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?


Microelectrode measurements

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O2

H2S

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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


• 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


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


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Depth (mm)

fraction of phylogenetic assignments per layer (%)

Unassigned Alphaproteobacteria

Cyanobacteria Deltaproteobacteria

Gammaproteobacteria Bacteroidetes

Blast


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Depth (mm)

fraction of phylogenetic assignments per layer

Alphaproteobacteria Bacteriodetes Chloroflexi

Cyanobacteria Planctomycetes

Marker genes


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Proteobacteria Bacteroidetes Chloroflexi Cyanobacteria Planctomycetes

Spirochaetes Verrucomicrobia

16S clone library


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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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Depth (mm)

fraction of phylogenetic assignments per layer

Alphaproteobacteria Bacteriodetes Chloroflexi

Cyanobacteria Planctomycetes

Marker genes


Functional content

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Sulfatases Methyltransferases Heterodisulfidereductase

ferredoxinoxidoreductase

DUF820 domain

Mat Gutless Worm Soil Whalefall Sludge AMD IMG


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Sulfatases Methyltransferases Heterodisulfidereductase

ferredoxinoxidoreductase

DUF820 domain

Mat

Gutless Worm

Soil

Whalefall

Sludge

AMD

IMG


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H2S

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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?


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


Amino acid usage

Aspartate content distribution

0102030405060708090

100

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


GC content distribution of contigs in HSM

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GC contnent

Occurence

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Conclusions

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

• Adaptations to salinity– Isoelectric point– Amino acid usage


Acknowledgments

Jeroen RaesKirk Harris

Natalia Ivanova John Spear

Christian Von MeringBrad Bebout Peer Bork

Norman PacePhilip Hugenholtz

sequencing the world of possibilities for energy & environment

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