FACE Soil Metagenome Comparisons in IMG

Melissa Dsouza, Peter Hallin, Craig Herbold, Rima Upchurch, & Paul Wilkinson

FACE Site OverviewFree-air and Carbon Dioxide Enrichment

Elevated levels of CO2 expected in future decadesMetagenome data in IMG/M

Forested sites Duke (Sweetgum forest) Oak Ridge (Loblolly pine)

Nevada sites Soil crust Cresote

Maryland Estuary

Metagenomes OverviewSoil samplesStandardized extraction across all sites

Cheryl Kuske (LANL)FastDNA Spin kit2007-2008 sampling

Soil samplesExtracted using FastDNA Spin kit454 Titanium sequencing at JGI

Metagenomes OverviewSimilar GCSimilar

sequencing effortScaffold data

65-75% - 1 geneLength of

scaffolds varied low as 100 to 630 450-550 median

Basically unassembled reads

Questions about FACEQuestion 1: Are there differences in the taxonomic

and functional distributions across all sites?

Question 2: Are there are any differences between taxonomy within each elevated and ambient sites?

Question 3: Are there any functional differences within each elevated and ambient sites?

Taxonomy across sitesSimilar phyla present overall, but there were a few

differences between sites Actinobacteria and Proteobacteria top phyla

Swapped top or second between sites

Other top groups shifted between sitesFirmicutes, Bacteroidetes, Cyanobacteria,

Acidobacteria and Verrucomicrobia

Functional overview across sites

Nitrogen fixationAdded KEGG pathway (ko00910) to “Function

Card” and compared in Metagenome samplesOutput is matrix containing gene countsUsing R (Sorry IMG team!) we created a 2-

dimensional clustering.Clearly, samples cluster according to site, rather

than CO2 level.

Maryland Estuary CO +/- lights up

Clustering of sitesPCA of all COGs in all sitesAxes 1 and 2 explain ~90% of the variation

Largest variation in COGs appeared related to site rather than treatment

Remaining analyses done by site to examine differences by treatment

Desert

Estuary

Forests

Site Function SummaryDid not see significant

differences in 3 of 5 sitesMaryland Estuary

Membrane biogenesis-related functions

Soil crustDid not see a pattern

Was there overlap in COG functionsOne: a transposase in both

COG Pfam TIGRfamForest

Oak Ridge 0 0 0North Carolina 0 0 0

EstuaryMaryland 5 0 0

DesertSoil crust 7 8 2Creosote 0 0 0

Site Function SummaryDid not see significant

differences in 3 of 5 sitesMaryland Estuary

Membrane biogenesis-related functions

Soil crustDid not see a pattern

Was there overlap in COG functionsOne: a transposase in both

COG Pfam TIGRfamForest

Oak Ridge 0 0 0North Carolina 0 0 0

EstuaryMaryland 5 0 0

DesertSoil crust 7 8 2Creosote 0 0 0

TransposaseMaryland Estuary

Dominant group was Alphaproteobacteria

Nevada soil crust Dominant group was

Cyanobacteria

Reflective of dominant members of each site

Eurya

rchae

ota

Actino

bacter

ia

Chlor

oflex

i

Cyan

obact

eriaBa

cilli

Clostr

idia

Alpha

proteo

bacte

ria

Betap

roteo

bacter

ia

Epsilo

nprote

obact

eria

Gammap

roteo

bacte

riaThe

rmi

Gammap

roteo

bacter

ia0%2%4%6%8%

10%

Actino

bacte

ria

Alpha

proteo

bacte

riaBa

cilli

Betap

roteob

acteri

a

Chlor

oflexi

Cyan

obact

eria

Eurya

rchae

ota

Gammap

roteo

bacter

ia

Planct

omyce

tes

Strept

ophyta

0.00%2.00%4.00%6.00%8.00%

10.00%

Maryland EstuaryCOGs that were significant

Uncharacterized conserved proteinTransposaseGlycoltransferaseSerine/Theronine protein kinaseMembrane-fusion protein

Maryland EstuaryCOGs that were significant

Uncharacterized conserved proteinTransposaseGlycoltransferaseSerine/Theronine protein kinaseMembrane-fusion protein

Cell membrane biogenesisStudied phylogenetic distribution of the genes within

these COGs revealed…

Maryland EstuaryDeltaproteobacteria

were the dominant group for the phylogenetic distribution

This group might be the driving force between ambient and elevated within this site

Conclusion

Acidob

acteri

a

Actino

bacter

ia

Chlor

oflex

i

Planct

omyce

tes

Alphap

roteo

bacte

ria

Betap

roteo

bacte

ria

Deltap

roteo

bacter

ia0.00%2.00%4.00%6.00%8.00%

10.00%12.00%14.00%16.00%18.00%

Phylogenetic dis-trubution of genes

within COGS

Nevada Desert Soil CrustCOGs that were

significantAcetyltransferasesPredicted hydrolases

or acyltransferasesTransposaseCytochrome b subunit

of the bc complexPredicted ATPase FOG: PAS/PAC domainMolybdenum cofactor

biosynthesis enzyme

Pfams COX1 SBP_bac_5 HAMP Hydrolase MoeA_C DUF349 Radical_SAM SH3_3

TIGRfam YD repeat (two copies) delta-60 repeat domain

Nevada Desert Soil CrustCOGs that were

significantAcetyltransferasesPredicted hydrolases

or acyltransferasesTransposaseCytochrome b subunit

of the bc complexPredicted ATPase FOG: PAS/PAC domainMolybdenum cofactor

biosynthesis enzyme

Pfams COX1 SBP_bac_5 HAMP Hydrolase MoeA_C DUF349 Radical_SAM SH3_3

TIGRfam YD repeat (two copies) delta-60 repeat domain

Nevada Desert Soil CrustActinobacteria,

Alphaproteobacteria and Cyanobacteria were the most dominate in the distribution of functional categories

In one case Chloroflexi (shown right) were most dominate

ConclusionsAct

inobac

teria

Bacte

roide

tes

Chlor

oflex

i

Cyan

obact

eria

Clostr

idia

Fusob

acteri

a

Deltap

roteo

bacter

iaThe

rmi

Verru

comicr

obia

0.00%

0.50%

1.00%

1.50%

2.00%

2.50%

3.00%

3.50%Predicted ATPase

Final ConclusionsImpact of elevated carbon dioxide

Did not see any specific function changeTaxon played a major part in our analyses for

determining significance in functionsSequence quality

Our datasets were mainly unassembled sequencesLow quality (shorter reads) affects annotationFrame shift questions for 454 sequencingNot seeing the “real” picture of these sites