Sequencing the World of Possibilities for Energy & Environment

Sequencing the World of Possibilities for Energy & Environment

Metagenomics with spatial resolution

When doing metagenomics, we take a sample from a

community and homogenise

The structure and complexityof a community are ignored

What about layers in microbial communities?

Sequencing the World of Possibilities for Energy & Environment

Microelectrode measurements

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Introduction

• Hypersaline mat, Guerrero Negro, Baja California

– ~6cm thick

• The most diverse community on Earth

– Probably :)– Tens of thousands of microbial species– Dozens of novel phyla

• Steep physico/chemical gradients with depth

– O2, H2S, light

• Highly stratified (layered) community

– Cyanobacteria on top– Cloroflexi, proteobacteria,

bacteroides below

Sequencing the World of Possibilities for Energy & Environment

Sequencing the World of Possibilities for Energy & Environment

• Sequenced 10 layers of the mat

• Samples are taken: 1mm top layers, 3mm lower layers

• A parallel project is aimed to identify the diversity, deep sequencing of 16S is under way in the Norm Pace’ lab.

• Sequencing, from each layer:– ~13,000 reads (between 11,574 and 15,663)– Assembly gives contigs less than 10kb, all analysis

done with reads– ~13,600 proteins (between 12,012 and 15,984) – Loaded into IMG/M`

Sequencing strategy

Sequencing the World of Possibilities for Energy & Environment

The questions

• How does the mat live?– Metabolism, interactions, chemotaxis

• Are there gradients observable from the data?– Species– Protein families– Functional families– Other characteristics?

• What is special about the mat as compared to other environments?

– Adaptations similar across many layers?– Adaptations that cross phylogenetic

boundaries?

• Stress management– Salinity– Heat

Sequencing the World of Possibilities for Energy & Environment

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Depth (mm)

fraction of phylogenetic assignments per layer (%)

Unassigned Alphaproteobacteria

Cyanobacteria Deltaproteobacteria

Gammaproteobacteria Bacteroidetes

Blast

Sequencing the World of Possibilities for Energy & Environment

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fraction of phylogenetic assignments per layer

Alphaproteobacteria Bacteriodetes Chloroflexi

Cyanobacteria Planctomycetes

Marker genes

Sequencing the World of Possibilities for Energy & Environment

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Proteobacteria Bacteroidetes Chloroflexi Cyanobacteria Planctomycetes

Spirochaetes Verrucomicrobia

16S clone library

Sequencing the World of Possibilities for Energy & Environment

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Proteobacteria Bacteroidetes Chloroflexi Cyanobacteria Planctomycetes

Spirochaetes Verrucomicrobia

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Depth (mm)

fraction of phylogenetic assignments per layer (%)

Unassigned Alphaproteobacteria

Cyanobacteria Deltaproteobacteria

Gammaproteobacteria Bacteroidetes

Species content - the discrepancies

16S clone library

Blast

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fraction of phylogenetic assignments per layer

Alphaproteobacteria Bacteriodetes Chloroflexi

Cyanobacteria Planctomycetes

Marker genes

Sequencing the World of Possibilities for Energy & Environment

Functional content

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Sulfatases Methyltransferases Heterodisulfidereductase

ferredoxinoxidoreductase

DUF820 domain

Mat Gutless Worm Soil Whalefall Sludge AMD IMG

Sequencing the World of Possibilities for Energy & Environment

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Sulfatases Methyltransferases Heterodisulfidereductase

ferredoxinoxidoreductase

DUF820 domain

Mat

Gutless Worm

Soil

Whalefall

Sludge

AMD

IMG

Sequencing the World of Possibilities for Energy & Environment

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Sequencing the World of Possibilities for Energy & Environment

Sequencing the World of Possibilities for Energy & Environment

Sequencing the World of Possibilities for Energy & Environment

Sequencing the World of Possibilities for Energy & Environment

Sequencing the World of Possibilities for Energy & Environment

Unifying features?

• Layers are different– Phylogenetically– Functionally

• Anything common to all layers– Hypersalinity is common– Adaptations to hypersalinity?– K/Na exchange, increasing intracellular K

• In Archaea• Salinibacter Ruber• In the mat?

Sequencing the World of Possibilities for Energy & Environment

Isoelectric profilesAverage isoelectric point distribution

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4.7 5.1 5.5 5.9 6.3 6.7 7.1 7.5 7.9 8.3 8.7 9.1 9.5 9.9

Isoelectric point

Fraction of dataset

Bacteria Archaea Mat Metagenomes Phages

Sequencing the World of Possibilities for Energy & Environment

Amino acid usage

Aspartate content distribution

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3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Proteomic aspartate fraction

Fraction of dataset

archaea bacteria mat microbiomes viruses

Sequencing the World of Possibilities for Energy & Environment

GC content distribution of contigs in HSM

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GC contnent

Occurence

12345678910

Sequencing the World of Possibilities for Energy & Environment

Conclusions

• Gradients found of– Phylogenetic groups– Protein families– Functional families

• Adaptations to salinity– Isoelectric point– Amino acid usage

Sequencing the World of Possibilities for Energy & Environment

Acknowledgments

Jeroen RaesKirk Harris

Natalia Ivanova John Spear

Christian Von MeringBrad Bebout Peer Bork

Norman PacePhilip Hugenholtz