metagenomes of native and electrode-enriched … · metagenomes of native and electrode-enriched...

Metagenomes of native and electrode-enriched microbial communities from the Soudan Iron MineJonathan P. Badalamenti and Daniel R. BondDepartment of Microbiology and BioTechnology Institute, University of Minnesota - Twin Cities, Saint Paul, Minnesota, USATwitter: @JonBadalamenti @wanderingbond

Approach - compare metagenomes from native and electrode-enriched deep subsurface microbial communitiesSummary

Results - long reads recover a closed genome from mine enrichements on electrodes

AcknowledgmentsConclusions

Results - native Soudan metagenomes

Introduction

Soudan Underground Iron Mine

Genome Comparison of Metal-Reducing Deltaproteobacteria

collect Soudan brine

inoculate electrode

biodreactors

enriched

unenrichedassembled metagenomes

enrich+0.24 V

20° C

de novo assembly

IDBA_UD

de novo assembly

IDBA_UD

hybridassembly

long readassembly

HGAP

N4 binning

harvest cellsfrom

electrodes

extract DNA

extractDNA

read trimming and filtering

reconstruct complete genome(s)

PacBio RS IIlong reads

Illumina HiSeqshort readstime (d)

curr

ent (µA

/cm

2 )

00

10

10

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40

N4 binning

TFF

filtrate

retentatereturn

0.1 µm

3 µm prefilter

boreholebrine

collection bottle

Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan Underground Iron Mine harbor active microbial communities . To characterize these assemblages, we performed shotgun metagenomics of native and enriched samples. Follwing enrichment on poised electrodes and long read sequencing, we recovered from the metagenome the closed, circular genome of a novel Desulfuromonas sp. with remarkable genomic features that were not fully resolved by short read assem-bly alone. This organism was essentially absent in unenriched Soudan communities, indicating that electrodes are highly selective for putative metal reducers. Native community metagenomes suggest that carbon cycling is driven by methyl-C1 me-tabolism, in particular methylotrophic methanogenesis. Our results highlight the promising potential for long reads in metagenomic surveys of low-diversity environ-ments.

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‘Ca. Desulfuromonas biwabikus DDH964’complete genome 3,924,648 bp circular62.23% G+C3,633 CDS54 tRNA

genome featuresglyoxlyate shunt nitrate respirationdegradation of aromaticsphosphonate transportevidence for conjugative mobule element transfer (tra genes) and chromosomal integrationheavy metal resistance; mercury resistance and methylation

c-type cytochromes

genes on + strand

phage DNA/transposonsrRNArepeat sequences >500 bprepeat sequences >2,000 bp

short read assembled contigsmapped reads > 6.5 kbpmapped long read coverage

genes on − strand

3536800 3538400 3540000

malate synthase aceB isoctrate lyase iclDBIWA_3278 DBIWA_3279

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eads

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datasets

Alpha-proteobacteria

Firmicutes

AlphaproteobacteriaGammaproteobacteria

Gamma-proteobacteria

other

UnclassifiedBacteriaEuryarchaeotaother

Delta-proteobacteria

IDBA_UD SPAdes SPAdes hybrid PBcR hybrid HGAPcontigs 2820 3816 3338 581 132total length 16,849,449 15,149,004 15,417,681 5,660,852 4,451,391N50 38,339 16,604 25,567 58,773 3,932,815L50 107 187 80 27 1

assembly comparison - electrode enrichment

adding PacBio long reads improves metagenomic assembly

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568genes

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Geoba

cter s

p. M

21

Pelob

acte

r car

binoli

cus

Geobacter argillaceus

Geopsychrobacter electrodophilus

Geobacter sp. GSS01Geob

acte

r sp.

M18

Geobacter daltonii FRC-32

Geob

acte

r pick

erin

gii G

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Geoalkalibacter ferrihydriticus Z-0531

Desulfuromonas acetoxidans

Pelobacter propionicus

Geoalkalibacter subterraneus Red1

Geobacter uraniireducens Rf4

Desulfuromonas sp. TF

Geobacter metallireducens

Geobacter bremensisGeobacter sp. OR-1

Desulfuvibrio vulgaris

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bact

er se

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‘Ca.

Geobacter sulfurreducens PCA

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romon

as su

bbitu

minosa

Geobacter bemidjiensis

Desulfuromonas thiophila

Geobacter lovleyi

‘Ca.

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3824 outgroup-rooted tree based on

alignment of concatenated set of 40 conserved single-copy genes generated using Phylosift (v. 1.0.1)

pan- and core genome analysis of avail-able Geobacter/Desulfuromonas clade genomes, with cytochrome plots re-ported using default clustering param-eters in get_homologues (3-6-2015 re-lease)

86multiheme* c-typecytochrome count

86multiheme* c-typecytochrome count

*3 or more heme-binding motifs

into carbon cycling the deep terrestrial biospherelow-complexity microbial communities which drive

fundamental biogeochemical cycles, including redox transformations of metals-

dance of putative metal reducers

reducers on insoluble metal oxides as electron acceptors, such as variability in crystallinity, redox potential, and adsorption of other compounds

microbes in natural communities, but at the expense of assembly contiguityThere is tremendous potential for long reads in improving metagenomic assem-blies and downstream phylogenetic, bioinformatic, and biochemical predic-tions

Family-level lineages, particularly among FirmicutesMethanolobus was the only Archaeon observed in

unenriched metagenomes, suggesting active methyl-C1 metabolism in situ-

ronments where they exist at extremely low relative abundance

However, for unenriched metagenomic datasets, additional long read cov-erage is required to address low abundance (<5%) community members

Duluth

Int’l Falls

Bemidji

O N T A R I O

L A K E S U P E R I O R

MI N

N E S O T A

40 km

C A N A D A

U N I T E D S T A T E S

mine transect massive veins of hematite embedded in an Archaean (2.7 Gya) banded iron formation

dry mine

reduced metals, suggesting active microbial metabolismSoudan is actively maintained as a Minne-sota state park, allowing year-round access to the deep terrestrial biosphere

We thank the Minnesota Supercomputing Institute (MSI) for developing, implementing, and main-taining the PacBio SMRT Analysis suite, and the Marine Biological Laboratory (MBL) for Illumina se-quencing under a seed grant from the Deep Carbon Observatory Census of Deep Life. Long read data was generously provided by Pacific Biosciences and we thank Karl Oles (Mayo Clinic Bioinformatics Core) for sample preparation. We also thank Chris O’Brien (Pall Life Sciences) for guidance with TFF

DEEP CARBONOBSERVATORY BIOINFORMATICS CORE

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multiheme* c-typecytochromes

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2060

100

multiheme* c-typecytochromes

DDH932

DDH944

DDH951

datasetsnatural, unenriched Soudan Mine communities

unclassifiedClostridiales



Desulfitibacter

Halothiobacillus Halothiobacillus

Marinobacter

Marinobacter

Marinobacter

DesulfopilaDehalobacterHalanaerobium

Halocella

Halocella HalocellaDesulfosporosinusDehalobacter Dehalobacter

unclassifiedHalanaerobiaceae

unclassifiedRhodobacteraceae

unclassifiedProteobacteria

unclassifiedRhodobacteraceae

unclassifiedRhodobacterales

unclassifiedBacteroidales

unclassifiedBacteroidales

unclassifiedAlteromonadales

unclassifiedPeptococcaceae



Methanolobus

Methanolobus

Methanolobus

GeobacterSphaerochaeta

Rhodovulum Rhodovulum

DemequinaDemequina

Roseovarius

inner rings novel lineagesFirmicutesFirmicutesBacteroidetes

AlphaproteobacteriaGammaproteobacteria

DeltaproteobacteriaEuryarchaeota

unclassifiedProteobacteria

Order levelClass level

Family level

deeper/more reduced

shallower/more oxidized samples collected along redox gradient

metagenomes of native and electrode-enriched … · metagenomes of native and electrode-enriched...

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