Albertsen, Mads

Karst, Søren Michael

McIlroy, Simon Jon

Nielsen, Per Halkjær

Stokholm-Bjerregaard, Mikkel

Examining the complete gene expression of unculturable bacteria in complex communities has been the dream of microbial ecologists for years. However, a major obstacle is the recovery of a reliable template, the relevant genomes from the communities. While metagenomics has been proposed to solve the problem, it is extremely rare to be able to recover high quality genomes from complex samples. This is not due to lack of sequencing depth, but the presence of multiple closely related species, which prevents decent genome assemblies.In this work we show that a successful approach is to make an enrichment of the target organisms in laboratory scale reactors under controlled conditions. We demonstrate that it is now easy and affordable to extract genomes of all the dominant organisms from reactors due to reduced micro-diversity and further use these to examine their individual gene expression profiles by metatranscriptomics. To demonstrate this we revisited the bacteria involved in enhanced biological phosphorus removal (EBPR) from wastewater treatment plants. The EBPR process is used all over the world, has a large body of information regarding the underlying microbiology, and is often studied using laboratory scale reactors. However, conflicting evidence is often obtained for laboratory scale studies, likely due to difference in the microbial community composition, which is often not defined. We investigated three laboratory scale reactors seeded with full-scale activated sludge and operated to enrich for bacteria contributing to phosphorus removal and their normal competitors. To extract complete genomes we generated two metagenomes from each reactor, taken approximately 1 month apart, using the Illumina HiSeq2000 platform. Due to low micro-diversity in the reactors (2-15 dominating species), most species assembled into few scaffolds that were subsequently binned to separate genomes using the two time-points through the multi-metagenome strategy. To investigate the individual gene expression of the dominant species in each reactor we designed a cycle study that mimicked the normal phosphorus removal process in the treatment plant, sampling every 20 min during the 9 hour experiment. Metratranscriptome data was generated from selected samples using the stranded RNAseq Illumina protocol.We were able to extract genomes from the model polyphosphate accumulating organism (PAO) Ca. Accumilibacter, the putative PAO Tetrasphaera and the model competitors Defluviicoccus and Ca. Competibacter. The metatranscriptomes were subsequently used to annotate the genomes and finally shed light on many of the previous literature-based hypotheses regarding their physiologies. Interestingly, we also recovered a genome of an abundant bacteria closely related to Ca. Accumulibacter, which did not accumulate phosphate, i.e. a potential new competitor. Surprisingly, the bacteria was also hit by the Accumulibacter group-defining FISH probe (PAOmix) and would thereby mistakenly lead to an overestimation of the abundance of PAOs in treatment plants. In full-scale metagenomes, the Accumulibacter-related putative competitor was often found in higher abundance than Ca. Accumulibacter. In conclusion, it is now feasible to generate genomes and subsequent individual transcriptomes from laboratory scale reactors, which provides an opportunity for unprecedented levels of insight into the competition and cooperation between unculturable species. <br/

    Aalborg UniversitetUsing metagenomics and metatranscriptomics to study specific bacterial speciesinvolved in biological phosphorus removal from wastewaterAlbertsen, Mads; McIlroy, Simon Jon; Stokholm-Bjerregaard, Mikkel; Karst, Søren Michael;Nielsen, Per HalkjærPublication date:2014Document VersionEarly version, also known as pre-printLink to publication from Aalborg UniversityCitation for published version (APA):Albertsen, M., McIlroy, S. J., Stokholm-Bjerregaard, M., Karst, S. M., & Nielsen, P. H. (2014). Usingmetagenomics and metatranscriptomics to study specific bacterial species involved in biological phosphorusremoval from wastewater. Poster session presented at 15th International Symposium on Microbial Ecology,Seoul, Korea, Republic of.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: April 25, 2017Using metagenomics and metatranscriptomics to study specific bacterial species involved in biological phosphorus removal from wastewaterAAUI n t r o d u c t i o nM e t h o d sC o n c l u s i o n sR e s u l t sEnrichmentMads Albertsen, Simon J. McIlroy, Mikkel Stokholm-Bjerregaard, Søren M. Karst & Per H. NielsenCenter for Microbial Communities, Aalborg University, Aalborg, DenmarkGenome recoveryma@bio.aau.dk                    @MadsAlbertsen85 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYWastewater treatment plant Sequencing Batch ReactorTranscriptomics2013-09-06Seed12-1912-1311-2511-05ExperimentSamples2013-09-0611-0511-2512-1312-19AssemblyMiSeq 2x300bpHiSeq 2x150bpTruSeq PENextera MPMetagenomesmmgenomeCLCCoverageGC contentTetranucleotide frequencyEssential genesTaxonomic classification (PhyloPythiaS+)rRNA genesEcotype specific genesPaired-end connectionsBinningA toolbox for reproducible genome extraction from metagenomesMadsAlbertsen/mmgenomeExamplerRNA removal (Ribo-Zero Bacteria)mRNA seq (TruSeq Stranded)HiSeq 1x50 bp9 timepoints27 samples400 million readsMapping to the metagenome bins enables transcriptomics of the individual species in the community.Amaricoccus kaplicensisgenus Defluviicoccusgenus TetrasphaeraMicrolunatus phosphovorusMicrolunatus glycogencia0.10AlphaproteobacteriaGammaproteobacteriaActinobacteriaBetaproteobacteriagenus “Ca. Contendobacter”genus “Ca. Competibacter”Plasticicumulans acidivoransAcinetobacter IwofiiDechloromonas agiatagenus Propionivibriogenus “Ca. Accumulibacter”Relative ExpressionMaximumMinimum**************************** > 2 fold change15        180A  B  C  A  B  CSamplesTime [min]A GAO disguised as a PAO. The putative GAOPropionivibrio (magenta) is hit by the probes used toquantify the PAO Accumulibacter (white). However, it doesnot accumulate excessive amounts of polyphosphate.Probes: PAO651 (FLUOS, Accumulibacter); PAO462 (Cy3,both); PAO846 (Cy3, both); EUBmix (Cy5, all bacteria).Phylogenetic tree (16S rRNA) of known PAOs and GAOs. Until nowthe PAOs and GAOs have been separated into distant phylogeneticlineages. The discovery of a GAO from the genus Propionivibriomakes interpretations more difficult. The binned Accumulibactergenome was closely related to Accumulibacter clade IIC based onphylogenetic analysis of the ppk1 gene.Accumulibacter transcriptomics. Relative expressionof the core genes of the Accumulibacter bin underanaerobic feast (15 min) and aerobic famine (180 min)conditions.Genome extraction from metagenomes. Differential coveragebinning is used as the first step in extracting individual genomesfrom the metagenomes. Each point represents a scaffold, coloredby essential single copy genes and scaled by scaffold length. Theplot was made using the mmplot function in the mmgenome Rpackage. Suprisingly a bacteria from the genus Propionivibrio wasenriched along with Accumulibacter.AccumulibacterPropionivibrio*Circular 1 Mbp genome from candidate Phylum SR1Accumulibacter PropionivibrioGenome size 4.7 Mbp 3.8 MbpScaffolds 30 88Completeness 100 % > 95 %Strains 1 2-3Plasmids 1 1-2Non-rRNA transcriptome reads 70 million 159 millionAbundance: Experiment 15 % 32 %Abundance: Seed WWTP 0.2 % 0.2 %Abundance: Other WWTPs* < 0.1 - 0.2 % 0.1 - 0.8 %*Estimated from 15 full scale WWTP metagenomes (2.3 bn reads).Time [min]Enhanced Biological Phosphorous Removal (EBPR)from wastewater treatment plants is managed byPolyphosphate Accumulating Organisms (PAOs). Themodel PAO is “Candidatus Accumulibacter phosphatis”(Accumulibacter) and despite a large body ofknowledge, their detailed physiology remains elusive.In this study we combined metagenomics,metatranscriptomics and laboratory scale enrichmentin order to examine the gene expression of theuncultured Accumulibacter and the co-enrichedassociated community.• Combining metagenomics, metatranscriptomics and laboratory scale enrichments enablestranscriptome studies of most individual species in the community.• For the first time we reveal the detailed transctriptome landscape of Accumulibacter during theanaerobic feast and aerobic famine conditions of the EBPR process.• A new GAO (competitor to PAOs with a negative impact on EBPR) was discovered. Propionivibrio isclosely related to Accumulibacter and hit by the current FISH probes used to define PAOs.Concentration [c-mmol/L]Concentration [p-mmol/L]

Using metagenomics and metatranscriptomics to study specific bacterial species involved in biological phosphorus removal from wastewater

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Using metagenomics and metatranscriptomics to study specific bacterial species involved in biological phosphorus removal from wastewater

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