Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities.

BACKGROUND: Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets. RESULTS: The analyses of ca. 250,000 sequence reads revealed that the number of detected Operational Taxonomic Units (OTUs) far exceeded previous richness estimates from the same sites based on clone libraries and Sanger sequencing. More than 90% of this diversity was represented by OTUs with less than 10 sequence tags. We detected a substantial number of taxonomic groups like Apusozoa, Chrysomerophytes, Centroheliozoa, Eustigmatophytes, hyphochytriomycetes, Ichthyosporea, Oikomonads, Phaeothamniophytes, and rhodophytes which remained undetected by previous clone library-based diversity surveys of the sampling sites. The most important innovations in our newly developed bioinformatics pipeline employ (i) BLASTN with query parameters adjusted for highly variable domains and a complete database of public ribosomal RNA (rRNA) gene sequences for taxonomic assignments of tags; (ii) a clustering of tags at k differences (Levenshtein distance) with a newly developed algorithm enabling very fast OTU clustering for large tag sequence data sets; and (iii) a novel parsing procedure to combine the data from individual analyses. CONCLUSION: Our data highlight the magnitude of the under-sampled 'protistan gap' in the eukaryotic tree of life. This study illustrates that our current understanding of the ecological complexity of protist communities, and of the global species richness and genome diversity of protists, is severely limited. Even though 454 pyrosequencing is not a panacea, it allows for more comprehensive insights into the diversity of protistan communities, and combined with appropriate statistical tools, enables improved ecological interpretations of the data and projections of global diversity.

The Diversity of Sulfide Oxidation and Sulfate Reduction Genes Expressed by the Bacterial Communities of the Cariaco Basin, Venezuela.

Qualitative expression of dissimilative sulfite reductase (dsrA), a key gene in sulfate reduction, and sulfide:quinone oxidoreductase (sqr), a key gene in sulfide oxidation was investigated. Neither of the two could be amplified from mRNA retrieved with Niskin bottles but were amplified from mRNA retrieved by the Deep SID. The sqr and sqr-like genes retrieved from the Cariaco Basin were related to the sqr genes from a Bradyrhizobium sp., Methylomicrobium alcaliphilum, Sulfurovum sp. NBC37-1, Sulfurimonas autotrophica, Thiorhodospira sibirica and Chlorobium tepidum. The dsrA gene sequences obtained from the redoxcline of the Cariaco Basin belonged to chemoorganotrophic and chemoautotrophic sulfate and sulfur reducers belonging to the class Deltaproteobacteria (phylum Proteobacteria) and the order Clostridiales (phylum Firmicutes).

The dynamics of the bacterial diversity in the redox transition and anoxic zones of the Cariaco Basin assessed by parallel tag sequencing.

Massively parallel tag sequencing was applied to describe the bacterial diversity in the redox transition and anoxic zones of the Cariaco Basin. In total, 14 samples from the Cariaco Basin were collected over a period of eight years from two stations. A total of 244 357 unique bacterial V6 amplicons were sequenced. The total number of operational taxonomic units (OTUs) found in this study was 4692, with a range of 511-1491 OTUs per sample. Approximately 95% of the OTUs found in the redox transition zone and anoxic layers of Cariaco are represented by less than 50 amplicons suggesting that only about 5% of the bacterial OTUs are responsible for the bulk of the microbial processes in the basin redox transition and anoxic zones. The same dominant OTUs were observed across all eight years of sampling although periodic fluctuations in their proportion were apparent. No distinctive differences were observed between the bacterial communities from the redox transition and anoxic layers of the Cariaco Basin water column. The largest proportion of amplicons belongs to Gammaproteobacteria represented mostly by sulfide oxidizers, followed by Marine Group A (originally described as SAR406; Gordon and Giovannoni 1996), a group of uncultured bacteria hypothesized to be involved in metal reduction, and sulfate-reducing Deltaproteobacteria. Gammaproteobacteria, Deltaproteobacteria and Marine Group A make up 67-90% of all V6 amplicons sequenced in this study. This strongly suggests that the basin's microbial communities are actively involved in the sulfur-related metabolism and coupling of the sulfur and carbon cycles. According to detrended canonical correspondence analysis, ecological factors such as chemoautotrophy, nitrate and oxidized and reduced sulfur compounds influence the structuring and distribution of the Cariaco microbial communities.

Bacterial community composition in a large marine anoxic basin: a Cariaco Basin time-series survey.

Redox transition zones play a crucial role in biogeochemical cycles of several major elements. Because microorganisms mediate many reactions of these cycles, they actively participate in establishing geochemical gradients. In turn, the geochemical gradients structure microbial communities. We studied the interrelationship between the bacterial community structure and the geochemical gradient in the Cariaco Basin, the largest truly marine anoxic basin. This study's dataset includes bacterial community composition in 113 water column samples as well as the data for environmental variables (gradients of oxygen, hydrogen sulfide, sulfite, thiosulfate, ammonia, nitrate, nitrite, dissolved manganese and iron, dark CO2 fixation, and bacterial abundance) collected between 1997 and 2006. Several prominent bacterial groups are present throughout the entire water column. These include members of Gamma-, Delta-, and Epsilonproteobacteria, as well as members of the Marine Group A, the candidate divisions OP11 and Car731c. Canonical correspondence analysis indicated that microbial communities segregate along vectors representing oxygenated conditions, nitrite, nitrate and anoxic environments represented by chemoautotrophy, ammonia, sulfite, and hydrogen sulfide.