microbial and functional diversity of a subterrestrial high ph

Microbial and functional diversity of a subterrestrialhigh pH groundwater associated to serpentinization

Igor Tiago1,2 and António Veríssimo1,2*1Department of Life Sciences, University of Coimbra,Apartado 3046, 3001-401 Coimbra, Portugal.2Center of Neuroscience and Cell Biology, University ofCoimbra, 3004-517 Coimbra, Portugal.

Summary

Microbial and functional diversity were assessed,from a serpentinization-driven subterrestrial alkalineaquifer – Cabeço de Vide Aquifer (CVA) in Portugal.DGGE analyses revealed the presence of a stablemicrobial community. By 16S rRNA gene librariesand pyrosequencing analyses, a diverse bacterialcomposition was determined, contrasting with lowarchaeal diversity. Within Bacteria the majorityof the populations were related to organismsor sequences affiliated to class Clostridia, butmembers of classes Acidobacteria, Actinobacteria,Alphaproteobacteria, Betaproteobacteria, Deinoc-occi, Gammaproteobacteria and of the phyla Bacter-oidetes, Chloroflexi and Nitrospira were alsodetected. Domain Archaea encompassed mainlysequences affiliated to Euryarchaeota. Only form IRuBisCO – cbbL was detected. Autotrophic carbonfixation via the rTCA, 3-HP and 3-HP/4H-B cyclescould not be confirmed. The detected APS reductasealpha subunit – aprA sequences were phylogeneti-cally related to sequences of sulfate-reducing bacte-ria belonging to Clostridia, and also to sequencesof chemolithoautothrophic sulfur-oxidizing bacteriabelonging to Betaproteobacteria. Sequences ofmethyl coenzyme M reductase – mcrA were phylo-genetically affiliated to sequences belonging toAnaerobic Methanotroph group 1 (ANME-1). Thepopulations found and the functional key markersdetected in CVA suggest that metabolisms related toH2, methane and/or sulfur may be the major drivingforces in this environment.

Introduction

Alkaline water associated with ophiolites and serpentini-zation activity has been considered a potential environ-ment for the emergence of life on the early Earth(Nealson et al., 2005; Schulte et al., 2006; Russell et al.,2010; Sleep et al., 2011). Serpentinization leads to therelease of H2, methane and low-molecular-weightorganic compounds (McCollom and Seewald, 2007;Proskurowski et al., 2008), constituting a potentialsource of reducing power and organic carbon for organ-isms inhabiting the ultramafic subsurface. Althoughactive serpentinization is occurring on all of the world’scontinents and comprise significant portions of the deepseafloor, such ecosystems are still some of the mostpoorly understood portions of the biosphere. Althoughmicrobial diversity in deep-sea ultramafic hydrothermalsystems has been determined by several studies(Schrenk et al., 2003; 2004; Kelley et al., 2005; Brazel-ton et al., 2006; 2011) to date, microbial diversitysurveys on terrestrial serpentine driven environmentsare rare. Despite a pioneer study on a spring water inthe ophiolitic complex of Semail in Oman by Bath andcolleagues (1987), the studies on Cabeço de Videaquifer in South-east of Portugal by Tiago and col-leagues (2004), on the Maraquin site in Jordan by Ped-ersen and colleagues (2004) and on the Del PuertoOphiolite in California by Blank and colleagues (2009),only recently Brazelton and colleagues (2012) reportedmetagenomic evidence for H2 oxidation and H2 produc-tion by microbial communities inhabiting a serpentine-hosted terrestrial environment in Canada. Cabeço deVide aquifer (CVA) in the south of Portugal had its originin an ophiolite-like context where serpentinization activityhas been recognized (Marques et al., 2008). Thegroundwater in CVA is associated with mafic/ultramaficrocks, has a distinct chemical composition (Na-Cl/Ca-OHtype waters) and pH value around 11.4. The serpentini-zation processes are very slow and occur at low tem-perature as a result of a 2790 � 40 years BP rechargeof the CVA system (Marques et al., 2008). Low-temperature geochemical processes, like serpentiniza-tion associated with mafic and ultramafic rocks on Earth,are considered geologically similar to those occurring onMars, and are discussed as Mars analogues (Schulteet al., 2006; Blank et al., 2009).

Received 9 March, 2012; accepted 25 October, 2012. *For corre-spondence. E-mail [email protected]; Tel. (+351) 239824024; Fax(+351) 239826798.

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Environmental Microbiology (2012) doi:10.1111/1462-2920.12034

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd

mailto:[email protected]

Analyses of 16S rRNA gene phylogeny combined withthe detection and phylogenetic analyses of genes encod-ing key enzymes of specific metabolic pathways (such asautotrophic carbon fixation pathways, and sulfur andmethane cycles) has been crucial in the study of somesubsurface microbial habitats (Campbell et al., 2003;Campbell and Cary, 2004; Blazejak et al., 2006; Brazeltonet al., 2006; 2011; 2012; Lin et al., 2006; Nakagawa andTakai, 2008; Alfreider et al., 2009). This approach enablesto predict which geochemical energy sources are power-ing the ecosystem and which biochemical processes aresustaining the microbial community (Nealson et al., 2005;Hoehler, 2007; Cardace and Hoehler, 2009).

Earlier we used cultured-based approaches to charac-terize the heterotrophic aerobic population isolatedfrom CVA. We found a cultivable microbial communityconstituted mainly of Gram-positive bacteria belongingto Actinobacteria, Bacillus and Staphylococcus. Only avery small fraction was Gram-negative (Tiago et al.,2004).

The purpose of this study was to perform a culture-independent determination of the microbial diversity andpopulation dynamics in a continental serpentinization-driven aquatic environment, by analysing groundwatersamples collected directly from CVA. Molecular tech-niques such as 16S rRNA gene clone libraries, pyrotagsequencing and DGGE analyses were employed. Fur-thermore, we performed a PCR survey and phylogeneticcharacterization of genes encoding key enzymes ofautotrophic CO2 fixation pathways: namely Calvin-Benson-Bassham cycle (CBB cycle), reductive tricarbo-xylic acid cycle (rTCA cycle), 3-hydroxypropionatecycle (3-HP cycle) and the 3-hydroxypropionate/4-hydroxybutyrate cycle (3-HP/4-HB cycle) and also ofgenes encoding key enzymes related with the sulfur andmethane cycles. The overall results obtained contributed

to a better understanding of this unique subterrestrialmicrobial ecosystem.

Results

Seven water samples were recovered from CVA, during a3-year period, processed and analysed as described inTable 1.

Chemical characterization of the groundwater and totalcell concentrations

The groundwater recovered during the last 20 years at theborehole AC3 in CVA showed a stable and distinctivechemical composition dominated by ions such as hydrox-ide, chloride, sodium and calcium; carbonate was alsopresent but in low concentrations (Supplementary TableS2). Dissolved CO2 and O2 levels were below the detec-tion limit; moreover the presence of CO2 is improbabledue to the high pH of the water. The resulting aqueoussolution was highly alkaline (pH 11.4).

During the sampling periods and in other occasionstotal coliforms, faecal coliforms, faecal streptococci, Pseu-domonas aeruginosa and Clostridium perfringens, werenever detected. The total cell concentrations determinedvaried between 5.6 ¥ 102 � 2.4 ¥ 102 cells ml-1 and6.4 ¥ 102 � 4.1 ¥ 102 cells ml-1.

Clone library construction

For each sample, bacterial and archaeal 16S rRNA geneclone libraries were constructed, but the phylogeneticanalysis of the microbial community was determined fromthe combined data sets of all clone libraries. Despiteseveral attempts, no amplification was obtained fordomain Eukarya.

Table 1. CVA water samples information regarding collection date, volume, pore sizes filter used for concentration and the culture-independentanalyses performed.

Samples 1 2 3 4 5 6 7

Collection date July 2006 January 2007 January 2007 July 2008 July 2008 December 2008 May 2009Volume (l) 6 24 24 24 24 24 24Filter pore Ø (mm) 0.2 0.2 0.1 0.2 0.1 0.1 0.1Cloninga Xb X X – – – –DGGEc –d X X X X X XPyrosequencinge – – X – X – XFunctional genesf – – – – – – X

a. Cloning – culture-independent analyses performed for the 16S rRNA gene for both domains Bacteria and Archaea: clone library construction.b. X, analysis performed.c. DGGE – denaturing gradient gel electrophoresis analyses performed for the 16S rRNA gene for both domains Bacteria and Archaea.d. ‘–’, analysis not performed.e. Pyrosequencing – pyrosequencing analyses performed for the 16S rRNA gene for both domains Bacteria and Archaea.f. Functional genes – culture-independent analyses performed for the genes encoding key enzymes of autotrophic CO2 fixation pathways: namelyCBB cycle, rTCA cycle and 3-HP and 3-HP/4-HB cycles, and also to the presence of genes encoding key enzymes related with the sulfur andmethane cycles: clone library construction.

2 I. Tiago and A. Veríssimo

© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology

Bacterial diversity assessed by 16S rRNA geneclone library

A total of 441 clones were screened by RFLP analyses.After phylogenetic analyses, of the representative clonesof each RFLP cluster, at a cut-off of 16S rRNA genesimilarity of � 97%, 45 different operational taxonomicunits (OTUs) were obtained (Table 2, Fig. 1, Supplemen-tary Figs S1 and S2). The 45 OTUs were distributedwithin 10 different phylogenetic groups, comprisingclasses Acidobacteria, Actinobacteria, Alphaproteobacte-ria, Betaproteobacteria, Clostridia, Deinococci, Gamm-aproteobacteria and phyla Bacteroidetes, Chloroflexi andNitrospira (Table 2). Class Clostridia encompassed 53.3%of the OTUs (corresponding to 42.8% of the clones).Class Betaproteobacteria encompassed 11.1% of theOTUs, corresponding to 36.7% of the clones (Table 2).The remaining OTUs were distributed in other phyloge-netic groups (Table 2).

Within the 45 determined OTUs, five major populationswere identified, four of them were closely related tochemolithoautotrophic organisms. OTU 1, comprising35.1% of total clones was closely affiliated (~ 97% simi-larity) with Hydrogenophaga flava a facultative chemo-lithoautotrophic Betaproteobacteria that can use oxidationof H2 as energy source and CO2 as carbon source(Willems et al., 1989). OTU 25 the second most retrieved(comprising 24.3% of the clones) was closely related(~ 96% similarity) to the chemolithoautotrophic Candida-tus ‘Desulforudis audaxviator’, belonging to classClostridia, retrieved from a very deep (2.8 km) groundwa-ter environment (Chivian et al., 2008). OTU 11 (compris-ing 9.8% of the clones) was related to clone sequencesaffiliated to phylum Bacteroidetes retrieved from differentsources (Ley et al., 2008; Perkins et al., 2009; Zhanget al., 2010). OTU 40 (comprising 3.6% of the clones) wasclosely related (~ 94% similarity) to the facultativeautotrophic bacteria Dethiobacter alkaliphilus (Sorokin

687188

53

88

70

56

74

97

99

6560

99

82

98

72

99

9847

40

79

98

63

89

53

99

98

95

45

99

99

98

97

94

3625

3549

64

0.1

uncultured bacterium clone CVCloAm1Ph2-OTU18 (AM777989)uncultured bacterium clone CVCloAm3Ph64-OTU18 (AM778018)

uncultured bacterium clone CVCloAm2Ph1-OTU18 (AM777982)uncultured Nitrospirae bacterium clone S15A-MN131 (AJ534688)

uncultured bacterium clone 2WB_25 (EU574661)uncultured bacterium clone MD2896-B96 (EU385709)



uncultured bacterium clone CVCloAm2Ph137-OTU22 (AM777956)



uncultured bacterium clone SGNY0215 (EU731004)




Dethiobacter alkaliphilus (EF422412)

DGGE band CVA Lane21CL81 (FN401554 )

uncultured bacterium clone B11-15_GoMY (AB476673)

uncultured bacterium clone dr36g (AY540845)uncultured bacterium clone KL441HWDS3.4 (DQ191818)

DGGE band CVA Lane19CL73 (FN401546)

Candidatus “Desulforudis audaxviator” MP104C (CP000860)

uncultured bacterium clone ANTXXIII_706-4_Bac71 FN429799)

uncultured bacterium clone SGNY0002 (EU730978)

uncultured bacterium clone CVCloAm2Ph80-OTU23 (AM777977)uncultured Gram-positive bacterium (AJ431345)uncultured low G+C Gram-positive bacterium clone ML-A-7 (DQ206408)

uncultured low G+C Gram-positive bacterium clone ML635J-4 (AF507887)

DGGE band CVA Lane17CL68 (FN401544 )uncultured bacterium clone CVCloAm1Ph72-OTU31 (AM777996)

uncultured bacterium clone BE325FW032701CTS_hole1-10 (DQ088769)


uncultured bacterium clone DR9IPCB16SCT8 (AY604055)DGGE band CVA Lane12Cl48 (FN401536)




DGGE band CVA Lane22CL88 (FN401557 )DGGE band CVA Lane3Cl12 (FN401564 )

DGGE band CVA Lane1Cl2 (FN401549 )DGGE band CVA Lane9Cl34 (FN401573 )

uncultured bacterium clone 11S_02e05 (FJ382718)

uncultured eubacterium OCG1 (AB047111)

uncultured bacterium clone pGXAR39 (DQ266903)



uncultured bacterium clone CVCloAm3Ph113-OTU1 (AM778004)Hydrogenophaga flava (AB021420)Hydrogenophaga pseudoflava (AF078770)Hydrogenophaga atypica (AJ585992) B

eta

pro

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Nit

rosp

ira

Clo

stri

dia

Ba

cter

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Gra

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Fig. 1. Neighbor-joining tree indicating thephylogenetic positions of the dominant CVAclones B-OTUs (encompassing more than 1%of the total sequences). A more throughphylogenetic analysis of the determinedpopulations belonging to domain Bacteria isshown in Supplementary Figs S1 and S2.Sequences in bold are from this study. Thescale bar represents 10% nucleotidesequence differences.

Microbial and functional diversity of subsurface aquifer 3


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obac

teria

Aci

neto

bact

erca

lcoa

cetic

us(E

U83

4256

)S

oil

9848

NA

DD

DD

DD

Act

inob

acte

riaP

ropi

onib

acte

rium

acne

s(E

U88

8516

)S

kin

9849

NA

DD

DD

DD

Clo

strid

iaC

lone

BE

325F

W03

2701

CT

S_h

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-69

(DQ

0887

59)

Gro

undw

ater

ina

deep

gold

min

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50N

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lost

ridia

Clo

neB

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7(D

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roun

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ade

epgo

ldm

ine

9651

NA

DD

––

––

Div

isio

nO

P11

Clo

neD

e210

4(H

Q18

4020

)Le

acha

tese

dim

ent

ecos

yste

ms

8552

NA

DD

DD

DD

Unc

lass

ified

bact

eria

Clo

neO

RS

FC

2_a1

1(E

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3413

)C

onso

rtia

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aero

bic

river

sedi

men

ts99

53N

A–

–D

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DU

ncla

ssifi

edba

cter

iaC

lone

OR

I-86

0-02

-P_S

221-

223_

024B

04(J

N12

3504

)M

etha

nese

ep84

a.T

hecl

oses

tcu

lture

dre

lativ

eis

give

nun

less

ther

ew

asle

ssth

an90

%se

quen

cesi

mila

rity

toan

ycu

lture

dba

cter

ium

:th

enth

ecl

oses

tre

late

dse

quen

cefr

omda

taba

seis

indi

cate

din

stea

d.b

.W

hen

seve

ralp

hylo

type

sin

the

sam

eO

TU

,th

elo

wer

valu

eof

sim

ilarit

yis

pres

ente

d.c.

D,

dete

cted

byD

GG

Ean

alys

es;

‘–’,

not

dete

cted

byD

GG

Ean

alys

es.

The

dist

ribut

ion

ofth

edi

ffere

ntO

TU

sde

tect

edby

DG

GE

anal

yses

are

also

pres

ente

d.N

A,

not

appl

ied.



et al., 2008). None of the populations detected by 16SrRNA clone libraries were related to previously isolatedbacteria from CVA (Tiago et al., 2004).

Archaeal diversity assessed by 16S rRNA geneclone library

A total of 270 clones were screened by RFLP analyses.After phylogenetic analyses and at a cut-off of 16S rRNAgene similarity of � 97%, five different archaeal OTUs(A-OTU) were determined (Table 3, Fig. 2, Supplemen-tary Fig. S3). None of the A-OTUs determined had a 16SrRNA gene similarity value higher than 90% with anyarchaeal isolate (Table 3).

A-OTU 1, affiliated with phylum Euryarchaeota, repre-senting 79% of the clones, had highest similarity ~ 97%with environmental sequences belonging to the SouthAfrica Gold Mine Euryarchaeota Group 2 (SAGMEG 2)first designated by Takai and colleagues (2001). ThisA-OTU was also related with environmental sequencesfound in subsurface water (Gihring et al., 2006; Löfflerand Edwards, 2006; T.M. Gihring et al., pers. comm.)and deep marine sediments (Inagaki et al., 2003; 2006;Sørensen and Teske, 2006). A-OTU 2 represented 9.3%of the clones, and was phylogenetically related, at simi-larities of ~ 94%, to environmental sequences of theSAGMEG lineage within Euryarchaeota, retrieved fromdeep environment (Fry et al., 2009). A-OTU 3 and A-OTU4, represented a small percentage of the clones, were theonly A-OTUs assigned to phylum Crenarchaeota andwere affiliated to class Thermoprotei. A-OTU 5 represent-ing 8.5% of the clones, had highest similarity (~ 97%) withenvironmental sequences belonging to Anaerobic Meth-anotrophs group 1 (ANME-1) (Gihring et al., 2006; T.M.Gihring et al., pers. comm.).

DGGE analyses

In order to determine the stability of the microbial popu-lations in CVA, DGGE analyses were performed over a3-year period. Despite minor differences in the finger-prints obtained, the vast majority of the dominant bandswere present in all profiles indicating a stable microbialcommunity (Fig. 3).

In the domain Bacteria, several OTUs comprisingrepresentative phylogenetic groups detected by the clonelibrary, namely Betaproteobacteria, Bacteroidetes andClostridia were also detected by DGGE analyses.Furthermore, eight additionally OTUs (46–53) were deter-mined only by DGGE analyses (Table 2).

With respect to the domain Archaea, all A-OTUs deter-mined by the 16S rRNA clone library were detected byDGGE analyses. Additionally, A-OTU 6 affiliated tophylum Euryarchaeota, was determined only by DGGEanalyses (Table 3). Ta

ble

3.P

hylo

gene

ticaf

filia

tion

ofth

ear

chae

alO

TU

sde

term

ined

from

Cab

eço

deV

ide

aqui

fer,

with

16S

rRN

Age

neda

taba

sese

quen

ces.

A-O

TU

sa

Per

cent

age

onto

tal

ofcl

ones

;(n

umbe

rof

phyl

otyp

es)

Det

ecte

dby

DG

GE

anal

yses

(sam

ples

)bP

hylo

gene

ticre

latio

nshi

p

23

45

67

Put

ativ

eaf

filia

tion

Clo

sest

rela

tive

retr

ieve

dfr

omda

taba

se(A

cces

sion

No.

)cO

rigin

ofth

eda

taba

sese

quen

ce% si

mila

rityd

179

.1;(

9)D

DD

DD

DP

hylu

mE

urya

rcha

eota

–S

AG

ME

GC

lone

NO

27F

W10

0501

SA

B19

0(D

Q23

0938

)S

ubsu

rfac

ew

ater

97%

29.

3;(2

)D

DD

––

DP

hylu

mE

urya

rcha

eota

–S

AG

ME

GC

lone

DE

BIT

S_1

1.2_

1F53

(AM

8830

15)

Coa

ldep

osit

1.6?

2.3

km94

%3

0.8;

(1)

–D

–D

D–

Phy

lum

Cre

narc

haeo

ta–

uncl

assi

fied

Clo

neA

0610

D00

2_K

09(A

B65

5539

)W

ater

man

agem

ent

inric

epa

ddy

soil

98%

42.

3;(1

)–

–D

D–

–P

hylu

mC

rena

rcha

eota

–un

clas

sifie

dC

lone

A7

(GU

3228

82)

Was

tew

ater

trea

tmen

tbi

orea

ctor

98%

58.

5;(1

)D

DD

DD

DP

hylu

mE

urya

rcha

eota

–A

NM

E-1

aC

lone

DR

9IP

CA

16S

CT

1(A

Y60

4063

)S

ubsu

rfac

ew

ater

97%

6N

A–

––

–D

Phy

lum

Eur

yarc

haeo

ta–

uncl

assi

fied

Clo

neC

P-A

127

(DQ

5211

94)

Col

dsa

line

pere

nnia

lspr

ing

89%

a.M

ajor

A-O

TU

sde

tect

edon

16S

rRN

Acl

one

libra

ryar

eon

bold

(ape

rcen

tage

valu

eup

per

than

3%of

the

tota

lclo

nesc

reen

edw

asco

nsid

ered

).b

.D

,de

tect

edby

DG

GE

anal

yses

;‘–

’,no

tde

tect

edby

DG

GE

anal

yses

.c.

The

clos

est

cultu

red

rela

tive

isgi

ven

unle

ssth

ere

was

less

than

90%

sequ

ence

sim

ilarit

yto

any

cultu

red

arch

aeon

:th

enth

ecl

oses

tre

late

dse

quen

cefr

omda

taba

seis

indi

cate

din

stea

d.d

.W

hen

seve

ralp

hylo

type

sin

the

sam

eO

TU

,th

elo

wer

valu

eof

sim

ilarit

yis

pres

ente

d.T

hedi

strib

utio

nof

the

diffe

rent

A-O

TU

sde

tect

edby

DG

GE

anal

yses

are

also

pres

ente

d.N

A,

not

appl

ied.



Bacterial diversity assessed by pyrosequencing

From a total of 17 385 reads obtained for samples 3, 5 and7 (~ 5800 reads per sample), 7897 pyrosequences, allcontaining the V1–V2 region of the 16S rRNA gene, wereused for further analyses. After analysing the rarefactioncurves (Supplementary Fig. S4) we decided to use a cut-off value of 0.03 (� 97% sequence similarity) for thedetermination of the pyrosequencing OTUs for domainBacteria (BP-OTU) for further analyses. The 7897 pyrose-quences were assigned to 5 major phylogenetic groups,namely phylum Bacteroidetes (17.6%) and classesBetaproteobacteria (21.9%), Gammaproteobacteria(2.0%), Clostridia (33,4%) and Actinobacteria (12.3%)(Fig. 4). The taxonomic classification of 7.3% of thepyrosequences could not be resolved by the mothurclassifier using Silva taxonomy classification nor by RDPtaxonomy classification and therefore were designatedas ‘unclassified Bacteria’ (Fig. 4). A total of 182 BP-OTUswere determined but 144 comprised only 22 or lesspyrosequences, representing 7.8% of the total. Thevast majority (92.2%) of the 7897 pyrosequenceswere distributed by 38 BP-OTUs (Table 4). The majorityof these 38 BP-OTUs corresponded to populationsdetected by 16S rRNA gene clone library and as inclone library, major BP-OTUs were closely affiliatedwith chemolithoautotrophic organisms, and/or with clonesequences retrieved from subsurface environments(Table 4). Twelve of these BP-OTUs did not correspondedto B-OTUs determined by clone library. Curiously, someBP-OTUs were closely related to organisms previously

isolated from CVA (Tiago et al., 2004), namely Dietzia spp.and Staphylococcus spp. (Table 4).

Archaeal diversity assessed by pyrosequencing

From a total of 12 151 reads obtained for the threesamples (~ 4000 reads per sample), 4983 sequences,

CVA A-OTU 1

CVA A-OTU 2 - unclassified group 1

84

9754

85

84

86

80

72

91

93

80

99

70

99

98

6974

52

79

97

56

99

0.10

uncultured archaeon clone CVA-2,3 phyl grBtot OTU5a (FN690968)uncultured archaeon DGGE band CVA 6 OTU5a (FN690955)uncultured archaeon clone DR9IPCA16SCT1 (AY604063)

uncultured archaeon clone MS149BH1062003_10 (DQ354742)uncultured euryarchaeote clone pIta-HW-4 (AB301860)

uncultured archaeon clone A163H03 (FJ455963)uncultured archaeon clone Arch125 (FN428824)

Methanosaeta harundinacea (AY970347)Methanomethylovorans hollandica (AY260433)

uncultured archaeon clone NO27FW100501SAB190 (DQ230938)

uncultured archaeon SAGMA-12 (AB050242)

uncultured archaeon SAGMA-H (AB050212)uncultured archaeon SAGMA-U (AB050226)

uncultured archaeon clone FeOrig_A_7 (GQ356919)uncultured archaeon clone TWP6-42 (GQ410922)

uncultured archaeon clone HDBW-WA14 (AB237747)uncultured archaeon clone MD3059V-68 (GQ927645)

uncultured archaeon clone NO27FW100501SAB175 (DQ230939)

Geogemma indica strain 296 (DQ492260)Pyrodictium occultum strain PL-19 (NR_025933)

uncultured archaeon DGGE band CVA 9+ OTU4a(FN690961)uncultured crenarchaeote clone MLSB_30m_2E_A (EF420185)

uncultured archaeon clone CVA-3 phyl 37 gr1Dtot OTU4a (FN690972)uncultured archaeon clone MLSB_30m_6G_A (EF420189)

uncultured archaeon clone ss016a (AJ969779)uncultured archaeon clone ASC37 (AB161337)

uncultured archaeon clone A7 (GU322882)anaerobic methanogenic archaeon ET1-8 (AJ244284)

anaerobic methanogenic archaeon ET1-10 (AJ244286)

Class Thermoprotei

unclassifiedgroup 1

SAGMEG 2

Phylum Euryarchaeota

ANME-1a

Phylum Crenarchaeota

Fig. 2. Neighbor-joining tree indicating the phylogenetic positions of the dominant CVA clones A-OTUs (encompassing more than 1% of thetotal sequences). A more through phylogenetic analysis of the determined populations belonging to domain Archaea is shown inSupplementary Fig. S3. Sequences in bold are from this study. The scale bar represents 10% nucleotide sequence differences.

Domain Bacteria Domain Archaea

2 3 4 75 6 2 3 4 75 6

selpmaSselpmaS

Fig. 3. DGGE profiles of the PCR-amplified 16S rRNA genefragments (domains Bacteria and Archaea) obtained from theaquifer, sample 2 (collected January 2007), sample 3 (collectedJanuary 2007), sample 4 (collected July 2008), sample 5 (collectedJuly 2008), sample 6 (collected December 2008) and sample 7(collected May 2009). Arrows indicate gel portions (lanes) that wereexcised from the gel for cloning and sequencing purposes.



all containing the V1–V2 region of the 16S rRNA gene,were used for further analyses. After analysing the rare-faction results (Supplementary Fig. S4) we decided touse a cut-off value of 0.03 (� 97% sequence similarity)for the determination of the pyrosequencing OTUs fordomain Archaea (AP-OTUs) in order to perform furtheranalyses. The vast majority of the 4983 pyrosequenceswere assigned to phylum Euryarchaeota (99.9%) andonly a single pyrosequence was assigned to phylumCrenarchaeota (Fig. 4). A total of 29 AP-OTUs weredetermined but only eight accounted for more than 1%of the pyrosequences (Table 5), the remaining AP-OTUsrepresented just 3.4% of the pyrosequences. The mostrepresentative AP-OTUs determined had high similarityto A-OTUs detected by 16S rRNA gene clone library(Table 5).

Statistical analyses

Microbial community diversity metrics calculated for thebacterial and archaeal 16S rRNA gene clone library andpyrosequence analyses were similar within each domain(Supplementary Table S3a). The coverage values indi-cated that a significant bacterial and archaeal diversity

was covered by both approaches. Furthermore the rare-faction analysis, of total OTUs determined at a cut-offvalue of 0.03, generated curves with tendency to satura-tion (Supplementary Fig. S4). The determined Shannonindexes indicated a higher diversity level in domain Bac-teria when compared with domain Archaea. The Shan-non’s equitability determined showed some evennessdegree within the bacterial populations, and the presenceof major archaeal populations. The Sørensen similarityindex was applied to the populations determined to eachDGGE profile and values obtained demonstrated the sta-bility of the populations in the different samples studied(Supplementary Table S3b).

Functional genes

Autotrophic CO2 fixation. Form II RuBisCO was notdetected in CVA despite multiple PCR assays undervarious conditions. Form I RuBisCO was detected, and allobtained translated sequences clustered in one mono-phyletic group with several protein phylotypes sharinghigh phylogenetic closeness and amino acid identities(93.5% to 100%). The translated sequences of CVA-cbbLhad high phylogenetic affiliation (Fig. 5) to translated cbbLsequences found in Betaproteobacteria sulfur-oxidizing

a)

b) SAGMEG, 85.6%

unclassified Euryarchaeota, 11.18%

ANME, 3.18%

unclassified Crenarchaeota, 0.02%

Phy

lum

Eur

yarc

haeo

ta

Phylum Crenarchaeota

Clostridia; 32.1%

Deltaproteobacteria; 0.4%

Gammaproteobacteria; 1.6%

KI-96; 0.6%

Staphylococcaceae;0.5%

unclassifiedActinobacteria; 10.2%

Dietziaceae; 1.2%

unclassifedBacteroidetes; 16.4%

Bacteroidales;0.3%

Betaproteobacteria; Comamonadaceae; 21.0%

Trueperaceae; 0.7%

unclassified bacteria; 7.3%

PhylumActinobacteria

PhylumBacteroidetes

PhylumProteobacteria

PhylumFirmicutes

PhylumChloroflexi

PhylumDeinococcus-Thermus

Fig. 4. Classification and distribution of the pyrosequences from domains Bacteria (A) and Archaea (B).



Tab

le4.

Phy

loge

netic

affil

iatio

nof

the

BP

-OT

Us

dete

rmin

edfr

omC

abeç

ode

Vid

eaq

uife

r,w

ith16

SrR

NA

gene

data

base

sequ

ence

s.

BP

-OT

Ua

Per

cent

age

onto

tal

pyro

sequ

ence

s

Equ

ival

ence

tocl

one

libra

ry

Clo

sest

rela

tive

retr

ieve

dfr

omda

taba

sec

Put

ativ

eph

ylog

enet

iccl

assi

ficat

ion

OT

Usb

14.

744

(95%

)A

Y54

0789

(100

%);

AY

5408

19(9

7%)

Act

inob

acte

ria2

5.6

44C

lone

SM

D-B

10(A

B47

8002

)A

ctin

obac

teria

31.

2N

AD

ietz

iasp

p.A

ctin

obac

teria

;D

ietz

iace

ae4

5.5

15,

16(9

7%)

Clo

ne11

S_0

2e05

(FJ3

8271

8)B

acte

roid

etes

55.

812

Clo

neT

68A

C-1

8,F

N70

6497

(99%

)B

acte

roid

etes

60.

313

Clo

neam

bien

t_al

kalin

e-9

(GU

4549

92)

Bac

tero

idet

es;

Bac

tero

idal

es7

5.1

14C

lone

Sak

i_aa

j64a

06(E

U47

5765

)B

acte

roid

etes

810

.01,

2,5

(97%

)H

ydro

geno

phag

asp

.B

etap

rote

obac

teria

;C

omam

onad

acea

e9

0.4

1(9

7%),

2(9

7%),

5(9

7%)

Hyd

roge

noph

aga

sp.

Bet

apro

teob

acte

ria;

Com

amon

adac

eae

100.

33

Met

hylib

ium

subs

axon

icum

(AM

7744

13)

Bet

apro

teob

acte

ria;

Com

amon

adac

eae

110.

3N

AH

ydro

geno

phag

asp

.(9

1%);

Lept

othr

ixoc

hrac

ea(9

2%)

Bet

apro

teob

acte

ria;

Com

amon

adac

eae

129.

91

(96%

),2

(97%

),5

(96%

)H

ydro

geno

phag

asp

.(9

7%);

Lept

othr

ixoc

hrac

ea(9

5%)

Bet

apro

teob

acte

ria;

Com

amon

adac

eae

130.

4N

AC

lone

FL0

428B

_PF

70,

FJ7

1643

5(9

8%);

clon

eD

F5I

PC

ant1

8c03

,G

Q92

1432

(97%

)D

elta

prot

eoba

cter

ia;

uncl

assi

fied

140.

66

Sila

nim

onas

lent

a(A

Y55

7615

)G

amm

apro

teob

acte

ria;

Xan

thom

onad

acea

e15

1.0

NA

Pse

udom

onas

aeru

gino

sa99

%G

amm

apro

teob

acte

ria;

Pse

udom

onad

acea

e16

0.6

45Is

olat

eba

cter

ium

Elli

n652

9(H

M74

8677

)C

hlor

oflex

i;K

D4-

9617

0.5

NA

Sta

phyl

ococ

cus

spp.

Firm

icut

es;

Sta

phyl

ococ

cace

ae18

2.3

28C

lone

BE

325F

W03

2701

CT

S_h

ole1

-27

1(D

Q08

8764

)C

lost

ridia

;un

clas

sifie

d19

1.1

34C

lone

:B

11-1

5_G

oMY

(AB

4766

73)

Clo

strid

ia;

uncl

assi

fied

200.

532

(98%

)G

eosp

orob

acte

rsu

bter

renu

s(D

Q64

3978

)C

lost

ridia

;C

lost

ridia

ceae

213.

432

Geo

spor

obac

ter

subt

erre

nus

(DQ

6439

78)

Clo

strid

ia;

Clo

strid

iace

ae22

5.2

26C

andi

datu

s‘D

esul

foru

dis

auda

xvia

tor’

,C

P00

0860

(95%

)C

lost

ridia

;un

clas

sifie

d23

0.3

NA

Fin

egol

dia

sp.

Clo

strid

ia;

Clo

strid

iale

s24

0.4

24C

rypt

anae

roba

cter

phen

olic

us(N

R_0

2575

7)C

lost

ridia

;P

epto

cocc

acea

e25

0.3

22C

lone

PS

2569

(FN

6673

67)

Clo

strid

ia;

uncl

assi

fied

261.

4N

AC

lone

BE

325F

W03

2701

CT

S_h

ole1

-77

DQ

0887

58,

(92%

);cl

one

MiJ

7,E

U64

5123

(86%

)C

lost

ridia

;un

clas

sifie

d27

0.5

NA

Clo

ne23

O56

,E

U64

4913

(85%

)C

lost

ridia

;un

clas

sifie

d28

1.0

26(9

4%)

Clo

neB

E32

5FW

0327

01C

TS

_hol

e1-2

7,D

Q08

8764

(93%

)C

lost

ridia

;P

epto

cocc

acea

e29

2.6

30(9

5%)

Clo

neF

4-B

15,

FJ2

0681

7(9

4%)

Clo

strid

ia;

Syn

trop

hom

onad

acea

e30

0.9

38,

40,

41,

42,

43(~

95%

)C

lone

F4-

B15

,F

J206

817

(91%

),D

ethi

obac

ter

alka

liphi

lus

(88%

)C

lost

ridia

;S

yntr

opho

mon

adac

eae

316.

338

,40

,41

,42

,43

Det

hiob

acte

ral

kalip

hilu

s(N

R_0

4420

5)C

lost

ridia

;S

yntr

opho

mon

adac

eae

325.

530

Clo

neF

4-B

15F

J206

817,

(91%

);cl

one

BE

325F

W03

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bacteria isolates, namely Thiomonas intermedia (Moreiraand Amils, 1997) and Thiobacillus ajanensis (Dul’tsevaet al., 2006).

Genes encoding key enzymes of the rTCA cycle,namely carbon dioxide-fixing enzymes: 2-oxoglutarate:ferredoxin oxidoreductase (oorA), and both forms of thepyruvate:ferredoxin oxidoreductase (por and nifJ) weredetected in CVA. However, the key enzyme ATP citratelyase (aclB) was never detected.

The gene encoding the biotin carboxylase subunit(accC) was detected in CVA. The accC translatedsequences formed four clusters. CVA-accC-Clusters-1,-3 and -4 were phylogenetically related to translatedaccC sequences from organisms and environmentalsequences belonging to domain Bacteria. The phylogenyof the sequences included in CVA-accC-Cluster-2 couldnot be resolved. They were possibly affiliated to twoArchaea species, Archaeoglobus profundus and Fer-

roglobus placidus, although with low amino acid identitiesvalues (57% and 63% respectively) and low bootstrapsupport (Supplementary Fig. S5).

Sulfur cycle. The presence of the APS reductase alphasubunit encoding gene (aprA) was detected in CVA. Thetranslated sequences formed six clusters, revealing thepresence of sulfate-reducing prokaryotes (SRP) andsulfur-oxidizing prokaryotes (SOP) in CVA. CVA-aprA-cluster-1, representing 66% of the clones sequenced,was phylogenetically related to Candidatus ‘D. audaxvia-tor’ (Fig. 6) and shared ~ 91% amino acid identitieswith the translated aprA sequence from this candidatus.CVA-aprA-cluster-2, comprised 13% of the clonessequenced, was phylogenetically related to anaerobicsulfur-reducing Desulfomaculum spp. isolates (Fig. 6) andtheir translated aprA sequences shared ~ 87% amino acididentities. The monophyletic CVA-aprA-cluster-3, was

Table 5. Phylogenetic affiliation of the AP-OTUs determined from Cabeço de Vide aquifer, with 16S rRNA gene database sequences

AP-OTUsa

Percentageon totalpyrosequences

Equivalence to clonelibrary A-OTUsb Closest relative retrieved from databasec

Putative phylogeneticclassification

1 47.3 A-OTU1 Clone NO27FW100501SAB190, DQ230938 (97%) Euryarchaeota – SAGMEG2 23.1 A-OTU2 Clone DEBITS_11.2_1F53, AM883015 (94%) Euryarchaeota – SAGMEG3 13.1 A-OTU1 (98–97%) Clone NO27FW100501SAB190, DQ230938 (95%) Euryarchaeota – SAGMEG4 4.1 A-OTU2 (98%) Clone A7, GU322882 (95%) Euryarchaeota – SAGMEG5 3.9 A-OTU1 (98–97%) Clone NO27FW100501SAB190, DQ230938 (97%) Euryarchaeota – SAGMEG6 2.8 A-OTU5 Clone DR9IPCA16SCT1, AY604063 (97%) Euryarchaeota – ANME-1a7 2.4 A-OTU1 (98–97%) Clone NO27FW100501SAB190, DQ230938 (94%) Euryarchaeota – SAGMEG8 1.0 A-OTU1 (97–96%) Clone NO27FW100501SAB19, DQ230938 (95%) Euryarchaeota – SAGMEG

a. Only AP-OTUs encompassing � 1.0% of the pyrosequencings are listed.b. The designation of the correspondent A-OTUs determined by 16S rRNA clone library is presented, on parentheses the average percentage ofsimilarity is shown when value is equal and/or lower than 98%.c. The closest cultured relative is given unless there was less than 90% sequence similarity to any cultured archaeon: then the closest relatedsequence from database is indicated instead. The equivalence to the A-OTUs determined by clone library is also presented.

cbbL-clone: ML23J-18R (AAP79249)cbbL-clone: ng3L492 (AAX21155)

cbbL-clone: ng2L543 (AAX21154)cbbL-clone: ng5L618 (AAX21163)

cbbL-clone: fg1L566 (AAX21113)cbbL-clone: fg5L762 (AAX21124)cbbL-clone: fg3L829 (AAX21121)cbbL-clone: fg7L883 (AAX21131)

cbbL-clone: 40 (ABD66346)cbbL-clone: 36 (ABD66345)

cbbL-clone: 64 (ABD66348)cbbL-clone: 42 (ABD66347)

cbbL-Thiomonas sp. 3As (CAO82080)cbbL-Thiomonas intermedia K12 (YP_003641858)

cbbL-Thiobacillus sajanensis (ABD52278)

CVA-cbbL-cluster

CVA-cbbL-clone 10 (HE686952)CVA-cbbL-clone 28 (HE686957)

CVA-cbbL-clone 13 (HE686954)CVA-cbbL-clone 31 (HE686958)

CVA-cbbL-clone 9 (HE686961)CVA-cbbL-clone 15 (HE686955)CVA-cbbL-clone 26 (HE686956)

CVA-cbbL-clone 12 (HE686953)CVA-cbbL-clone 32 (HE686959)CVA-cbbL-clone 33 (HE686960)

cbbL-Nitrosomonas eutropha C9 (YP_747036)cbbL-Nitrobacter sp. Nb-311 (YP_571759)cbbL- Nitrobacter winogradskyi (ZP 01046073)

100

96

88

92

90

27

80

41

37

24

9873

0.01

Fig. 5. Phylogenetic diversity of translatedsequences of RuBisCO detected in clonelibrary from CVA. CVA sequences are in bold.



most phylogenetically related to a bacterial translatedaprA sequence retrieved from mouse gastrointestinaltract (Deplancke et al., 2000) but was also related to theknown SOP Chlorobium and Pelodictyon belongingto class Chlorobia (Fig. 6). The other three clusters,CVA-aprA-cluster-4, -5 and -6, were phylogeneticallyrelated, sharing 86–93% amino acid identities betweenthem. All were phylogenetic related to SOP translatedaprA sequences from Betaproteobacteria (CVA-aprA-Cluster-4) and Gammaproteobacteria (CVA-aprA-clusters, -5 and -6).

Methane cycle. Despite several attempts, no amplifica-tion was obtained for the gene encoding the membrane-associated form of the methane monooxygenase-encoding gene, pmoA.

The methyl coenzyme M reductase-encoding gene(mcrA) was detected in CVA and the respective translatedsequences formed a single cluster, with severalphylotypes, with amino acid identity similarity valuesranging from 89% to 99% (Fig. 7). This cluster had highestphylogenetic affiliation to several translated mcrAsequences classified as belonging to the ANME-1 (Lloydet al., 2011). The CVA cluster sequences had highestphylogenetic affiliation and amino acid identity values(85%) to environmental mcrA translated sequences

retrieved from the serpentinite-hosted ecosystem LostCity Hydrothermal Field (LCHF) (Kelley et al., 2005).

Discussion

The microbial community present in CVA was most likelyrestricted to bacterial and archaeal populations sincemicroeukaryotes were never detected. DGGE analysesand the Sørensen similarity values determined pro-vided strong evidence of the stability of this microbialcommunity. By 16S rRNA gene clone libraries and pyro-sequencing analyses, we determined a diverse bacterialcomposition, contrasting with low archaeal diversity.

In this environment the primary production appears tobe dependent on chemolithoautotrophic microorganisms.Indeed the major population found was related to H. flavaa hydrogen-oxidizing bacteria, others fell within Nitrospiragroup, probably representing chemolithoautotrophicnitrite oxidizers, and some other populations determinedwere related to species Dethiobacter alkaliphilus, a bac-terium that may grow chemolithotrophically with H2

(Sorokin et al., 2008). Additionally, several populationswere closely related to Candidatus ‘D. audaxviator’.The genome of this candidatus indicates a sporulating,sulfate-reducing chemoautotrophic thermophilic organismcapable of carbon and nitrogen fixation (Chivian et al.,

SRP

CVA-aprA-clone: 32 (HE686944)CVA-aprA-clone: 38 (HE686948)

CVA-aprA-clone: 25 (HE686940)CVA-aprA-clone: 13 (HE686935)CVA-aprA-clone: 43 (HE686951)

CVA-aprA-clone: 37 (HE686947)CVA-aprA-clone: 22 (HE686939)CVA-aprA-clone: 33 (HE686945)CVA-aprA-clone: 42 (HE686950)

Candidatus “Desulforudis audaxviator” MP104C (YP_001718010)Desulfotomaculum thermobenzoicum (AAL57428)

Desulfotomaculum kuznetsovii DSM 6115 (AAL57419)Desulfotomaculum sp. DSM 8775 (ABR92586)


clone: apsA40B9 (ADD84888)clone: bcs 3.41 (CAT03614)

Desulfotomaculum geothermicum DSM 3669 (ABR92566)Thermodesulfovibrio islandicus (AAL57380)

Thermodesulfovibrio yellowstonii (ABR92418)Thermacetogenium phaeum (ABR92597)

clone: APS7.3 (AAF16948)CVA-aprA-clone: 28 (HE686941)

clone: DGGE gel band SLB-W-10 (ABV00946)Chlorobium phaeobacteroides strain BS1 (YP_001960235)

Chlorobium tepidum strain TLS (NP_661759)Pelodictyon phaeoclathratiforme strain BU-1 (YP_002017013)

clone: C6507_aprA_12B207 (ACM47801)clone: bcs 5.6 (CAT03587)clone: aprE6 (CBH30869)

CVA-aprA-clone: 30 (HE686942)Thiobacillus denitrificans strain ATCC 25259 (YP_316040)

Thiobacillus thioparus (ABV80092)

CVA-aprA-clone: 39 (HE686949)clone: bcs 2.23 (CAT03594)CVA-aprA-clone: 21 (HE686938)CVA-aprA-clone: 31 (HE686943)

isolate sulfur-oxidizing bacterium strain DIII5 (ABV80086)clone: bcs 6.17 (CAT03549)clone: bcs 3.16 (CAT03583)clone: DR9IPCAPSCT1 (AAT37614)clone: S6CL3 (ABJ90325)clone: S8CL5 (ABJ90331)clone: S8CL3 (ABJ90330)


99

5772

100

99

9278

98

33

23

22

25

99

8398

77

65

98

97

99

42

60

21

57

55

42

45

39

40

47

30

18

18

10

20

100

0.05

CVA-aprA-cluster-3

CVA-aprA-cluster-2

CVA-aprA-cluster-1

CVA-aprA-cluster-5

CVA-aprA-cluster-6

CVA-aprA-cluster-4

SOP

Fig. 6. Phylogenetic diversity of translatedsequences of APS reductase alpha subunitydetected in clone library from CVA. CVAsequences are in bold.



2008). The remaining populations were associatedwith chemoorganotrophic organisms (including a greatnumber of anaerobic populations) or to environmentalclone sequences from unknown organisms.

A less diverse community was determined for thedomain Archaea, dominated by populations belonging tophylum Euryarchaeota. The major archaeal population,belonged to the euryarchaeotal SAGMEG lineage, anuncultured lineage of unknown metabolic role discoveredin South Africa gold mines (Takai et al., 2001).

None of the OTUs determined by clone libraries orDGGE analyses was closely affiliated with the hetero-trophic aerobic strains isolated from the same environ-ment in previous occasions (Tiago et al., 2004). However,high-throughput pyrosequencing allowed the determina-tion of BP-OTUs that closely matched isolates from CVA,for example Dietzia natronolimnaea (one major popula-tion isolated) and Staphylococcus spp., but other isolatedpopulations escaped even detection by pyrosequencing.This result may be related to the fact that only aerobicheterotrophic populations were screened by culture,whereas the majority of the community was most likelyautotrophic or anaerobic or microaerophilic, as deter-mined by 16S rRNA analyses. Indeed the concentration of

dissolved oxygen in CVA is below 0.05 mM (the detectionlimit of the method used). Thus, the aerobic heterotrophicpopulations would most likely persist as seed banks(Pedrós-Alió, 2006) that remain beyond the reach of mostmolecular tools.

The only autotrophic CO2 fixation activity detected inCVA was related with the CBB cycle. Indeed the presenceof the rTCA cycle could not be confirmed since aclB wasnever detected. Since the rTCA cycle requires the com-bined use of all key enzymes for reductive carboxylationto occur (Campbell et al., 2003; Campbell and Cary, 2004;Nakagawa and Takai, 2008), it is probable that organismswith this CO2 fixation pathway were not present in CVA.Nevertheless, its absence was not surprising given thatthe rTCA cycle has been detected mainly in Chlorobium,the Aquificales and the Epsilonproteobacteria (Berg,2011), and none of these organisms were detected inCVA. The biotin carboxylase subunit gene (accC) of theAcetyl CoA carboxylase (ACCase) enzyme, recognizedas the key marker for the 3-HP and 3-HP/4H-B cycle wasdetected in CVA. However, in Bacteria and Eukarya,ACCase catalyses the first step of fatty acid biosynthesisand, since Archaea do not contain fatty acids, ACCasehas a different metabolic role in these organisms – most

100

100

79

81

8071

100

100

97

100

100

72

60

100

56

0.1

CVA-mcrA-clone: 46 (HE686964)

CVA-mcrA-clone: 7 (HE686966)CVA-mcrA-clone: 32 (HE686963)

CVA-mcrA-clone: 47 (HE686965)

CVA-mcrA-clone: 27 (HE686962)CVA-mcrA-clone: 8 (HE686967)

Lost City clone: LCM1443-12 (AY760635)Lost City clone: LCM1557-12 (AY760638)Lost City clone: LCM1446-4 (AY760639)

CVA-mcrA-cluster

Holocene sediment clone: AHH21mcr_14 (AB560814)

Guaymas Basin clone 4483 ANME1mcr 29 (JF937800)hydrothermal sediments clone Guaymas 50enr mcrA 54 (FR682817)methane seep of the Nankai Trough clone KM-m-1.13 (AB233459)

sediment clone 4486 ANME1mcr 11 (JF937820)

Pearl River Estuary clone: TopMcrA17 (EU681939)

Natural Asphalt Lake clone: PL-D2_15_2_D08 (GU447226)

sub-surface mud volcano sediments clone: 1-579C (FN553435)Lake Kivu water clone: DGGE gel band mcrA_180m_p1_4_TypeM (FJ952121)

marine sediments clone: 1327C20-3 (AB525707)Kazan mud volcano sediments clone: AN07BC1_15_33 (AY883172)

tidal creek bank sediments clone: AUGCONTROLC09 (EU302050)tidal creek bank sediments clone: D07AUGControlb (EU302010)

mud volcano clone: MV1_6_7_E02 (GU447206)

clone: GZfos_17_30.54 (AY324369)

Black Sea microbial mat clone: 0418F12 (BX649197)Guaymas Basin sediments clone 4483 ANME1mcr 04 (JF937799)

Guaymas Basin hydrothermal sediments clone B09 (AY837774)Methanococcoides methylutens mcr gene partial cds (U22235)

Methermicoccus shengliensis strain ZC-1 mcrA gene partial cds (EF026570)Cascadia Margin marine sediment clone: ODP8-ME1 (AF121099)Blake Ridge PC26 clone BR 42bB08 (AY324367)

Methanobacterium formicicum strain DSM 1312 mcrA gene partial cds (AF414050)

ANME-1

Methanosarcinales

Fig. 7. Phylogenetic diversity of translated sequences of methyl coenzyme M reductase detected in clone library from CVA. CVA sequencesare in bold.



probably related to carbon fixation (Berg et al., 2010).Therefore, only accC sequences with archaeal affiliationcan be considered indicators of the presence of the 3-HPor 3-HP/4-HB cycles. CVA translated accC sequenceswere distributed into four distinct clusters. Three of thesephylogenetic clusters were affiliated with bacterial groups,and could therefore not be considered as indicators forthe presence of the 3-HP and 3-HP/4-HB cycles. TheCVA-accC-Cluster-2 could not be clearly positioned, butthese sequences showed some relatedness with sometranslated accC sequences belonging to archaeal organ-isms. This could suggest that the sequences forming thiscluster could probably represent a yet uncharacterizedform of accC gene.

Only cbbL sequences (classified as belonging to form IRuBisCO) were detected in CVA. Form I RuBisCO hashigher affinity to CO2 contrasting with the low affinitydetermined for form II RuBisCO (Tabita et al., 2007). Thedetected form I RuBisCO may be very suitable to organ-isms in an environment like CVA where CO2 may beextremely scarce due to the existing physicochemicalcharacteristics, namely the high pH values (Marqueset al., 2008). The RuBisCO diversity detected in CVA wasvery low and the translated cbbL sequences formeda monophyletic cluster comprising highly similarsequences. Indeed, CVA-cbbL-cluster could be the resultof severe selective pressures that lead to natural selectionof organisms with only this form of the enzyme. This couldmean that a very few organisms are adapted to fix CO2 inCVA (via CBB cycle) and/or that a specific version ofform I RuBisCO is more suitable to accomplish that task.Moreover, CVA-cbbL-cluster showed phylogenetic affilia-tion to translated cbbL sequences retrieved from isolatesbelonging to Betaproteobacteria. Since, one of the majorpopulations detected belonged to Betaproteobacteriaand was phylogenetically affiliated with species H. flava afacultative chemolithoautotrophic (Willems et al., 1989),one could putatively assign this carbon fixation pathwayto that major population. Furthermore carbon isotopicfractionation-d13C value determined by Marques and col-leagues (2008) in CVA (-22.9‰) strongly suggestsautotrophic activity linked to the CBB cycle that producesd13C values in the range of -20‰ to -30‰ (Sirevåg et al.,1977; McNevin et al., 2007), while the activity of otherautotrophic pathways generates carbon isotopic frac-tionation in other range of values (Sirevåg et al., 1977;Berg et al., 2010) reinforcing the conviction that CBBwas the major carbon fixation mechanism operating inCVA.

The presence of both SRP and SOP was determinedin CVA by the detection of aprA. Two clusters of SRP-related sequences could be identified, and were phylo-genetically related to translated aprA sequencesbelonging to class Clostridia, one of the most frequently

detected phylogenetic groups in CVA. Indeed, CVA-aprA-Cluster-1 was phylogenetically related and shared91% of amino acid identities with translated aprAsequence from Candidatus ‘D. audaxviator’, one of themajor populations detected in CVA. This fact could leadus to foresee the importance of ‘D. audaxviator’ namelyon the energetic flow in this environment, probablylinked to the sulfur cycling. The translated aprAsequences phylogenetically related with SOP sequenceswere distributed by three small clusters phylogeneticallyrelated with translated sequences of aprA belonging toProteobacteria. However no 16S rRNA clones exhibitedany significant sequence similarity to organisms previ-ously reported to be associated to the oxidation of theavailable sulfur.

Genes encoding methyl coenzyme M reductase (mcrA)were detected in CVA; without exception, they formed asingle cluster that was phylogenetically affiliated withseveral translated mcrA sequences belonging to ANME-1(Kelley et al., 2005) most probably involved in anaerobicoxidation of methane (AOM). During AOM, methane isoxidized with sulfate as the terminal electron acceptor:CH4 + SO4

2- → HCO3- + HS- + H2O. This process has

been hypothesized has an enzymatic reversal of themethanogenesis pathway (Hallam et al., 2004; Meyer-dierks et al., 2010). AOM is considered to be mediatedby a syntrophic consortium that may be constituted bymethanotrophic archaea and sulfate-reducing bacteria(Hoehler et al., 1994; Meyerdierks et al., 2010), althoughthe process is still poorly understood (Lloyd et al., 2011).It has also been observed that some SOP, namely sym-bionts, can take advantage of the hydrogen sulfide that isproduced during AOM (Caldwell et al., 2008; Lloyd et al.,2010).

The Lost City hydrothermal field (LCHF), a well-studiedserpentinization driven marine environment, is dominatedby organisms similar to sulfur-oxidizing, sulfate-reducingand methane-oxidizing Bacteria; as well as methanogenicand anaerobic methane-oxidizing Archaea indicating thatmicrobial cycling of sulfur and methane are dominantbiogeochemical processes (Brazelton et al., 2006). InCVA, metabolisms related with sulfur and methane alsoseem to play a crucial role in the environment; however,the microbial populations acting in each habitats arerather different. Moreover in CVA methanogenic markerswere never found, but the CVA mcrA sequences belong-ing to ANME-1 were closely related with LCHF mcrAsequences determined in the lower temperature ventingsites (Kelley et al., 2005). Although both ecosystems areserpentinization driven the physical and chemical charac-teristics of both environments are dramatically differentshaping the microbial diversity.

Surface waters also associated with serpentinizationactivity have been studied to some extent and the



presence of Proteobacteria (Pedersen et al., 2004),Cyanobacteria, Bacteroidetes and Archaea, includingmethanogens (Blank et al., 2009) have been reported butno obvious similarities were found to the CVA sequences.

Curiously, the microbial populations detected on CVA,had highest phylogenetic similarities with environmentalsequences retrieved from others subsurface water envi-ronments with no serpentinization activity (Lin et al., 2006;Shimizu et al., 2006; Chivian et al., 2008; T.M. Gihringet al., pers. comm.). Indeed, one of the major populationsdetected was phylogenetically related to Candidatus ‘D.audaxviator’, which dominates deep, rock-hosted aquifersin South African gold mines (Chivian et al., 2008) to thealmost complete exclusion of other organisms; in con-trast, its relatives in CVA are part of a diverse microbialcommunity. Further studies in CVA environment may helpto understand the role of these organisms in highly oligo-trophic subterrestrial habitats, under long-term isolationfrom the photosynthetic biosphere.

More recently a metagenomic survey performed byBrazelton and colleagues (2012) in a serpentine-hostedsubterrestrial environment, at Tablelands Ophiolite inCanada (TOC), presented evidences for the activity of H2

oxidizers belonging to Burkholderiales, probably Hydro-genophaga spp., and anaerobic organisms, probablyClostridia-related, capable of H2 production. Like in TOC,in CVA these two bacterial groups constitute major popu-lations, and their recognized complementary activitiesreinforces the importance of metabolic interactionsdepending on these and other bacterial groups as impor-tant mediators of carbon and energy exchange in theseenvironments. Additionally, in CVA the presence ofavailable CO2 is unlikely to occur since precipitationunder x-CO3 form is the most probable occurrence, con-straining primary production. Again close metabolicinteractions between the putative heterotrophic CO2 pro-ducers with the autotrophic CO2-fixing organisms mayplay an important role in this environment. In this hostileconditions the existence of consortia of well adaptedcells with strict interactions could be envisage as vital forthe maintenance of this particular ecosystem. Moreover,the well-documented existence of biofilms and syntrophicrelationships in other aquatic environments namely inLCHF are also considered of crucial importance for themaintenance of these ecosystems (Brazelton et al.,2006; 2011).

In conclusion, the extreme conditions in CVA (e.g. highpH, unavailable free CO2) surely select for specificallyadapted, stable microbial populations of unexpectedlyhigh diversity. Furthermore, the populations found andthe functional key markers detected in CVA stronglysuggest that metabolisms related to H2, methane and/orsulfur may be the major driving forces in this specificenvironment.

Experimental procedures

The samples and the sampling site

Groundwater samples were recovered at Cabeço de Vide,South-east of Portugal. The groundwater was extracted fromone borehole designated AC3, as previously described byTiago and colleagues (2004), and transported to the labora-tory at 4°C and assayed within 8 h of sampling. Before re-covering the water samples the water was allowed to run forat least 30 min to ensure that only water from the deepaquifer was recovered.

A total of seven water samples were recovered (during a3-year period), concentrated and used as described inTable 1.

Determination of total microorganisms was performed bythe ethidium bromide staining (Ferreira et al., 1994) and thecounts were made using a Leitz Laborlux epifluorescentmicroscope equipped with a fluorescence filter block I3 (blueexcitation filter BP 450–490 nm, dichromatic mirror 510,emission filter LP 515 nm).

Results of chemical analysis performed three times a yearbetween 1991 and 2011 by a recognized State laboratory(from the Admistração Regional de Saúde do Alentejo, Por-tugal) using standard methods (Clesceri et al., 1998) arepresented here by permission.

DNA extraction from water samples

Samples 1, 2 and 4 were concentrated by filtrationthrough a 0.2-mm-pore-size sterile membrane filter (GelmanSupor® 200; 47 mm diameter), whereas all other sampleswere filtered through 0.1-mm-pore-size sterile membranefilters (Gelman Supor® 100; 47 mm diameter), using a fil-tration unit with a low-pressure vacuum. Total DNA wasextracted using the UltraClean™ Water DNA Isolation Kit(Mo Bio Laboratories) according to manufacturer’s instruc-tions. One additional step of three freeze (-80°) and heat(+60°) cycles was performed during the lysis step of theoriginal protocol. The resulting DNA was preserved at-20°C until further use.

DNA amplification and clone library construction

PCR amplification from purified genomic DNA from samples1, 2 and 3 was carried out with set of primers specific fordomains Bacteria, Archaea and Eukarya. Bacteria clonelibraries were prepared from amplification products obtainedwith the set of primers 27F/1525R (Supplementary TableS1). DNA extracted from Microcella alkaliphila was used aspositive control for PCR amplification. PCR reactions wereperformed in a total volume of 50 ml, with 1.5 U of PlatiniumTaq-DNA polymerase (Invitrogen) and 2 mM MgCl2 finalconcentration, in an automated thermal cycler (MJ MINI;Bio-Rad) as followed: initial step at 94°C for 5 min followedby 30 cycles at: 94°C for 1 min, 55°C for 1 min, 72°C for3 min; and a final step at 72°C for 10 min. A set of primerswas used in PCR reactions for the amplification of 16SrRNA gene of the domain Archaea: 21F/958R (Supplemen-tary Table S1). PCR conditions were as described above.



DNA extracted from Pyrococcus furiosus was used aspositive control for PCR amplification. For detection ofmicroeukaryotes, PCR reactions for amplification of theinternal transcribed spacer region (including the 5.8S rRNAgene) and the 26S/28S rRNA gene D1/D2 domain wereperformed using the set of primers ITS1/ITS4 and NL1/NL4respectively (Supplementary Table S1). Modifications of thestandard PCR reaction conditions were implemented: 35cycles using annealing temperatures ranging from 48°C to55°C for 1 min. DNA from Candida sp. was used as positivecontrol for PCR amplification.

The presence of PCR products and respective concentra-tions were checked by agarose gel electrophoresis.

To generate nearly full-length 16S rRNA gene clone librar-ies, the PCR products were cloned using TOPO TA Cloning®Kit (Invitrogen) according to the manufacturer’s instructions.Positive clones were incubated overnight in selective media,and plasmidic DNA extracted using Jetquick Plasmid Mini-prep Spin Kit (Genomed GmbH). The inserts were amplifiedby PCR using primers flanking the cloning sites of the vector.PCR products were purified using Jetquick PCR PurificationSpin Kit (Genomed GmbH) according to the manufacturer’sinstructions.

RFLP analysis, sequencing and phylogenetic analysis

For grouping the clones, RFLP analysis was performed byovernight digestion of the purified PCR products at 37°Csimultaneously with restriction enzymes HinfI and HaeIII(Takara). RFLP patterns were obtained by electrophoresesusing 3% LM SIEVE Agarose (Pronadisa) gels. From eachRFLP group determined representative clones weresequenced as described previously (Rainey et al., 1996).Purified reaction mixtures were electrophoresed using amodel 310 Genetic Analyser (Applied Biosystems).

The resulting ~ 1490 bp and ~ 980 bp sequences, fordomains Bacteria and Archaea respectively, were phyloge-netically classified according to the Naive Bayesian rRNAClassifier (Version 1.0) of the Ribosomal Database Project II(http://rdp.cme.msu.edu/) and aligned primarily using SILVAWebaligner (Pruesse et al., 2007). The alignment was manu-ally checked against the closely related sequences present inthe ARB database and sequences obtained from the Ribos-omal Database Project-RDP II (Cole et al., 2003) and fromGenBank database using BLAST-N (http://www.ncbi.nlm.nih.gov/BLAST/). Furthermore, all sequences were subjected tosimilarity matrix analysis included in ARB software (Ludwiget al., 2004), and clustered into operational taxonomic units(OTUs) at a level of sequence similarity of � 97% in order toquantify diversity.

The Bellerophon program (Huber et al., 2004) and Check-_Chimaera program available on RDP server were used forsearches of chimera artefacts.

Trees topologies were obtained by neighbor-joining algo-rithm (Saitou and Nei, 1987) included in the ARB software.Filters were used to selectively exclude uninformative orambiguous alignment portions. Cultured organisms wereincluded as phylogenetic references if they shared at least90% sequence similarity to clones obtained in this study. Inthe absence of cultured relatives, the closest environmentalsequences were included.

DGGE analyses

PCR amplification from purified genomic DNA from samples2 to 7 was carried out with set of primers specific for thedomain Bacteria, 341f-GC clamped/534r (SupplementaryTable S1). PCR reactions were performed as describedabove but 35 cycles were used. The amplicons size waschecked by electrophoresis. For domain Archaea, a nestedPCR methodology was employed as previously described byVissers and colleagues (2009). The PCR products obtainedusing the set of primers 21F/958R (above) were used astemplate for the nested PCR reactions for the six samples,using the set of primers by Coolen and colleagues (2004),namely Parch519/Arch915-GC clamped (SupplementaryTable S1).

DGGE was performed with the Bio-Rad DCodeTM UniversalMutation Detection System, essentially as described previ-ously (Muyzer et al., 1993) with minor modifications. Samplescontaining approximately equal amounts of PCR ampliconswere loaded into vertical gels containing 8% (w/v) polyacry-lamide and a linear gradient of denaturants: urea and forma-mide. Denaturing gradient of 35–80% of denaturant [100%denaturant corresponds to 7 M urea plus 40% (v/v) of deion-ized formamide] were used for separation of the 16S rRNAgene fragments obtained. Gradients were created with aMSE (CBS Scientific) gradient maker and peristaltic pump.The gels were prepared in 0.5¥ TAE buffer [20 mM Tris,10 mM acetic acid, 0.5 mM ethylenediaminetetraacetic acid(EDTA), pH 8.0], which was also used as the electrophoresisbuffer. Electrophoresis was performed at a constant voltageof 70 V and a temperature of 60°C for 17 h. After electro-phoresis, gels were incubated for 15 min in Milli-Q watercontaining ethidium bromide (0.5 mg l-1), rinsed for 10 minwith Milli-Q water, and photographed with UV transillumina-tion (302 nm) with Gel Doc XR System (Bio-Rad). Replicationof the PCR and DGGE steps was performed to confirm theconsistency of the DGGE profiles.

The selected dominant bands from DGGE profiles wereexcised with a scalpel, placed into sterile microcentrifugetubes containing 20 ml of sterile water and stored at 4°Covernight. Two microlitres of the resulting elution suspensionwere used as templates for PCR using the primers previouslyused for direct environmental amplification, and a new DGGEwas performed in order to confirm the position and purity ofthe excised bands. After confirmation, an additional PCRreaction was performed with the primers described abovebut without the GC clamp. The size of the amplicons wascheck by electrophoresis, and were ligated into pCR® 2.1vectors (Invitrogen) and transformed into competent cells(Escherichia coli TOP10; Invitrogen) following the supplier’sinstructions. At least four positive clones of each dominantDGGE band were sequenced and the phylogenetic classifi-cation and analyses of the partial 16S rRNA gene fragmentssequenced was performed as described above.

Pyrosequencing runs

Pyrosequencing runs covering the V1–V2 region of the 16SrRNA gene of domains Bacteria and Archaea were performedon Samples 3, 5 and 7 as previously described (Dowd et al.,2008; Callaway et al., 2010) at the Research and Testing



http://rdp.cme.msu.edu/

http://www.ncbi.nlm.nih.gov/BLAST/

http://www.ncbi.nlm.nih.gov/BLAST/

Laboratory Lubbock, TX, USA. The set of primers used aredescribed on Supplementary Table S1.

Pyrosequencing data sets and phylogenetic analyses

The raw data from the pyrosequencing reactions yielded atotal of 17 385 reads for domain Bacteria and 12 151 readsfor domain Archaea. Conservative control measures wereemployed, using mothur software (http://www.mothur.org;Schloss et al., 2009). Low-quality reads with ambiguousbases, sequences smaller than 250 bp, sequence tags, chi-meras and non-ribosomal sequences were removed from thedata sets. The obtained data sets (for both domains Bacteriaand Archaea) were additionally screened using the V-Xtractorsoftware (Hartmann et al., 2010) in order to obtain final pyro-sequence data containing the V1–V2 region of the 16S rRNAgene. Finally, a total of 7897 and 4986 pyrosequences wereobtained for domains Bacteria and Archaea respectively.Alignments and clustering in pyrosequencing operationaltaxonomic units (P-OTUs) were performed using the RDPpyrosequencing pipeline (http://pyro.cme.msu.edu/). Thephylogenetic classification of the 16S rRNA gene pyrose-quences was performed using the ARB-Silva taxonomyon mothur software. The linkage between the determinedP-OTUs and the pyrosequences phylogenetic classificationwas performed using a Windows interface program specifi-cally programmed for that purpose in R (http://www.r-project.org/). Only P-OTUs containing more than 0.3% of the totalpyrosequencing data set were considered for determinationof phylogenetic affiliation by using the standalone BLAST

queries (Camacho et al., 2009) using at least three repre-sentative pyrosequences. P-OTUs with similarity values� 98% with OTUs (determined for domains Bacteria andArchaea by 16S rRNA gene clone libraries) were consideredequivalent, with the same phylogenetic affiliation.

Statistical analysis – 16S rRNA gene clone libraries andpyrosequence data sets

The screening process used to construct the 16S rRNA geneclone libraries, was tested by statistical analyses to evaluatewhether total diversity was covered by overall clones. Cov-erage values (Good, 1953) were calculated using the follow-ing equation: C = (1 - n/N) ¥ 100, where n is the number ofunique OTUs and N is the total number of clones examined.Rarefaction analysis (Hughes et al., 2001) was performed todetermine the number of unique OTUs as a proportion of theestimated total diversity. Calculations were executed usingthe freeware program Analytic Rarefaction version 1.3 (byS. M. Holland; http://www.uga.edu/strata/software/Software.html). The Shannon-Weaver index (H′) and Shannon even-ness (E) value were calculated using standard equations (Hillet al., 2003). The same statistical analyses were performed tothe 16S rRNA gene pyrosequencing data sets.

Statistical analysis – DGGE analyses

To evaluate the stability or similarity of the microbial commu-nities detected through time on the various samples usingDGGE analyses, Sørenson’s similarity indexes (QS) were

determined using the following equation: QS = 2C/(A + B)where A and B are the species numbers in samples A and B,respectively, and C is the number of species shared by thetwo samples (Sørensen, 1948).

PCR amplification of functional genes and librariesconstruction

Environmental DNA extracted from sample 7 was used forPCR detection of genes encoding key enzymes of variousautotrophic CO2-fixing pathways, and sulfur and methanecycles, using specific set of primers (SupplementaryTable S1).

The PCR conditions for the detection of both forms ofRuBisCO, form I (cbbL) and form II (cbbM), both forms ofpyruvate:ferredoxin oxidoreductase gene (por and nifJ),ATP citrate lyase beta subunit (aclB), and 2-oxoglutarate:ferredoxin oxidoreductase gene (oorA) were as follows: initialstep of 94°C for 2 min, followed by two cycles at: 94°C for2 min, 37°C for 2 min and 72°C for 3 min, followed by 35cycles at: 94°C for 1 min, (annealing temperature specific foreach gene listed below) for 1 min, and 72°C for 1 min; and afinal step at 72°C for 10 min. The following changes inannealing temperatures for genes: 53°C for cbbL, 58°C forcbbM, 49°C for porA, 55°C for nifJ, 54°C for aclB and 52°C foroorA, were implemented as previously described by Camp-bell and Cary (2004). The PCR conditions for: the detection ofthe biotin carboxylase-encoding gene (accC) was as previ-ously described by Auguet and colleagues (2008); the detec-tion of APS reductase alpha subunity-encoding gene (aprA)were performed as previously described by Blazejak andcolleagues (2006); and for the detection of methyl coenzymeM reductase gene (mcrA) and methane monooxygenasegene (pmoA) were as previously described by Luton andcolleagues (2002) and Costello and Lidstrom (1999)respectively.

PCR reactions were performed in a total volume of 65 ml,with 1.5 U of Platinium Taq-DNA polymerase (Invitrogen) and2.5 mM MgCl2 final concentration, in an automated thermalcycler (MJ MINI; Bio-Rad). The presence of PCR products,size confirmation and concentration were checked byagarose gel electrophoresis. Clone libraries construction,positive clone growth and plasmid DNA extraction were per-formed as previously described above on the clone libraryconstruction section.

Functional gene sequencing and phylogenetic analysis

Seventy clones of each gene were sequenced and thesequences quality was checked. DNA sequences obtainedfrom the clone libraries were translated and aligned againstsimilar sequences obtained from the GenBank database. Thealignments were used to construct phylogenetic trees onMEGA 5 software (Tamura et al., 2011). Trees topologieswere obtained by neighbor-joining algorithm (Saitou and Nei,1987) included in the MEGA 5 software. Insertions and dele-tions were not included in the calculations.

Nucleotide sequence accession numbers

The 16S rRNA gene sequences determined in this studyobtained from 16S rRNA gene clone library, DGGE analysis



http://www.mothur.org

http://pyro.cme.msu.edu/

http://www.r-project.org/

http://www.r-project.org/

http://www.uga.edu/strata/software/Software.html

http://www.uga.edu/strata/software/Software.html

and from the pyrosequencing analyses for domain Bacteria,were deposited in EMBL/EBI data library under AccessionNos: AM777947 to AM778028, FN401530 to FN401573 andHE672168 to HE680064 respectively. Those obtained for thedomain Archaea were deposited under Accession Nos:FN690937 to FN690961 (DGGE analysis), FN690962 toFN690977 (16S rRNA gene clone library) and HE681934 toHE686919 (pyrosequencing analysis).

All nucleotide sequences (cbbL, accC, aps and MCR) havebeen deposited in GenBank and assigned Accession Nos:HE686920 to HE686967.

Acknowledgements

This research was funded by FCT/FEDER project POCTI/BSE/42732/2001, and I. Tiago acknowledge a fellowshipfrom FCT reference SFRH/BPD/75296/2010. We thank F.Campelo for programming the windows interface softwarein R. We are indebted to Mr Fontainhas and Junta deFreguesia de Cabeço de Vide for the permission to collectthe samples and use the chemical analysis data presentedhere.

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Supporting information

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Fig. S1. Neighbor-joining tree indicating the phylogeneticpositions of the Gram-positive CVA clones belonging toclasses Clostridia, Acidobacteria, Actinobacteria and phylaNitrospira and Chloroflexi and to Gram-positive unclassifiedbacteria groups determined by clone library and DGGEanalyses from CVA. Sequences in bold are from thisstudy. The scale bar represents 10% nucleotide sequencedifferences.Fig. S2. Neighbor-joining tree indicating the phylogeneticpositions of the Gram-negative CVA clones belonging toclasses Alphaproteobacteria, Betaproteobacteria, Deinoc-occi and Gammaproteobacteria and phylum Bacteroidetesdetermined by clone library and DGGE analyses from CVA.Sequences in bold are from this study. The scale bar repre-sents 10% nucleotide sequence differences.Fig. S3. Neighbor-joining tree indicating the phylogeneticpositions of the archaeal CVA clones belonging to phylumEuryarchaeota and class Thermoprotei determined by clone

library and DGGE analyses from CVA. Sequences in bold arefrom this study. The scale bar represents 10% nucleotidesequence differences.Fig. S4. Rarefection curves at a cut-off of 0.05 for: (a)total clones screened (Bacteria �, n = 441; Archaea�, n = 270) and at cut-off values between 0.01 and 0.1 for(b) total pyrosequences (Bacteria, n = 7897; Archaea,n = 4983).Fig. S5. Phylogenetic diversity of translated sequences ofbiotin carboxylase detected in clone library from CVA. CVAsequences are in bold.Table S1. Primer sets used for detection of genes encoding16S rRNA SSU for both domains Bacteria and Archaea andgenes encoding key enzymes of CO2 metabolic pathways,sulphur and methane cycles.Table S2. Chemical composition, temperature and pH of theCabeco de Vide aquifer.Table S3. Statistical analysis results.



microbial and functional diversity of a subterrestrial high ph

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