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Microbial Metagenomics: A Tale of the Dead and the Living
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is a microbial world we live in: microbes outnumber other organisms by several orders of magnitude, and they have great importance for the environment. However, environmental microbes are notoriously difficult to grow in the laboratory, and using culture independent techniques is necessary to expand our view. In this thesis, I apply metagenomics and single-cell genomics to environmental samples from ancient human remains and lakes.

First, I used metagenomics to learn about bacteria from a Neanderthal’s bone and the gut of Ötzi, a frozen natural mummy. Both were exploratory studies where the main question was what kind of bacteria are present. I found out that Streptomyces dominated this particular Neanderthal fossil, and the DNA lacked the damage that is often seen in ancient samples. Ötzi's gut sample was dominated by Clostridia and fungi belonging to Basidiomycota.

Second, ten single-cell amplified genomes of freshwater Alphaproteobacterium LD12 and three metagenomes from Swedish lakes were sequenced. Comparative metagenomics allowed hypothesizing about which functions are important for microbe proliferation in freshwater, pointing to osmoregulation and transport proteins and, possibly, to different strategies of metabolizing sugars. I also focused on SAR11 sister-groups in oceans and lakes. Phylogenies and sequence evolutionary distance estimates indicated the existence of microclusters within LD12, showing variation in abundance between lakes. The most striking difference was the relative amount of recombination compared to mutation, the estimated r/m ratio. SAR11 marine and their freshwater cousins are found at the opposite extremes of the r/m range, lowest and highest, respectively. The genetic background or sequence diversity did not explain the observed dramatic difference, so it is possibly connected to environmental adaptation or population dynamics.

In addition, I have spent a substantial amount of effort benchmarking available metagenomic methods, for example fragment recruitment of metagenomes to reference genomes.

In conclusion, my exploratory metagenomic studies have shed some light on the bacteria present in ancient human remains; comparative metagenomics has suggested the importance of substrate preference on functional differences between lakes and oceans; finally, single-cell genomes have allowed some insight into molecular evolutionary processes taking place in the freshwater LD12 bacterium. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 108 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1067
Keyword [en]
single-cell genomics, single cell, fossil metagenome, freshwater metagenome, LD12
National Category
Microbiology Bioinformatics and Systems Biology
Identifiers
URN: urn:nbn:se:uu:diva-206702ISBN: 978-91-554-8742-3 (print)OAI: oai:DiVA.org:uu-206702DiVA: diva2:645075
Public defence
2013-10-18, B41, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-09-25 Created: 2013-09-03 Last updated: 2014-01-23
List of papers
1. No Ancient DNA Damage in Actinobacteria from the Neanderthal Bone
Open this publication in new window or tab >>No Ancient DNA Damage in Actinobacteria from the Neanderthal Bone
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, e62799- p.Article in journal (Refereed) Published
Abstract [en]

Background: The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized. Principal Findings: Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences. Conclusions/Significance: Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-205391 (URN)10.1371/journal.pone.0062799 (DOI)000321202100042 ()
Available from: 2013-08-29 Created: 2013-08-16 Last updated: 2017-12-06Bibliographically approved
2. Single cell genomics reveals low recombination frequencies in freshwater bacteria of the SAR11 clade
Open this publication in new window or tab >>Single cell genomics reveals low recombination frequencies in freshwater bacteria of the SAR11 clade
Show others...
2013 (English)In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 14, no 11, R130Article in journal (Refereed) Published
Abstract [en]

Background: The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt-and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet. Results: We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules. Conclusions: We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-206203 (URN)10.1186/gb-2013-14-11-r130 (DOI)000330616200009 ()24286338 (PubMedID)
Funder
Swedish Research Council, 349-2007-831 621-2008-3259 621-2011-4669-4669 2009-3784 2008-1923 2012-3892EU, European Research CouncilGöran Gustafsson Foundation for promotion of scientific research at Uppala University and Royal Institute of TechnologyKnut and Alice Wallenberg Foundation, KAW-2011.0148 KAW-2012.0075Swedish National Infrastructure for Computing (SNIC), p2006019 p2009043
Available from: 2013-09-03 Created: 2013-08-29 Last updated: 2017-12-06Bibliographically approved
3. Productivity and salinity structuring of the microplankton revealed by comparative freshwater metagenomics
Open this publication in new window or tab >>Productivity and salinity structuring of the microplankton revealed by comparative freshwater metagenomics
Show others...
2014 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 16, no 9, 2682-2698 p.Article in journal (Refereed) Published
Abstract [en]

Little is known about the diversity and structuring of freshwater microbial communities beyond the patterns revealed by tracing their distribution in the landscape with common taxonomic markers such as the ribosomal RNA. To address this gap in knowledge, metagenomes from temperate lakes were compared to selected marine metagenomes. Taxonomic analyses of rRNA genes in these freshwater metagenomes confirm the previously reported dominance of a limited subset of uncultured lineages of freshwater bacteria, whereas Archaea were rare. Diversification into marine and freshwater microbial lineages was also reflected in phylogenies of functional genes and there were also significant differences in functional beta-diversity. The pathways and functions that accounted for these differences are involved in osmoregulation, active transport, carbohydrate and amino acid metabolism. Moreover, predicted genes orthologous to active transporters and recalcitrant organic matter degradation were more common in microbial genomes from oligotrophic versus eutrophic lakes. This comparative metagenomic analysis allowed us to formulate a general hypothesis that oceanic- compared to freshwater-dwelling microorganisms, invest more in metabolism of amino acids and that strategies of carbohydrate metabolism differ significantly between marine and freshwater microbial communities.

National Category
Microbiology Natural Sciences
Identifiers
urn:nbn:se:uu:diva-206205 (URN)10.1111/1462-2920.12301 (DOI)000341579700006 ()24118837 (PubMedID)
Note

Published in 2013 as Epub ahead of print

Available from: 2013-09-03 Created: 2013-08-29 Last updated: 2017-12-06Bibliographically approved
4. Metagenomic analysis of a distal gut sample from the Tyrolean Iceman
Open this publication in new window or tab >>Metagenomic analysis of a distal gut sample from the Tyrolean Iceman
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-206206 (URN)
Available from: 2013-09-03 Created: 2013-08-29 Last updated: 2014-01-23
5. Testing fragment recruitment methods by simulating environmental communities of different complexities
Open this publication in new window or tab >>Testing fragment recruitment methods by simulating environmental communities of different complexities
(English)Manuscript (preprint) (Other academic)
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:uu:diva-206208 (URN)
Available from: 2013-09-03 Created: 2013-08-29 Last updated: 2014-01-23

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