Thousands of primer-free, high-quality, full-length SSU rRNA sequences from all domains of life
Karst SM, Dueholm SM, McIlroy SJ, Kirkegaard RH, Nielsen PH & Albertsen M. BioRxiv 2016 (in review).
Abstract: Ribosomal RNA (rRNA) genes are the consensus marker for determination of microbial diversity on the planet, invaluable in studies of evolution and, for the past decade, high-throughput sequencing of variable regions of ribosomal RNA genes has become the backbone of most microbial ecology studies. However, the underlying reference databases of full-length rRNA gene sequences are underpopulated, ecosystem skewed, and subject to primer bias, which hamper our ability to study the true diversity of ecosystems. Here we present an approach that combines reverse transcription of full-length small subunit (SSU) rRNA genes and synthetic long read sequencing by molecular tagging, to generate primer-free, full-length SSU rRNA gene sequences from all domains of life, with a median raw error rate of 0.17%. We generated thousands of full-length SSU rRNA sequences from five well-studied ecosystems (soil, human gut, fresh water, anaerobic digestion, and activated sludge) and obtained sequences covering all domains of life and the majority of all described phyla. Interestingly, 30% of all bacterial operational taxonomic units were novel, compared to the SILVA database (less than 97% similarity). For the Eukaryotes, the novelty was even larger with 63% of all OTUs representing novel taxa. In addition, 15% of the 18S rRNA OTUs were highly novel sequences with less than 80% similarity to the databases. The generation of primer-free full-length SSU rRNA sequences enabled eco-system specific estimation of primer-bias and, especially for eukaryotes, showed a dramatic discrepancy between the in-silico evaluation and primer-free data generated in this study. The large amount of novel sequences obtained here reaffirms that there is still vast, untapped microbial diversity lacking representatives in the SSU rRNA databases and that there might be more than millions after all. With our new approach, it is possible to readily expand the rRNA databases by orders of magnitude within a short timeframe. This will, for the first time, enable a broad census of the tree of life.
Complete nitrification by Nitrospira bacteria
Daims H, Lebedeva EV, Pjevac P, Han P, Herbold C, Albertsen M, Jehmlich N, Palatinszky M, Vierheilig J, Bulaev A, Kirkegaard RH, von Bergen M, Rattei T, Bendinger B, Nielsen PH and Wagner M. Nature 2015.
Abstract: Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.
Complete nitrification by a single microorganism
van Kessel MAHJ, Speth DR, Albertsen M, Nielsen PH, Op den Camp HJM, Kartal B, Jetten MSM and Lücker S. Nature 2015.
Abstract: Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms. Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.
Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes
M Albertsen, Hugenholtz P, Skarshewski A, Nielsen KL, Tyson GW & Nielsen PH. Nature Biotechnology 2013.
Abstract: Reference genomes are required to understand the diverse roles of microorganisms in ecology, evolution, human and animal health, but most species remain uncultured. Here we present a sequence composition–independent approach to recover high-quality microbial genomes from deeply sequenced metagenomes. Multiple metagenomes of the same community, which differ in relative population abundances, were used to assemble 31 bacterial genomes, including rare (<1% relative abundance) species, from an activated sludge bioreactor. Twelve genomes were assembled into complete or near-complete chromosomes. Four belong to the candidate bacterial phylum TM7 and represent the most complete genomes for this phylum to date (relative abundances, 0.06–1.58%). Reanalysis of published metagenomes reveals that differential coverage binning facilitates recovery of more complete and higher fidelity genome bins than other currently used methods, which are primarily based on sequence composition. This approach will be an important addition to the standard metagenome toolbox and greatly improve access to genomes of uncultured microorganisms.