competition by micro-niche adaptation and response to viral predation as putative major forces that drive microevolution within these Salinibacter strains. In addition, this work highlights the extent of bacterial functional diversity and environmental adaptation, beyond the resolution of the 16S rRNA and internal transcribed spacers regions.

Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism.

Marinobacter adhaerens HP15 is the type strain of a newly identified marine species, which is phylogenetically related to M. flavimaris, M. algicola, and M. aquaeolei. It is of special interest for research on marine aggregate formation because it showed specific attachment to diatom cells. In vitro it led to exopolymer formation and aggregation of these algal cells to form marine snow particles. M. adhaerens HP15 is a free-living, motile, rod-shaped, Gram-negative gammaproteobacterium, which was originally isolated from marine particles sampled in the German Wadden Sea. M. adhaerens HP15 grows heterotrophically on various media, is easy to access genetically, and serves as a model organism to investigate the cellular and molecular interactions with the diatom Thalassiosira weissflogii. Here we describe the complete and annotated genome sequence of M. adhaerens HP15 as well as some details on flagella-associated genes. M. adhaerens HP15 possesses three replicons; the chromosome comprises 4,422,725 bp and codes for 4,180 protein-coding genes, 51 tRNAs and three rRNA operons, while the two circular plasmids are ~187 kb and ~42 kb in size and contain 178 and 52 protein-coding genes, respectively.

Extremely halophilic microbial communities in anaerobic sediments from a solar saltern.

The prokaryotic communities inhabiting hypersaline sediments underlying a crystallizer pond of a Mediterranean solar saltern have been studied in a polyphasic approach including 16S rRNA and dsrAB gene libraries analysis [the last encoding for dissimilatory (bi)sulfite reductase], most probable number of cultivable counts, and metabolic measurements of sulfate reduction. The samples studied here represent one of the most hypersaline anoxic environments sampled worldwide that harbour a highly diverse microbial community different from those previously reported in other hypersaline sediments. Both bacterial and archaeal types are present but, contrarily to the overlying brine system, the former dominates. Molecular analyses indicated that the bacterial fraction is highly diverse and mostly composed by groups related to sulfate-reducing bacteria (SRB). In good agreement with this, sulfate-reducing activity was detected in the sediment, as well as the metabolic diversity within SRB (as indicated by the use of different electron donors in enrichments). On the other hand, the archaeal fraction was phylogenetically homogeneous and, surprisingly, strongly affiliated with the MBSl-1 candidate division, an euryarchaeotal group only reported in deep-sea hypersaline anoxic basins of the Western Mediterranean, for which a methanogenic metabolism was hypothesized. The hypersaline studied samples constitute a valuable source of new prokaryotic types with metabolisms adapted to the prevalent in situ extreme conditions.

Occurrence of Halococcus spp. in the nostrils salt glands of the seabird Calonectris diomedea.

The nostrils of the seabird Calonectris diomedea are endowed with a salt-excreting gland that could produce a suitable environment for the colonization of extreme halophilic prokaryotes. We have studied in this organ the presence of extreme halophiles by means of culturing techniques. We could easily cultivate members of haloarchaea, and all cultures studied were identified as members of one of the two species Halococcus morrhuae and Hcc. dombrowskii. In order to reveal the diversity of these colonizers, we undertook a taxonomic study. Altogether, the results indicated that members of the genus Halococcus may constitute a part of the natural epizootic microbiota of C. diomedea, and that they exhibit such an important degree of taxonomic variability that appeals for a pragmatic species definition. This seabird nests in the west Mediterranean coasts, but its migratory habits, reaching locations as distant from the Mediterranean as the South Atlantic, may help in the dispersal mechanisms of haloarchaea through the Earth's surface.