Design of 16S rRNA-targeted oligonucleotide probes for detecting cultured and uncultured archaeal...

Environmental Microbiology (2004)

6

(2) 170ndash182 doi101046j1462-2920200400560x

copy 2004 Blackwell Publishing Ltd

Blackwell Science LtdOxford UKEMIEnvironmental Microbiology1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd 20036

2170182

Original Article

16S rRNA probes for Archaea thriving in hot habitatsO Nercessian et al

Received 29 August 2003 revised 4 November 2003accepted 4 November 2003 For correspondence E-mailjeanthonuniv-brestfr Tel (+33) 298 498 751 Fax (+33) 298 498705

dagger

Present address Department of Chemical Engineering Box352125 University of Washington Seattle WA 98195 USA E-mailnerceouwashingtonedu

Design of 16S rRNA-targeted oligonucleotide probes for detecting cultured and uncultured archaeal lineages in high-temperature environments

Olivier Nercessian

1dagger

Maria Prokofeva

2

Alexander Lebedinski

2

Steacutephane LrsquoHaridon

1

Craig Cary

3

Daniel Prieur

1

and Christian Jeanthon

1

1

UMR 6539 Centre National de la Recherche Scientifique and Universiteacute de Bretagne Occidentale Institut Universitaire Europeacuteen de la Mer Technopole Brest-Iroise Place Nicolas Copernic 29280 Plouzaneacute France

2

Institute of Microbiology Russian Academy of Sciences Prospect 60 Let Oktyabrya 72 117811 Moscow Russia

3

University of Delaware College of Marine Studies 700 Pilottown Road Lewes DE 19958 USA

Summary

In order to facilitate the evaluation of archaeal com-munity diversity and distribution in high-temperatureenvironments 14 16S rRNA oligonucleotide probeswere designed Adequate hybridization and washconditions of the probes encompassing most knownhyperthermophilic Archaea members of the ordersThermococcales Desulfurococcales and Sulfolo-bales of the families MethanocaldococcaceaePyrodictiaceae and Thermoproteaceae of the genera

Archaeoglobus

Methanopyrus

and

Ignicoccus

andof the as yet uncultured lineages Korarchaeota Cre-narchaeota marine group I deep-sea hydrothermalvent euryarchaeotic group 2 (DHVE 2) and deep-seahydrothermal vent euryarchaeotic group 8 (DHVE 8)were determined by dot-blot hybridization from targetand non-target reference organisms and environmen-tal clones The oligonucleotide probes were also usedto evaluate the archaeal community composition innine deep-sea hydrothermal vent samples All probesexcept those targeting members of SulfolobalesThermoproteaceae Pyrodictiaceae and Korarchaeotagave positive hybridization signals when hybridizedagainst 16S rDNA amplification products obtainedfrom hydrothermal DNA extracts The results con-

firmed the widespread occurrence of Thermococ-cales Desulfurococcales Methanocaldococcaceaeand Archaeoglobus in deep-sea hydrothermal ventsand extended the known ecological habitats ofuncultured lineages Despite their wide coverage theprobes were unable to resolve the archaeal commu-nities associated with hydrothermally influenced sed-iments suggesting that these samples may containnovel lineages This suite of oligonucleotide probesmay represent an efficient tool for rapid qualitativeand quantitative characterization of archaeal commu-nities Their application would help to provide newinsights in the future into the composition distribu-tion and abundance of Archaea in high-temperatureenvironments

Introduction

Analysis of archaeal 16S rRNA sequences has enabledthe phylogeny of Archaea to be described and majorgroups to be identified (Woese

et al

1990 Hugenholtz2002) So far composed of 12 recognized orders 69 gen-era and more than 210 characterized species Archaeaare divided into two phyla (Garrity and Holt 2001) Thephylum Euryarchaeota consists of extreme halophilesthermoacidophiles methanogens hyperthermophilic sul-phate andor sulphite reducers and sulphur metabolizersThe phylum Crenarchaeota is primarily composed ofhyperthermophiles most of which are able to metabolizesulphur (Garrity and Holt 2001)

With the advent of modern molecular biological tech-niques the diversity and widespread distribution of Eur-yarchaeota and Crenarchaeota in previously unsuspectedhabitats have been recognized (DeLong 1992 Bintrim

et al

1997 Vetriani

et al

1999 Takai

et al

2001a) Athird archaeal phylum Korarchaeota has been postulatedon the basis of environmental 16S rRNA sequencesretrieved from microbial communities from the YellowstonePark (Barns

et al

1996) However as no representativesof this group have been isolated in pure culture the phy-lum status of this lineage cannot currently be assessed

Assessment of microbial diversity in diverse high-temperature environments has led to the discovery ofmetabolically diverse Archaea that are thought to contrib-

16S rRNA probes for Archaea thriving in hot habitats

171


Environmental Microbiology

6

170ndash182

ute significantly to the biogeochemical cycles within thesehabitats (Takai and Horikoshi 1999 Orphan

et al

2000Takai

et al

2001b) The analysis of 16S rRNA genes ofhydrothermal vent microbial communities revealed a widediversity of sequences with no close relatives in culture(Barns

et al

1996 Takai and Horikoshi 1999 Takai andSako 1999 Reysenbach

et al

2000 Huber

et al

2002Nercessian

et al

2003) Owing to the lack of determina-tive molecular tools the qualitative and quantitative deter-mination of archaeal assemblages in high-temperatureenvironments still remains poorly assessed In this studywe report on the development of a suite of 14 16S rRNA-targeted oligonucleotide probes for different archaeal phy-logenetic levels of cultured and uncultured organismsretrieved from deep-sea hydrothermal systems (Takai andHorikoshi 1999 Takai and Sako 1999 Reysenbach

et al

2000 Huber

et al

2002 Teske

et al

2002Nercessian

et al

2003) In the context of a preliminaryapplication we analysed the composition of archaealcommunities associated with diverse deep-sea hydrother-mal vent samples

Results and discussion

Probe design

The design of oligonucleotide probes was based on com-parative analysis of 11 143 complete and partial 16 rRNAsequences of the

ARB

database using the

PROBE

DESIGN

option of the

ARB

package In addition to automatic designof probes alignments of 16S rRNA sequences werescreened to find signatures that allow the distinction oforders families and genera of cultured archaeal thermo-philes as well as currently uncultured lineages of Archaeaknown to thrive in hydrothermal ecosystems (Takai andHorikoshi 1999 Takai and Sako 1999 Reysenbach

et al

2000 Takai

et al

2001b Huber

et al

2002 Ner-cessian

et al

2003) Oligonucleotide probes specific toArchaea and Korarchaeota have been designed overrecent years (Stahl and Amann 1991 Burggraf

et al

1997) However the screening of the GenBank RDP andARB databases revealed that several recently deposited16S rRNA sequences retrieved from hydrothermal envi-ronments were not targeted by the existing probes (S-D-Arch-0915-a-A-20 S--Kor-0546-a-A-20 S--Kor-0604-a-A-20 S--Kor-1135-a-A-20) justifying the development ofupdated probes

Probes encompassing thermophilic cultured Archaea

Three order- three family- and three genus-level probeswere designed to target most of the thermophilic Archaea(Table 1) The sequence of S-O-Tcl-1408-a-A-18 perfectlymatched the sequences of all members of the order Ther-mococcales (group 2 in Fig 1) that includes chemoorga-

noheterotrophic hyperthermophiles and encompasses thegenera

Thermococcus

Pyrococcus

and

Palaeococcus

(Takai

et al

2000 Boone

et al

2001) The probe S-O-Sulf-1045-a-A-18 perfectly matched the sequences of allthermoacidophilic chemoorganotrophs of the order Sul-folobales (group 11 in Fig 1) (Boone

et al

2001) Theprobe S-O-Dsfc-0736-a-A-21 was designed to targetrepresentatives of the order Desulfurococcales thatencompass chemoorganoheterotrophic and chemolitho-autotrophic hyperthermophiles (group 8 in Fig 1) (Boone

et al

2001) It exhibited perfect matches with all Desulfu-rococcales sequences except those of species of thegenus

Desulfurococcus

[one GA mismatch at position747 (

Escherichia coli

numbering Brosius

et al

1978)]This probe was also found to match perfectly somesequences that are not affiliated with the order Desulfu-rococcales such as species of the genera

Thermocladium

and

Vulcanisaeta

(Boone

et al

2001) and the environ-mental clone pJP89 (Barns

et al

1994) The probe S-O-Dsfc-0736-a-A-21 was however retained for furtherexperiments because of the potential utility of its widecoverage

Three family- (S-F-Prd-0488-a-A-16 S-F-Thp-1225-a-A-22 and S-F-Mcc-1109-b-A-20) and three genus-levelprobes (S-G-Mp-0431-a-A-20 S-G-Ign-0463-a-A-16 andS-G-Agb-0431-a-A-21) were designed to target most of16S rRNA sequences of the Pyrodictiaceae Thermopro-teaceae and Methanocaldococcaceae

Methanopyrus

Ignicoccus

and

Archaeoglobus

respectively The probe S-F-Prd-0488-a-A-16 (group 9 in Fig 1) perfectly matchedsequences of all species of the family Pyrodictiaceaecomposed of hyperthermophilic chemolithoautotrophs orfermenters (Boone

et al

2001) Despite several attemptswe were unable to design a probe that targeted all mem-bers of the order Thermoproteales (group 12 in Fig 1)(Boone

et al

2001) with a T

m

lower than 76

infin

C andwithout altering the

in silico

specificity We thereforedesigned probe S-F-Thp-1225-a-A-22 that perfectlymatched all 16S rRNA sequences of the genera

Pyro-baculum

Thermoproteus

Caldivirga

and

Vulcanisaeta

known as thermoacidophilic chemoorganoheterotrophsusing sulphur O

2

or nitrate as electron acceptors (Boone

et al

2001 Itoh

et al

2002) However it had one mis-match with the sequences of members of genera

Ther-mocladium

(CT at position 1244

E coli

numbering) and

Thermofilum

(CA at position 1225

E coli

numbering)Contrary to the probe MCC1109 developed by Raskin

et al

(1994) the degenerate probe S-F-Mcc-1109-b-A-20perfectly matched the sequences of all hyperthermophilicmethanogenic species of the family Methanocaldococ-caceae (genera

Methanocaldococcus

and

Methanotorris

)(group 4 in Fig 1) (Boone

et al

2001)

It contained aslightly destabilizing GT mismatch at position 1121 (

Ecoli

numbering) with sequences of thermophilic and

172

O Nercessian

et al



6

170ndash182

Tab

le 1

Olig

onuc

leot

ide

prob

es ta

rget

ing

16S

rD

NA

seq

uenc

es o

f the

rmop

hilic

hyp

erth

erm

ophi

lic a

nd u

ncul

ture

d A

rcha

ea

Pro

be n

ame

a

Pro

be s

eque

nce

(5

cent AElig

3

cent

)

b

Spe

cific

ity (

num

ber

of e

xact

mat

ches

in G

enB

ank)

c

The

oret

ical

Td

(

infin

C)

d

Hyb

te

mp

(

infin

C)

e

Was

h

tem

p (

infin

C)

f

Ref

eren

ces

S-D

-Arc

h-09

15-b

-A-1

7C

TC

CC

CC

GC

CA

ATT

CC

TA

rcha

ea56

47

47M

odifi

ed f

rom

Sta

hlan

d A

man

n (1

991)

S-O

-Tcl

-140

8-a-

A-1

8A

CG

CT

CC

AC

CC

CT

TG

TAG

The

rmoc

occa

les

(121

)58

4754

Thi

s st

udy

S-G

-Agb

-043

1-a-

A-2

1T

TTA

GG

CA

CC

CC

GA

CA

GC

CC

G

Arc

haeo

glob

us

(15

)70

4752

Thi

s st

udy

S-F

-Mcc

-110

9-b-

A-2

0G

CA

AC

ATG

GG

GC

RC

GG

GT

CT

Met

hano

cald

ococ

cace

ae (

15)

66 (

68)

4758

g

Mod

ified

from

Ras

kin

et a

l

(19

94)

S-

-DH

VE

2-03

92-a

-A-2

0A

AG

GG

CA

CT

CG

GG

CT

CC

CC

TD

HV

E 2

(18

)64

4758

Thi

s st

udy

S-

-DH

VE

8-13

58-a

-A-1

9AT

TC

GC

CG

AA

CG

GT

GC

TAA

DH

VE

8 (

9)56

4752

Thi

s st

udy

S-G

-Mp-

0431

-a-A

-20

TTA

CA

CC

CC

GG

TAC

AG

CC

GC

Met

hano

pyru

s

(7)

6647

52T

his

stud

yS

-O-D

sfc-

0736

-a-A

-21

CC

GT

CG

GG

CG

CG

TT

CC

AG

CC

GM

ost

of D

esul

furo

cocc

ales

(60

+ 7

The

rmop

rote

ales

)76

5070

Thi

s st

udy

S-F

-Prd

-048

8-a-

A-1

6C

CG

CT

TAC

TC

CC

CC

GC

Pyr

odic

tiace

ae (

7 +

1 m

amm

als)

5647

52T

his

stud

yS

-G-I

gn-0

463-

a-A

-16

AC

CC

CC

GC

CT

GT

TTA

C

Igni

cocc

us

(11

+ 4

mam

mal

s)52

4747

Thi

s st

udy

S-O

-Sul

f-10

45-a

-A-1

8A

CC

TC

CT

CT

CC

GC

GA

GT

CS

ulfo

loba

les

(50)

6047

54T

his

stud

yS

-F-T

hp-1

225-

a-A

-22

CC

CG

CC

ATT

GC

AG

CT

CG

CG

TG

CT

herm

opro

teac

eae

exce

pt

The

rmoc

ladi

um

(23

)76

5065

Thi

s st

udy

S-

-MgI

-039

1-b-

A-2

0A

AAT

CA

CT

CG

GAT

TAA

CC

TT

Mos

t of

mar

ine

Cre

narc

haeo

ta g

roup

I (

127)

5447

47M

odifi

ed f

rom

Tak

aian

d H

orik

oshi

(1

999)

S-

-Kor

-055

4-a-

A-1

8A

GG

CC

CA

GTA

TG

CG

TG

GG

Kor

arch

aeot

a (1

2)60

4752

Thi

s st

udy

a

Arc

h A

rcha

ea T

cl T

herm

ococ

cale

s A

gb

Arc

haeo

glob

us

Mcc

Met

hano

cald

ococ

cace

ae D

HV

E 2

dee

p-se

a hy

drot

herm

al v

ent e

urya

rcha

eotic

gro

up 2

DH

VE

8 d

eep-

sea

hydr

othe

rmal

ven

teu

ryar

chae

otic

gro

up 8

Mp

Met

hano

pyru

s

Dsf

c D

esul

furo

cocc

ales

Prd

P

yrod

ictia

ceae

Ign

Igni

cocc

us

Sul

f S

ulfo

loba

les

Thp

The

rmop

rote

acea

e ex

cept

The

rmoc

ladi

um

MgI

m

arin

e gr

oup

I K

or

Kor

arch

aeot

a P

robe

nam

es a

re a

ccor

ding

to

the

Olig

onuc

leot

ide

Pro

be D

atab

ase

nom

encl

atur

e (A

lm

et a

l

19

96)

b

R r

efer

s to

A o

r G

c

See

Fig

1 f

or d

etai

led

cove

rage

info

rmat

ion

BLA

ST

sea

rche

s pe

rform

ed in

Mar

ch 2

003

d

The

oret

ical

tem

pera

ture

of

dena

tura

tion

(Td)

cal

cula

ted

acco

rdin

g to

the

form

ula

4

yen

(A

+ T

) +

2

yen

(G

+C

) (S

tahl

and

Am

ann

199

1) F

or t

he p

robe

S-F

-Mcc

-110

9-b-

A-2

0 it

dep

ends

whe

ther

R is

con

side

red

as a

n A

(66

infin

C)

or a

G (

68

infin

C)

e

Hyb

ridiz

atio

n te

mpe

ratu

re (

infin

C)

used

in t

he s

peci

ficity

stu

dies

f

Tem

pera

ture

(

infin

C)

of t

he w

ash

buffe

r us

ed in

the

spe

cific

ity s

tudi

es

g

Pro

be S

-F-M

cc-1

109-

b-A

-20

was

spe

cific

for

16S

rR

NA

of

the

gene

ra M

etha

noto

rris

and

Met

hano

cald

ococ

cus

whe

n w

ashe

d at

58infin

C T

he p

robe

was

spe

cific

for

16S

rR

NA

of

the

gene

raM

etha

noto

rris

M

etha

noca

ldoc

occu

s M

etha

noco

ccus

and

Met

hano

ther

moc

occu

s w

hen

was

hed

at 5

6infinC

16S rRNA probes for Archaea thriving in hot habitats 173

copy 2004 Blackwell Publishing Ltd Environmental Microbiology 6 170ndash182

mesophilic methanogenic species of the genera Methan-othermococcus and Methanococcus respectively Theprobes S-G-Mp-0431-a-A-20 and S-G-Ign-0463-a-A-16(groups 7 and 10 in Fig 1 respectively) perfectly boundall sequences of the genera Methanopyrus and Ignicoc-cus (Boone et al 2001) Contrary to other members ofthe order Desulfurococcales species of Ignicoccus areobligate chemolithoautotrophic sulphur reducers (Booneet al 2001) With the hyperthermophilic methanogensthese organisms probably represent the main primaryproducers in high-temperature marine environmentsThe probe S-G-Agb-0431-a-A-21 perfectly matched allsequences of the hyperthermophilic mixotrophic sulphate-or sulphite- and thiosulphate-reducing organisms of thegenus Archaeoglobus and environmental clones (ieVC21 Arc8 VC21Arc4 and pEPR796) retrieved fromdeep-sea hydrothermal vents (Reysenbach et al 2000Boone et al 2001 Nercessian et al 2003) (group 3 inFig 1) However it contained major mismatches withsequences of species of genera Ferroglobus (AA at posi-tion 444 E coli numbering) and Geoglobus and withenvironmental clones VC21 Arc2 (CA at position 434 Ecoli numbering) VC21Arc36 (AA at position 444 E colinumbering) and pMC2A228 (CC at position 440 E colinumbering) retrieved from deep-sea hydrothermal vents(Hafenbradl et al 1996 Takai and Horikoshi 1999 Rey-senbach et al 2000 Kashefi et al 2002)

Probes encompassing uncultured organisms

In addition to probes targeting cultured Archaea wedeveloped four oligonucleotide probes specific to as yetuncultured organisms With the exception of marine groupI Crenarchaeota (group 13 in Fig 1) retrieved from variousmarine ecosystems (Vetriani et al 1999 Massana et al2000 Huber et al 2002) these uncultured organismshave only been detected in hydrothermal systems (Takaiand Horikoshi 1999 Takai and Sako 1999 Reysenbachet al 2000 Marteinsson et al 2001 Takai et al 2001bHuber et al 2002 Nercessian et al 2003) The probeS--DHVE2-0392-a-A-20 (group 5 in Fig 1) matched per-fectly all sequences belonging to the deep-sea hydrother-mal vent euryarchaeotic group 2 (DHVE 2 Takai andHorikoshi 1999) The probe S--DHVE8-1358-a-A-19(group 6 in Fig 1) matched perfectly all sequences fromthe recently discovered environmental clade deep-seahydrothermal vent euryarchaeotic group 8 (DHVE 8 Takaiand Horikoshi 1999) Burggraf et al (1997) designedprobes specific to the Korarchaeota (Barns et al 1996)However recently deposited lsquokorarchaealrsquo 16S rRNAsequences retrieved from coastal and deep-sea hydro-thermal vents contained several mismatches with thelatter probes We therefore designed the probe S--Kor-0554-a-A-18 to encompass most of the 16S rRNA

sequences of Korarchaeota available in the databases(group 14 in Fig 1) The probe S--MgI-0391-b-A-20matched most sequences belonging to the marine groupI Crenarchaeota (group 13 in Fig 1) retrieved from variousmarine ecosystems (Vetriani et al 1999 Massana et al2000 Huber et al 2002) However some sequences con-tained a slightly destabilizing TG mismatch at positions398 or 407 (E coli numbering)

Finally a new general archaeal probe was developed inorder to include the new archaeal lineage DHVE8 (Ner-cessian et al 2003) (group 1 in Fig 1) In contrast to theArchaea-specific probe S-D-Arch-0915-a-A-20 developedby Stahl and Amann (1991) the probe S-D-Arch-0915-a-A-17 (group 1 in Fig 1) perfectly matched the 16S rRNAfrom the DHVE8 lineage However similar to the probe S-D-Arch-0915-a-A-20 the new probe still contained severalstrongly destabilizing mismatches with some DHVE2 (CAat position 928) and all Korarchaeota sequences (CA andTG at positions 923 and 930)

Specificity studies

The specificity of selected oligonucleotide sequencesrevealed by comparison with available rRNA sequencedatabases was ensured by optimization of experimentalhybridization conditions The hybridization and post-hybridization washing temperatures ensuring specificitywere experimentally determined for the 14 probes char-acterized in this study (Table 1) The 14 identical mem-branes containing nucleic acids from the reference strainsand environmental clones mentioned in Table 2 are shownin Fig 2 Dot-blot hybridization experiments generallyconfirmed the in silico specificity analysis Probe S-D-Arch-0915-a-A-17 gave positive signals for most of thearchaeal nucleic acids Confirming the in silico analysisno hybridization signals were obtained for clonespEPR193 pEPR152 and pEPR153 (Fig 2a blots C4 G2and G3 respectively) that belonged to the lineagesDHVE2 or Korarchaeota The organisms targeted byprobes S-O-Tcl-1408-a-A-18 (Fig 2b) S-G-Agb-0431-a-A-21 (Fig 2c) S--DHVE2-0392-a-A-20 (Fig 2e)S--DHVE8-1358-a-A-19 (Fig 2f) S-G-Mp-0431-a-A-20(Fig 2g) S-G-Ign-0463-a-A-16 (Fig 2i) S-F-Prd-0463-a-A-16 (Fig 2j) S-O-Sulf-1045-a-A-18 (Fig 2k) andS--MgI-0391-a-A-20 (Fig 2m) were unambiguouslydiscriminated from non-target strains The probe S-F-Mcc-1109-b-A-20 was found to be specific for mesophilic ther-mophilic and hyperthermophilic methanogens from theorder Methanococcales when washed at 56infinC (data notshown) It was specific for hyperthermophilic methano-gens only when washed at 58infinC (Fig 2d) This differencein specificity resulted from a slightly destabilizing GT mis-match at position 1121 (E coli numbering) in the se-quences of Methanothermococcus thermolithotrophicus

174 O Nercessian et al




and Methanococcus voltae Under the conditions usedthe probe S-F-Thp-1225-a-A-22 was specific for membersof the families Thermoproteaceae and ThermofiliaceaeHowever lower signal intensities probably due to thepresence of a single weakly destabilizing mismatch were

observed for Thermocladium (family Thermoproteaceae)and Thermofilum (family Thermofiliaceae) (Fig 2l) Ourexperimental conditions confirmed that probe S-O-Dsfc-0736-a-A-16 matched perfectly nearly all sequences ofthe order Desulfurococcales and some of the order Ther-

Table 2 Reference strains and environmental clones used in this study

Reference strains or clonesa Position on blotb Reference

Methanocaldococcus jannaschii (DSM 2661T) A1 Jones et al (1983)Methanotorris igneus (DSM 5666T) A2 Burggraf et al (1990a)Methanothermococcus thermolithotrophicus (DSM 2095T) A3 Huber et al (1982)Methanococcus voltae (DSM 1537T) A4 Balch et al (1979)Thermococcus celer (DSM 2476T) A5 Zillig et al (1983b)Pyrococcus abyssi strain GE5 A6 Erauso et al (1993)Archaeoglobus profundus (DSM 5631T) A7 Burggraf et al (1990b)Methanopyrus kandleri (DSM 6324T) B1 Kurr et al (1991)Methanoculleus marisnigri (DSM 1498T) B2 Romesser et al (1979)Methanohalophilus mahii (DSM 5219T) B3 Paterek and Smith (1985)pEPR809 (Methanocaldococcus spp) B4 Nercessian et al (2003)pEPR743 (Thermococcus spp) B5 Nercessian et al (2003)pEPR145 (Pyrococcus spp) B6 Nercessian et al (2003)pEPR796 (Archaeoglobus spp) B7 Nercessian et al (2003)pEPR829 (Methanopyrus spp) C1 Nercessian et al (2003)pEPR717 (DHVE 2) C2 Nercessian et al (2003)pEPR719 (DHVE 2) C3 Nercessian et al (2003)pEPR193 (DHVE 2) C4 Nercessian et al (2003)pEPR824 (DHVE 8) C5 Nercessian et al (2003)pEPR895 (DHVE 8) C6 Nercessian et al (2003)pEPR731 (DHVE 8) C7 Nercessian et al (2003)Pyrodictium abyssi (DSM 6158T) D1 Pley et al (1991)Pyrolobus fumari (DSM 11204T) D2 Blochl et al (1997)Ignicoccus pacificus (DSM 13166T) D3 Huber et al (2000)Staphylothermus marinus (DSM 3639T) D4 Fiala et al (1986)Aeropyrum pernix (DSM 11879T) D5 Sako et al (1996)Thermococcus profundus (JCM 9378T) D6 Kobayashi et al (1994)Desulfurococcus mobilis (DSM 2161T) D7 Zillig et al (1982)Acidilobus aceticus (DSM 11585T) E1 Prokofeva et al (2000)Sulfolobus shibatae (DSM 5389T) E2 Grogan et al (1990)Metallosphaera sedula (DSM 5348T) E3 Huber et al (1989)Acidianus brierleyi (DSM 1651T) E4 Zillig et al (1980)Thermoproteus tenax (DSM 2078T) E5 Zillig et al (1981)Thermocladium modestius (JCM 0088T) E6 Itoh et al (1998)Thermofilum pendens (DSM 2475T) E7 Zillig et al (1983a)Pyrobaculum organotrophum (DSM 4185T) F1 Huber et al (1987)pEPR940 (Pyrodictium spp) F2 Nercessian et al (2003)pEPR936 (Ignicoccus spp) F3 Nercessian et al (2003)pEPR805 (Staphylothermus spp) F4 Nercessian et al (2003)pEPR985 (Aeropyrum spp) F5 Nercessian et al (2003)pEPR853 (marine Crenarchaeota group I) F6 Nercessian et al (2003)pEPR624 (marine Crenarchaeota group I) F7 Nercessian et al (2003)pEPR161 (marine Crenarchaeota group I) G1 Nercessian et al (2003)pEPR152 (Korarchaeota) G2 Nercessian et al (2003)pEPR153 (Korarchaeota) G3 Nercessian et al (2003)Desulfovibrio giganteus (DSM 4123T) G4 Esnault et al (1988)

a Collection numbers of species or phylogenetic relatives of environmental clones pEPR are indicated in brackets DSM Deutsche Sammlungvon Mikroorganismen und Zellkulturen (Braunschweig Germany) JCM Japanese Collection of Microorganisms (Saitama Japan)b See Fig 2 For example 16S rDNA of Methanocaldococcus jannaschii is located on dot A1 (lane A column 1 in Fig 2)

Fig 1 16S rDNA phylogenetic tree showing the archaeal groups targeted by the newly designed probes The tree was constructed using the neighbour-joining method (Saitou and Nei 1987) and the correction of Jukes and Cantor (1969) Archaeal lineages marked group 1 to group 14 were targeted by the following probes S-D-Arch-0915-b-A-17 (group 1) S-O-Tcl-1408-a-A-18 (group 2) S-G-Agb-0431-a-A-21 (group 3) S-F-Mcc-1109-b-A-20 (group 4) S--DHVE2-0392-a-A-20 (group 5) S--DHVE8-1358-a-A-19 (group 6) S-G-Mp-0431-a-A-20 (group7) S-O-Dsfc-0736-a-A-21 (group 8) S-F-Prd-0488-a-A-16 (group 9) S-G-Ign-0463-a-A-16 (group 10) S-O-Sulf-1045-a-A-18 (group 11) S-F-Thp-1225-a-A-22 (group 12) S--MgI-0391-b-A-20 (group 13) S--Kor-0554-a-A-18 (group 14) Bold sequences were used in the specificity studies (see Table 2 and Fig 2)



moproteales (Fig 2h blots E6 and E7) but not those ofthe genus Desulfurococcus (Fig 2h blot D7) Under low-stringency washing conditions (65infinC) signal intensities oftargeted organisms were strong but a faint positive signalwas also observed for the clones pEPR152 and pEPR153(Korarchaeota) Using higher stringency washing condi-tions (70infinC) poor fluorescence intensities (Fig 2h)were obtained for targeted organisms but Korarchaeotasequences were efficiently discriminated [probably be-cause of the presence of a single weak destabilizingmismatch (GT at position 749 E coli numbering)] ProbeS--Kor-0554-a-A-18 gave a positive signal only when hy-bridized with nucleic acids of clone pEPR153 but failedto hybridize with clone pEPR152 [16S rRNA sequence ofthe latter archaeal clone had a CT mismatch atposition 565 (E coli numbering)]

Detection of Archaea subgroups in environmental samples

Archaeal 16S rDNA amplicons were obtained by poly-merase chain reaction (PCR) from DNA isolated fromdeep-sea hydrothermal samples (Table 3) The amplifica-tion products were transferred onto positively chargednylon membranes DNA fixed to membranes was thenhybridized against the 14 designed and validated probesunder the conditions mentioned in Table 1 (Fig 3) ProbeS-D-Arch-0915-a-A-17 gave strong positive signals for allamplification products All other probes except those tar-geting members of Sulfolobales Pyrodictiaceae Thermo-proteaceae and Korarchaeota gave positive signals withdifferent intensities depending on the sample Our resultsconfirmed the apparent absence of thermoacidophiles ofthe order Sulfolobales and Thermoproteaceae in deep-sea hydrothermal vent environments Although end-

member hydrothermal fluid pH is usually below pH 45Sulfolobales may not tolerate large fluctuations in pH thatprobably occur in the zones of mixing of sea water andhydrothermal fluids (Jannasch 1995) The absence ofmembers of Thermoproteaceae is more likely to resultfrom their low tolerance of the high ionic strength of seawater and hydrothermal fluid mixtures Conversely iso-lates andor 16S rRNA sequences of Pyrodictiaceae andKorarchaeota have been retrieved from deep-sea hydro-thermal environments (Boone et al 2001 Teske et al

Fig 3 Dot-blot hybridizations of archaeal amplicons from diverse deep-sea hydrothermal samples The sample codes (A to I) are those reported in Table 3 The 16S rDNAs were hybridized with the following probes D-Arch-0915-b-A-17 (1) S-O-Tcl-1408-a-A-18 (2) S-G-Agb-0431-a-A-21 (3) S-F-Mcc-1109-b-A-20 (4) S-G-Mp-0431-a-A-20 (5) S-O-Dsfc-0736-a-A-21 (6) S-G-Ign-0463-a-A-16 (7) S--MgI-0391-b-A-20 (8) S--DHVE2-0392-a-A-20 (9) S--DHVE8-1358-a-A-19 (10) See Table 1 and Fig 1 for specificity and coverage

Fig 2 Dot-blot analyses of probe specificities The layout of the 46 target and non-target 16S rDNA sequences on blots is shown in Table 2 The blots were hybridized with the following probes S-D-Arch-0915-b-A-17 (a) S-O-Tcl-1408-a-A-18 (b) S-G-Agb-0431-a-A-21 (c) S-F-Mcc-1109-b-A-20 (d) S--DHVE2-0392-a-A-20 (e) S--DHVE8-1358-a-A-19 (f) S-G-Mp-0431-a-A-20 (g) S-O-Dsfc-0736-a-A-21 (h) S-F-Prd-0488-a-A-16 (i) S-G-Ign-0463-a-A-16 (j) S-O-Sulf-1045-a-A-18 (k) S-F-Thp-1225-a-A-22 (l) S--MgI-0391-b-A-20 (m) S--Kor-0554-a-A-18 (n) As a control the 16S rDNA of Desulfovibrio giganteus (blot G4) yielded a positive signal when hybridized with the general bacterial probe S-D-Bact-0388-a-A-18 (data not shown)



2002 Nercessian et al 2003) This may suggest that ifpresent they were probably too low in abundance in oursample to be detected

Probes targeting Thermococcales Archaeoglobus sppand Methanocaldococcaceae gave positive signals inmost of the samples confirming their widespread distri-bution in deep-sea hydrothermal ecosystems (Booneet al 2001) Hybridization signals specific to Methanopy-rus were obtained only in a few samples from EPR AsMethanopyrus- and Methanocaldococcus-like organismswere enriched from the MAR sediments (C Jeanthonunpublished data) but not or poorly detected by theirspecific probes it is presumed that hyperthermophilicchemolithoautotrophic methanogens were present in lownumbers in these samples

Although Desulfurococcales were present in all sam-ples the probes targeting lower phylogenetic levelsyielded no (family Pyrodictiaceae) or few (genus Ignicoc-cus) signals Major discrepancies (compare dots 6E to 6Iwith 7E to 7I in Fig 3) could indicate that other knowninhabitants of deep-sea hydrothermal vents such as Sta-phylothermus spp Aeropyrum spp and Thermodiscusspp (Takai and Sako 1999 Boone et al 2001 Takaiet al 2001b Nercessian et al 2003) might be presentin the corresponding samples However we cannotexclude the possibility that as yet unidentified Desulfuro-coccales reacted with the probe S-O-Dsfc-0736-a-A-16

The as yet uncultured organisms targeted by the otherprobes developed in this study were present in most sam-ples Marine group I sequences have often been recov-ered in libraries from deep-sea and coastal hydrothermalvent samples (Moyer et al 1998 Takai and Horikoshi1999 Huber et al 2002 Nercessian et al 2003) Severalstudies suggest that these non-thermophilic organismsmay contribute significantly to the mesopelagic microbialcommunity (Karner et al 2001) and that their occurrencein hydrothermal vent samples may be attributed to theirpresence in deep bottom water and their entrainment dur-ing subsurface mixing of sea water and hydrothermal flu-ids (Huber et al 2002 Nercessian et al 2003) Ourresults are in agreement with these hypotheses as repre-sentatives of marine group I Crenarchaeota were mostlydetected in sediments and in situ samplers but not inchimney samples Inversely sequences from unculturedEuryarchaeota (DHVE 2 and DHVE 8 groups) were notdetected in sediments Based on the high G+C contentsof their 16S rRNA gene sequences a possible thermo-philic lifestyle has been proposed for these organisms(Takai et al 2001b Nercessian et al 2003) Their pref-erential distribution in the chimney environment supportsthis hypothesis

Although our set of probes encompassed most of theknown thermophilic archaeal lineages few and weak sig-nals were generally obtained with amplification productsTa

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from MAR sediments To elucidate the composition ofthese archaeal communities we constructed 16S rDNAlibraries from the sediment DNA extracts Analysis of thecloned sequences revealed that except for a few clonesrelated to marine Crenarchaeota group I all belonged tonovel archaeal lineages (O Nercessian Y Fouquet CPierre D Prieur and C Jeanthon submitted)

Because of the recognized biases introduced by usingPCR for 16S rRNA gene amplification (von Wintzingerodeet al 1997) we cannot assume that the hybridizationsignal intensities reflect the natural abundance of eachtargeted group However keeping in mind these con-straints the EPR archaeal community appeared to begenerally more diverse than the MAR samples As differ-ent DNA extraction procedures were performed on Pacificand Atlantic samples we cannot exclude the possibilitythat they could have affected the observed compositionsof archaeal communities In addition given that distinctarchaeal communities were retrieved from in situ sam-plers chimneys and hydrothermal fluid samples (Takaiand Horikoshi 1999 Reysenbach et al 2000 Takaiet al 2001b Huber et al 2002 Nercessian et al 2003)the nature of the sample type may also have influencedthe composition of archaeal communities sampled Anal-yses of higher numbers of comparable samples are there-fore clearly needed to compare archaeal communities atboth vent fields

Investigations of archaeal community diversity andstructure have generally been achieved by cloning andsequence determination of 16S rDNA genes obtained byPCR amplification of DNA isolated from the samples Thesequencing of large numbers of cloned sequences whichis often required to detect the minor members in a givenenvironmental sample is expensive time-consuming andlabour intensive In the course of this study oligonucle-otide probes targeting 16S rRNAs of defined groups ofArchaea known to thrive in high-temperature environ-ments were developed They were subsequently used toscreen samples in order rapidly to obtain indications ofthe presence of distinct lineages of Archaea This allowedus (i) to confirm the widespread distribution of Thermo-coccales Desulfurococcales Methanocaldococcaceaeand Archaeoglobus in deep-sea hydrothermal vent habi-tats and the apparent absence of Sulfolobales and Ther-moproteaceae (ii) to give new insights into the distributionof uncultured lineages and (iii) to guide us in the identifi-cation of samples suitable for further extensive studiesWe demonstrated that this suite of oligonucleotide probesrepresents an efficient tool for qualitative characterizationof archaeal communities after 16S rDNA PCR amplifica-tion Further experiments should be conducted to deter-mine the conditions needed for their application inquantitative analyses These options should be particu-larly valuable if large numbers of samples are to be anal-

ysed to study spatial and temporal variations in archaealcommunities in high-temperature habitats

Experimental procedures

Organisms and culture conditions

The 26 reference strains and 20 recombinant clones usedin this study are listed in Table 2 Most of the referencestrains were obtained as active cultures from the Deut-sche Sammlung von Mikroorganismen und Zellkulturen(Braunschweig Germany) and the Japanese Collection ofMicroorganisms (Saitama Japan) Pyrococcus abyssi strainGE5 was isolated in the laboratory Methanoculleus marisn-igri (DSM 1498T) and Methanohalophilus mahii (DSM 5219T)were kindly provided by B Ollivier and M-L Fardeau (Lab-oratoire IRD de Microbiologie des Anaeacuterobies Universiteacute deProvence Marseille France) The reference organisms werecultured as described in the references cited in Table 2 Envi-ronmental archaeal 16S rDNA inserts cloned in the pCR-21TOPO vector (Invitrogen) were obtained previously from sev-eral deep-sea hydrothermal vent DNA samples collected at13infinN on the East Pacific Rise (EPR) (Nercessian et al2003)

Design and validation of oligonucleotide probes

Design The oligonucleotide probes designed in this studyare listed in Table 1 16S rRNA sequences from targeted andnon-targeted organisms were aligned using the functionFASTALIGNER version 30 of the software ARB (httpwwwarb-homede) The oligonucleotide probes were designed manu-ally or automatically with the PROBE_DESIGN function of ARBIn silico specificities were tested using the PROBE_MATCHBLAST search and PROBE_MATCH functions of ARB Gen-Bank (httpwwwncbinlmnihgov) and the RDP (httprdpcmemsuedu) respectively The self-probe dimers andhairpin formations were controlled with the PRIMERSELECT

311 software (DNASTAR) When possible several criteriawere applied to select suitable oligonucleotide probes includ-ing (i) a length between 15 and 25 nucleotides (ii) a G+Cmol content between 50 and 70 (iii) internal positionsof major mismatches with non-targeted organisms and (iv)absence of self-probe dimers and hairpins

Probe optimization and specificity studies Pure cultures ofthe reference strains (10ndash25 ml) and recombinant clones(5 ml) were centrifuged (5000 g for 10 min at 4infinC) and thepellets were stored at -20infinC until they were used for nucleicacid extraction Nucleic acids from reference strains andrecombinant plasmids of environmental clones wereextracted using the methods described by Charbonnier et al(1995) and Sambrook et al (1989) respectively The 16SrRNA genes from reference strains were amplified byPCR using the universal reverse primer 1407R (5cent-GACGGGGGGTGWGTRCAA-3cent) in conjunction with thearchaeal forward primer 4F (5cent-TCCGGTTGATCCTGCCRG-3cent) or the bacterial forward primer 8F (5cent-AGAGTTTGATYMTGGCTCAG-3cent) The 16S rDNA genes from environ-mental clones were amplified using M13F and M13Rprimers Amplification mixtures consisted of (as finalconcentration) 1yen DNA polymerase buffer 15 mM MgCl2



025 mM each dATP dCTP dGTP and dTTP 02 mM eachprimer and 2 U of Taq DNA polymerase (Promega) in a finalvolume of 50 ml PCR cycles were performed in a Robocycler(Stratagene) as follows one cycle at 95infinC for 5 min 30cycles at 95infinC for 15 min 53infinC for 15 min 72infinC for 25 minand one cycle at 72infinC for 8 min Amplification products werechecked for quality and quantity after electrophoresis on a08 agarose gel containing 05 mg ml-1 ethidium bromide

The oligonucleotide probes were tested for specificity indot-blot hybridization assays Approximately 100 ng of 16SrDNA amplicons was suspended into 50 ml of sterile waterdenatured for 5 min at 95infinC and immediately placed on icefor 5 min Amplified products were blotted onto positivelycharged nylon membrane (Hybond-N+ Amersham Bio-sciences) using a Minifold I dotslot system (Schleicher andSchuell) and immobilized by cross-linking after 2 min expo-sure to UV light The oligonucleotide probes were 3cent end-labelled with fluorescein-11dUTP using Gene Images 3cent-oligolabelling module (Amersham Biosciences) according tothe manufacturerrsquos instructions Membranes were first incu-bated for 45 min at the appropriate hybridization temperature(Table 2) in hybridization buffer consisting of 5yen SSC 01SDS 20yen diluted blocking reagent (Amersham Biosciences)and 05 (wv) dextran sulphate in order to prevent non-specific hybridizations Specific oligonucleotide probes werethen added at a final concentration of 5 ng ml-1 and hybrid-ized overnight at the appropriate temperature The washingsteps consisted of three stringency washes (1yen SSC 01SDS) for 20 min at the wash temperature (Table 2) Fluores-cein-11dUTP-labelled DNAs were then detected with an alka-line phosphatase-conjugated antibody The fluorescent signalintensity was detected with a Storm 860 (Amersham Bio-sciences) after 3ndash6 h of incubation at room temperature withthe detection reagent Pictures were acquired using the soft-ware package IMAGEQUANT (Amersham Biosciences) andassembled with Adobe PHOTOSHOP version 50

Application of probes on 16S rDNAs obtained from hydrothermal samples

Sampling and chemical analyses Nine deep-sea hydrother-mal vent samples collected during the cruises Iris [June2001 Rainbow vent field at 36infin13cent8le N and 33infin54cent1le W onthe Mid-Atlantic Ridge (MAR)] and Extreme2001 (October2001 9infin50cent8le N and 104infin17cent5le W on the EPR) were used assources of environmental archaeal 16S rDNAs Samplesfrom 9infinN EPR were obtained from in situ samplers (Nerces-sian et al 2003) designed to collect microorganisms dis-charged by hydrothermal fluid emitted by active vents Thesamplers were deployed for 2ndash5 days on two different hydro-thermal active areas by the submersible Alvin (Table 3) Sam-ples from the Rainbow vent field consisted of cores ofhydrothermally influenced sediments and fragments of activediffuse vents collected by the ROV Victor (Table 3)

For 9infinN EPR samples small volumes of fluids were col-lected using the Sipper sampler (Di Meo et al 1999) forshipboard chemical analyses using voltammetric and colori-metric methods Aliquots of the samples were separated fordissolved Fe(II) and Fe(total) [defined as Fe(total) = dissolvedFe(III) + dissolved Fe(II)] and analysed by colorimetry usinga Spectronic 601 (Milton Roy) according to the ferrozine

method (Stookey 1970) Electrochemical analyses used astandard three-electrode cell The working electrode was agold amalgam (AuHg) electrode of 01 mm diameter madein commercially available polyethyl ether ketone (PEEK) tub-ing sealed with epoxy as described by Brendel and Luther(1995) Counter (Pt) and reference (AgAgCl) electrodeseach of 05 mm diameter were made similarly For the volta-mmetric measurements the voltage range scanned was from-01 V to -20 V In linear sweep voltammetry (LSV) and cyclicvoltammetry (CV) scan rates of 200 500 or 1000 mV-1 wererun depending on targeted chemical species The parame-ters for square wave voltametry (SWV) were as follows pulseheight 24 mV step increment 1 mV frequency 100 Hz scanrate 200 mV-1 LSV and CV were used to measure oxygenand sulphur species while SWV was used for detection ofmetal redox species Electrochemically conditioning the elec-trode between scans removed any chemical species from thesurface of the electrode restoring it for the next measure-ment To remove any deposited Fe or Mn the working elec-trode was conditioned at a potential of -01 V for 10 s(Brendel and Luther 1995) Before sample measurementsstandard curves were produced for O2 Mn and sulphur spe-cies as described previously (Luther et al 2001)

DNA extraction 16S rDNA amplification and dot-blothybridizations Nucleic acids from EPR samples wereextracted as described previously (Nercessian et al 2003)whereas those from MAR were obtained using the UltraCleanDNA kit (Mobio Laboratories) according to the manufacturerrsquosinstructions

The 16S rDNA genes were primarily amplified from DNAextracts using the conditions used before A semi-nestedPCR with the archaeal-specific primers 341F and 1407R wasthen performed as described previously (Nercessian et al2003) to obtain the desirable amounts of PCR productsneeded for hybridization experiments Dot-blot hybridizationswith 16S rRNA oligonucleotide probes were conducted usingthe experimental conditions determined before

Acknowledgements

The authors are grateful to Yves Fouquet (chief scientist ofthe Iris cruise) for inviting us to participate in the Iris cruiseand analysis of the mineralogy of MAR samples Brian Glazeris also acknowledged for the chemical analyses of the 9infinNdiffuse vent fluids The authors also thank Barbara Campbellfor scientific discussion and facilities during the cruiseExtreme2001 The Iris cruise was organized by IFREMERwith the RV LrsquoAtalante and the ROV Victor The Extreme2001cruise was organized by Woods Hole Institute with RV Atlan-tis and the DSV Alvin We thank the captains and the crewsof LrsquoAtalante and Atlantis and the pilots of DSV Alvin and ROVVictor for their skilful operations Our thanks also go to Marie-Laure Fardeau and Bernard Ollivier for providing referencestrains We thank Erwan Corre Isabelle Mary and FabriceNot for scientific discussion This work was supported by theprogrammes Dorsales CNRSRhocircne-Poulenc and Intas 99-1250 and a PRIR from the Conseil Reacutegional de BretagneThe work performed at Plouzaneacute was made possible by aFEMS young researcher fellowship awarded to M Prokofevain 2001 O Nercessian is supported by a grant from theCommunauteacute Urbaine de Brest



References

Alm EW Oerther DB Larsen N Stahl DA and RaskinL (1996) The Oligonucleotide Probe Database Appl Envi-ron Microbiol 65 270ndash277

Balch WE Fox GE Magrum CJ Woese CR andWolfe RS (1979) Methanogens reevaluation of a uniquebiological group Microbiol Rev 43 260ndash296

Barns SM Fundyga RE Jeffries MW and Pace NR(1994) Remarkable archaeal diversity detected in a Yellow-stone National Park hot spring environment Proc NatlAcad Sci USA 91 1609ndash1613

Barns SM Delwiche CF Palmer JD and Pace NR(1996) Perspectives on archaeal diversity thermophily andmonophyly from environmental rRNA sequences ProcNatl Acad Sci USA 93 9188ndash9193

Bintrim SB Donohue TJ Handelsman J Roberts GPand Goodman RM (1997) Molecular phylogeny ofArchaea from soil Proc Natl Acad Sci USA 94 277ndash282

Blochl E Rachel R Burggraf S Hafenbradl D Jann-asch HW and Stetter KO (1997) Pyrolobus fumariigen and sp nov represents a novel group of Archaeaextending the upper temperature limit for life to 113degrees C Extremophiles 1 14ndash21

Boone DR Castenholz RW and Garrity GM (2001)Bergeyrsquos Manual of Systematic Bacteriology Vol 1 2ndedn New York Springer-Verlag

Brendel PJ and Luther GW (1995) Development of agold amalgam voltammetric microelectrode for the deter-mination of dissolved Fe Mn O2 and S(-II) in porewatersof marine and freshwater sediments Environ Sci Technol29 751ndash761

Brosius J Palmer JL Kennedy JP and Noller HF(1978) Complete nucleotide sequence of a 16S ribosomalRNA gene from Escherichia coli Proc Natl Acad Sci USA75 4801ndash4805

Burggraf S Fricke H Neuner A Kristjansson J RouvierP Mandelco L et al (1990a) Methanococcus igneus spnov a novel hyperthermophilic methanogen from a shal-low submarine hydrothermal system Syst Appl Microbiol13 263ndash269

Burggraf S Jannasch HW Nicolaus B and Stetter KO(1990b) Archaeoglobus profundus sp nov represents anew species within the sulfate-reducing archaebacteriaSyst Appl Microbiol 13 24ndash28

Burggraf S Heyder P and Eis N (1997) A pivotal Archaeagroup Nature 385 780

Charbonnier F Forterre P Erauso G and Prieur D(1995) Purification of plasmids from thermophilic andhyperthermophilic Archaea In Thermophiles Archaea aLaboratory Manual Robb FT and Place AR (eds)Cold Spring Harbor NY Cold Spring Harbor LaboratoryPress pp 87ndash90

DeLong EF (1992) Archaea in coastal marine environ-ments Proc Natl Acad Sci USA 89 5685ndash5689

Di Meo CA Wakefield JR and Cary SC (1999) A newdevice for sampling small volumes of water from marinemicro-environments Deep-Sea Res I 46 1279ndash1287

Erauso G Reysenbach AL Godfroy A Meunier JRCrump B Partensky F et al (1993) Pyrococcus abyssisp nov a new hyperthermophilic archaeon isolated from

a deep-sea hydrothermal vent Arch Microbiol 160 338ndash349

Esnault G Caumette P and Garcia JL (1988) Charac-terization of Desulfovibrio giganteus sp nov a sulfatereducing bacterium isolated from a brackish coastallagoon Syst Appl Microbiol 10 147ndash151

Fiala G Stetter KO Jannasch HW Langworthy TAand Madon J (1986) Staphylothermus marinus sp novrepresents a novel genus of extremely thermophilic sub-marine heterotrophic archaebacteria growing up to 98infinCSyst Appl Microbiol 8 106ndash113

Garrity GM and Holt JG (2001) The road map to themanual In Bergeyrsquos Manual of Systematic BacteriologyVol 1 2nd edn Boone DR Castenholz RW and Gar-rity GM (eds) New York Springer-Verlag pp 119ndash166

Grogan D Palm P and Zillig W (1990) Isolate B12 whichharbours a virus-like element represents a new species ofthe archaebacterial genus Sulfolobus Sulfolobus shibataesp nov Arch Microbiol 154 594ndash599

Hafenbradl D Keller M Dirmeier R Rachel R Rossna-gel P Burggraf S et al (1996) Ferroglobus placidusgen nov sp nov a novel hyperthermophilic archaeumthat oxidizes Fe2+ at neutral pH under anoxic conditionsArch Microbiol 166 308ndash314

Huber G Spinnler C Gambacorta A and Stetter KO(1989) Metallosphaera sedula gen and sp nov representsa new genus of aerobic metal-mobilizing thermoaceto-philic archaebacteria Syst Appl Microbiol 12 38ndash47

Huber H Thomm M Koumlnig H Thies G and Stetter KO(1982) Methanococcus thermolithotrophicus a novel ther-mophilic lithotrophic methanogen Arch Microbiol 132 47ndash50

Huber H Burggraf S Mayer T Wyschkony I RachelR and Stetter KO (2000) Ignicoccus gen nov anovel genus of hyperthermophilic chemolithoautotrophicArchaea represented by two new species Ignicoccusislandicus sp nov and Ignicoccus pacificus sp nov Int JSyst Evol Microbiol 50 2093ndash2100

Huber JA Butterfield DA and Baross JA (2002) Tem-poral changes in archaeal diversity and chemistry in a mid-ocean ridge subseafloor habitat Appl Environ Microbiol68 1585ndash1594

Huber R Kristjansson JK and Stetter KO (1987) Pyro-baculum gen nov a new genus of neutrophilic rod-shaped archaebacteria from continental solfataras growingoptimally at 100infinC Arch Microbiol 149 95ndash101

Hugenholtz P (2002) Exploring prokaryotic diversity in thegenomic area Genome Biol 3 1ndash8

Itoh T Suzuki K and Nakase T (1998) Thermocladiummodestius gen nov sp nov a new genus of rod-shapedextremely thermophilic crenarchaeote Int J Syst Bacteriol48 879ndash887

Itoh T Suzuki K and Nakase T (2002) Vulcanisaetadistributa gen nov sp nov and Vulcanisaeta souniana spnov novel hyperthermophilic rod-shaped crenarchaeotesisolated from hot springs in Japan Int J Syst Evol Microbiol52 1097ndash1104

Jannasch HW (1995) Microbial interactions with hydro-thermal fluids In Seafloor Hydrothermal SystemsPhysical Chemical Biological and Geological Interac-tions Humphris SE Zierenberg RA Mullineaux LS



and Thomson RE (eds) Washington American Geo-physical Union pp 273ndash296

Jones WJ Leigh JA Mayer F Woese CR and WolfeRS (1983) Methanococcus jannaschii sp nov anextremely thermophilic methanogen from a submarinehydrothermal vent Arch Microbiol 136 254ndash261

Jukes TH and Cantor CR (1969) Evolution of proteinmolecules In Mammalian Protein Metabolism MunroHN (ed) New York Academic Press pp 21ndash132

Karner MB DeLong EF and Karl DM (2001) Archaealdominance in the mesopelagic zone of the Pacific OceanNature 409 507ndash510

Kashefi K Tor JM Holmes DE Gaw Van Praagh CVReysenbach AL and Lovley DR (2002) Geoglobusahangari gen nov sp nov a novel hyperthermophilicarchaeon capable of oxidizing organic acids and growingautotrophically on hydrogen with Fe(III) serving as the soleelectron acceptor Int J Syst Evol Microbiol 52 719ndash728

Kobayashi T Kwak YS Akiba T Kudo T and HorikoshiK (1994) Thermococcus profundus sp nov a new hyper-thermophilic archaeon isolated from a deep-sea hydrother-mal vent Syst Appl Microbiol 17 232ndash236

Kurr M Huber R Koumlnig H Jannasch HW Fricke HTrincone A et al (1991) Methanopyrus kandleri gen andsp nov represents a novel group of hyperthermophilicmethanogens growing at 110infinC Arch Microbiol 156 239ndash247

Luther GW Glazer BT Hohmann L Popp JI TaillefertM Rozan TF et al (2001) Sulfur speciation monitoredin situ with solid state gold amalgam voltammetric micro-electrodes polysulfides as a special case in sedimentsmicrobial mats and hydrothermal vent waters J EnvironMonit 3 61ndash66

Marteinsson VT Kristjansson JK Kristmannsdottir HDahlkvist M Saemundsson K Hannington M et al(2001) Discovery and description of giant submarine smec-tite cones on the seafloor in Eyjafjordur northern Icelandand a novel thermal microbial habitat Appl Environ Micro-biol 67 827ndash833

Massana R DeLong EF and Pedros-Alio C (2000) A fewcosmopolitan phylotypes dominate planktonic archaealassemblages in widely different oceanic provinces ApplEnviron Microbiol 66 1777ndash1787

Moyer CL Tiedje JM Dobbs FC and Karl DM(1998) Diversity of deep-sea hydrothermal vent Archaeafrom Loihi Seamount Hawaii Deep-Sea Res II 45 303ndash317

Nercessian O Reysenbach AL Prieur D and JeanthonC (2003) Archaeal diversity associated with in situ sam-plers deployed on hydrothermal vents on the East PacificRise (13infinN) Environ Microbiol 5 492ndash502

Orphan VJ Taylor LT Hafenbradl D and Delong EF(2000) Culture-dependent and culture-independentcharacterization of microbial assemblages associated withhigh-temperature petroleum reservoirs Appl EnvironMicrobiol 66 700ndash711

Paterek JR and Smith PH (1985) Isolation and charac-terization of a halophilic methanogen from Great Salt LakeAppl Environ Microbiol 50 877ndash881

Pley U Schipka A Gambacorta A Jannasch HWFricke H Rachel R and Stetter KO (1991) Pyrodictium

abyssi sp nov represents a novel heterotrophic marinearchaeal hyperthermophile growing at 110infinC Syst ApplMicrobiol 14 245ndash253

Prokofeva MI Miroshnichenko ML Kostrikina NAChernyh NA Kuznetsov BB Tourova TP and Bonch-Osmolovskaya EA (2000) Acidilobus aceticus gen novsp nov a novel anaerobic thermoacidophilic archaeonfrom continental hot vents in Kamchatka Int J Syst EvolMicrobiol 50 2001ndash2008

Raskin L Stromley JM Rittmann BE and Stahl DA(1994) Group-specific 16S rRNA hybridization probes todescribe natural communities of methanogens Appl Envi-ron Microbiol 60 1232ndash1240

Reysenbach AL Longnecker K and Kirshtein J (2000)Novel bacterial and archaeal lineages from an in situgrowth chamber deployed at a Mid-Atlantic Ridge hydro-thermal vent Appl Environ Microbiol 66 3798ndash3806

Romesser JA Wolfe RS Mayer F Spiess E andWalther-Mauruschat A (1979) Methanogenium a newgenus of marine methanogenic Bacteria and characteriza-tion of Methanogenium cariaci sp nov and Methanoge-nium marisnigri sp nov Arch Microbiol 121 147ndash153

Saitou N and Nei M (1987) The neighbour joining methoda new tool for reconstructing phylogenetic trees Mol BiolEvol 4 406ndash425

Sako Y Nomura N Uchida A Ishida Y Morii H KogaY et al (1996) Aeropyrum pernix gen nov sp nov anovel aerobic hyperthermophilic archaeon growing at tem-peratures up to 100 degrees C Int J Syst Bacteriol 461070ndash1077

Sambrook J Fritsch EF and Maniatis T (1989) Molecu-lar Cloning a Laboratory Manual 2nd edn Cold SpringHarbor NY Cold Spring Harbor Laboratory Press

Stahl DA and Amann R (1991) Development and appli-cation of nucleic acid probes In Nucleic Acids Techniquesin Bacterial Systematics Stackebrandt E and Goodfel-low E (eds) Chichester John Wiley amp Sons pp 205ndash248

Stookey LL (1970) Ferrozine ndash a new spectrophotometricreagent for iron Anal Chem 42 779ndash781

Takai K and Horikoshi K (1999) Genetic diversity ofArchaea in deep-sea hydrothermal vent environmentsGenetics 152 1285ndash1297

Takai K and Sako Y (1999) A molecular view of archaealdiversity in marine and terrestrial hot water environmentsFEMS Microbiol Ecol 28 177ndash188

Takai K Sugai A Itoh T and Horikoshi K (2000) Palae-ococcus ferrophilus gen nov sp nov a barophilic hyper-thermophilic archaeon from a deep-sea hydrothermal ventInt J Syst Evol Microbiol 50 489ndash500

Takai K Moser DP DeFlaun M Onstott TC and Fre-derickson JK (2001a) Archaeal diversity in waters fromdeep South African gold mines Appl Environ Microbiol 673618ndash3629

Takai K Komatsu T Inagaki F and Horikoshi K (2001b)Distribution of Archaea in a black smoker chimney struc-ture Appl Environ Microbiol 67 3618ndash3629

Teske A Hinrichs KU Edgcomb V de Vera Gomez AKysela D Sylva SP et al (2002) Microbial diversity ofhydrothermal sediments in the Guaymas Basin evidencefor anaerobic methanotrophic communities Appl EnvironMicrobiol 68 1994ndash2007



Vetriani C Jannasch HW MacGregor BJ Stahl DAand Reysenbach AL (1999) Population structure andphylogenetic characterization of marine benthic Archaea indeep-sea sediments Appl Environ Microbiol 65 4375ndash4384

von Wintzingerode F Gobel UB and Stackebrandt E(1997) Determination of microbial diversity in environmen-tal samples pitfalls of PCR-based rRNA analysis FEMSMicrobiol Rev 21 213ndash229

Woese CR Kandler O and Wheelis ML (1990) Towardsa natural system of organisms proposal for the domainsArchaea Bacteria and Eucarya Proc Natl Acad Sci USA87 4576ndash4579

Zillig W Stetter KO Wunderl S Schulz W Priess Hand Scholz I (1980) The SulfolobusndashlsquoCaldariellarsquo grouptaxonomy on the basis of the structure of DNA dependentRNA polymerases Arch Microbiol 125 259ndash269

Zillig W Stetter KO Schaumlfer W Janekovic D Wunderl

S Holz J and Palm P (1981) Thermoproteales a noveltype of extremely thermoacidophilic anaerobic archaebac-teria isolated from Icelandic solfataras Zentbl BakteriolMikrobiol Hyg 1 Abt Orig C 2 205ndash227

Zillig W Stetter KO Prangishvilli D Schaumlfer WWunderl S Jankovic D et al (1982) Desulfurococ-caceae the second family of the extremely thermophilicanaerobic sulfur-respiring Thermoproteales Zentbl Bakte-riol Hyg Abt Orig C 3 304ndash317

Zillig W Gierl A Schreiber G Wunderl S Janekovic DStetter KO and Klenk HP (1983a) The archaebacte-rium Thermofilum pendens represents a novel genus of thethermophilic anaerobic sulfur respiring ThermoprotealesSyst Appl Microbiol 4 79ndash87

Zillig W Holz L Janekovic D Schaumlfer W and ReiterWD (1983b) The archaebacterium Thermococcus celerrepresents a novel genus within the thermophilic branch ofthe archaebacteria Syst Appl Microbiol 4 88ndash94

16S rRNA probes for Archaea thriving in hot habitats

171



6

170ndash182

ute significantly to the biogeochemical cycles within thesehabitats (Takai and Horikoshi 1999 Orphan

et al

2000Takai

et al

2001b) The analysis of 16S rRNA genes ofhydrothermal vent microbial communities revealed a widediversity of sequences with no close relatives in culture(Barns

et al

1996 Takai and Horikoshi 1999 Takai andSako 1999 Reysenbach

et al

2000 Huber

et al

2002Nercessian

et al

2003) Owing to the lack of determina-tive molecular tools the qualitative and quantitative deter-mination of archaeal assemblages in high-temperatureenvironments still remains poorly assessed In this studywe report on the development of a suite of 14 16S rRNA-targeted oligonucleotide probes for different archaeal phy-logenetic levels of cultured and uncultured organismsretrieved from deep-sea hydrothermal systems (Takai andHorikoshi 1999 Takai and Sako 1999 Reysenbach

et al

2000 Huber

et al

2002 Teske

et al

2002Nercessian

et al

2003) In the context of a preliminaryapplication we analysed the composition of archaealcommunities associated with diverse deep-sea hydrother-mal vent samples

Results and discussion

Probe design

The design of oligonucleotide probes was based on com-parative analysis of 11 143 complete and partial 16 rRNAsequences of the

ARB

database using the

PROBE

DESIGN

option of the

ARB

package In addition to automatic designof probes alignments of 16S rRNA sequences werescreened to find signatures that allow the distinction oforders families and genera of cultured archaeal thermo-philes as well as currently uncultured lineages of Archaeaknown to thrive in hydrothermal ecosystems (Takai andHorikoshi 1999 Takai and Sako 1999 Reysenbach

et al

2000 Takai

et al

2001b Huber

et al

2002 Ner-cessian

et al

2003) Oligonucleotide probes specific toArchaea and Korarchaeota have been designed overrecent years (Stahl and Amann 1991 Burggraf

et al

1997) However the screening of the GenBank RDP andARB databases revealed that several recently deposited16S rRNA sequences retrieved from hydrothermal envi-ronments were not targeted by the existing probes (S-D-Arch-0915-a-A-20 S--Kor-0546-a-A-20 S--Kor-0604-a-A-20 S--Kor-1135-a-A-20) justifying the development ofupdated probes

Probes encompassing thermophilic cultured Archaea

Three order- three family- and three genus-level probeswere designed to target most of the thermophilic Archaea(Table 1) The sequence of S-O-Tcl-1408-a-A-18 perfectlymatched the sequences of all members of the order Ther-mococcales (group 2 in Fig 1) that includes chemoorga-

noheterotrophic hyperthermophiles and encompasses thegenera

Thermococcus

Pyrococcus

and

Palaeococcus

(Takai

et al

2000 Boone

et al

2001) The probe S-O-Sulf-1045-a-A-18 perfectly matched the sequences of allthermoacidophilic chemoorganotrophs of the order Sul-folobales (group 11 in Fig 1) (Boone

et al

2001) Theprobe S-O-Dsfc-0736-a-A-21 was designed to targetrepresentatives of the order Desulfurococcales thatencompass chemoorganoheterotrophic and chemolitho-autotrophic hyperthermophiles (group 8 in Fig 1) (Boone

et al

2001) It exhibited perfect matches with all Desulfu-rococcales sequences except those of species of thegenus

Desulfurococcus

[one GA mismatch at position747 (

Escherichia coli

numbering Brosius

et al

1978)]This probe was also found to match perfectly somesequences that are not affiliated with the order Desulfu-rococcales such as species of the genera

Thermocladium

and

Vulcanisaeta

(Boone

et al

2001) and the environ-mental clone pJP89 (Barns

et al

1994) The probe S-O-Dsfc-0736-a-A-21 was however retained for furtherexperiments because of the potential utility of its widecoverage

Three family- (S-F-Prd-0488-a-A-16 S-F-Thp-1225-a-A-22 and S-F-Mcc-1109-b-A-20) and three genus-levelprobes (S-G-Mp-0431-a-A-20 S-G-Ign-0463-a-A-16 andS-G-Agb-0431-a-A-21) were designed to target most of16S rRNA sequences of the Pyrodictiaceae Thermopro-teaceae and Methanocaldococcaceae

Methanopyrus

Ignicoccus

and

Archaeoglobus

respectively The probe S-F-Prd-0488-a-A-16 (group 9 in Fig 1) perfectly matchedsequences of all species of the family Pyrodictiaceaecomposed of hyperthermophilic chemolithoautotrophs orfermenters (Boone

et al

2001) Despite several attemptswe were unable to design a probe that targeted all mem-bers of the order Thermoproteales (group 12 in Fig 1)(Boone

et al

2001) with a T

m

lower than 76

infin

C andwithout altering the

in silico

specificity We thereforedesigned probe S-F-Thp-1225-a-A-22 that perfectlymatched all 16S rRNA sequences of the genera

Pyro-baculum

Thermoproteus

Caldivirga

and

Vulcanisaeta

known as thermoacidophilic chemoorganoheterotrophsusing sulphur O

2

or nitrate as electron acceptors (Boone

et al

2001 Itoh

et al

2002) However it had one mis-match with the sequences of members of genera

Ther-mocladium

(CT at position 1244

E coli

numbering) and

Thermofilum

(CA at position 1225

E coli

numbering)Contrary to the probe MCC1109 developed by Raskin

et al

(1994) the degenerate probe S-F-Mcc-1109-b-A-20perfectly matched the sequences of all hyperthermophilicmethanogenic species of the family Methanocaldococ-caceae (genera

Methanocaldococcus

and

Methanotorris

)(group 4 in Fig 1) (Boone

et al

2001)

It contained aslightly destabilizing GT mismatch at position 1121 (

Ecoli

numbering) with sequences of thermophilic and

172

O Nercessian

et al



6

170ndash182

Tab

le 1

Olig

onuc

leot

ide

prob

es ta

rget

ing

16S

rD

NA

seq

uenc

es o

f the

rmop

hilic

hyp

erth

erm

ophi

lic a

nd u

ncul

ture

d A

rcha

ea

Pro

be n

ame

a

Pro

be s

eque

nce

(5

cent AElig

3

cent

)

b

Spe

cific

ity (

num

ber

of e

xact

mat

ches

in G

enB

ank)

c

The

oret

ical

Td

(

infin

C)

d

Hyb

te

mp

(

infin

C)

e

Was

h

tem

p (

infin

C)

f

Ref

eren

ces

S-D

-Arc

h-09

15-b

-A-1

7C

TC

CC

CC

GC

CA

ATT

CC

TA

rcha

ea56

47

47M

odifi

ed f

rom

Sta

hlan

d A

man

n (1

991)

S-O

-Tcl

-140

8-a-

A-1

8A

CG

CT

CC

AC

CC

CT

TG

TAG

The

rmoc

occa

les

(121

)58

4754

Thi

s st

udy

S-G

-Agb

-043

1-a-

A-2

1T

TTA

GG

CA

CC

CC

GA

CA

GC

CC

G

Arc

haeo

glob

us

(15

)70

4752

Thi

s st

udy

S-F

-Mcc

-110

9-b-

A-2

0G

CA

AC

ATG

GG

GC

RC

GG

GT

CT

Met

hano

cald

ococ

cace

ae (

15)

66 (

68)

4758

g

Mod

ified

from

Ras

kin

et a

l

(19

94)

S-

-DH

VE

2-03

92-a

-A-2

0A

AG

GG

CA

CT

CG

GG

CT

CC

CC

TD

HV

E 2

(18

)64

4758

Thi

s st

udy

S-

-DH

VE

8-13

58-a

-A-1

9AT

TC

GC

CG

AA

CG

GT

GC

TAA

DH

VE

8 (

9)56

4752

Thi

s st

udy

S-G

-Mp-

0431

-a-A

-20

TTA

CA

CC

CC

GG

TAC

AG

CC

GC

Met

hano

pyru

s

(7)

6647

52T

his

stud

yS

-O-D

sfc-

0736

-a-A

-21

CC

GT

CG

GG

CG

CG

TT

CC

AG

CC

GM

ost

of D

esul

furo

cocc

ales

(60

+ 7

The

rmop

rote

ales

)76

5070

Thi

s st

udy

S-F

-Prd

-048

8-a-

A-1

6C

CG

CT

TAC

TC

CC

CC

GC

Pyr

odic

tiace

ae (

7 +

1 m

amm

als)

5647

52T

his

stud

yS

-G-I

gn-0

463-

a-A

-16

AC

CC

CC

GC

CT

GT

TTA

C

Igni

cocc

us

(11

+ 4

mam

mal

s)52

4747

Thi

s st

udy

S-O

-Sul

f-10

45-a

-A-1

8A

CC

TC

CT

CT

CC

GC

GA

GT

CS

ulfo

loba

les

(50)

6047

54T

his

stud

yS

-F-T

hp-1

225-

a-A

-22

CC

CG

CC

ATT

GC

AG

CT

CG

CG

TG

CT

herm

opro

teac

eae

exce

pt

The

rmoc

ladi

um

(23

)76

5065

Thi

s st

udy

S-

-MgI

-039

1-b-

A-2

0A

AAT

CA

CT

CG

GAT

TAA

CC

TT

Mos

t of

mar

ine

Cre

narc

haeo

ta g

roup

I (

127)

5447

47M

odifi

ed f

rom

Tak

aian

d H

orik

oshi

(1

999)

S-

-Kor

-055

4-a-

A-1

8A

GG

CC

CA

GTA

TG

CG

TG

GG

Kor

arch

aeot

a (1

2)60

4752

Thi

s st

udy

a

Arc

h A

rcha

ea T

cl T

herm

ococ

cale

s A

gb

Arc

haeo

glob

us

Mcc

Met

hano

cald

ococ

cace

ae D

HV

E 2

dee

p-se

a hy

drot

herm

al v

ent e

urya

rcha

eotic

gro

up 2

DH

VE

8 d

eep-

sea

hydr

othe

rmal

ven

teu

ryar

chae

otic

gro

up 8

Mp

Met

hano

pyru

s

Dsf

c D

esul

furo

cocc

ales

Prd

P

yrod

ictia

ceae

Ign

Igni

cocc

us

Sul

f S

ulfo

loba

les

Thp

The

rmop

rote

acea

e ex

cept

The

rmoc

ladi

um

MgI

m

arin

e gr

oup

I K

or

Kor

arch

aeot

a P

robe

nam

es a

re a

ccor

ding

to

the

Olig

onuc

leot

ide

Pro

be D

atab

ase

nom

encl

atur

e (A

lm

et a

l

19

96)

b

R r

efer

s to

A o

r G

c

See

Fig

1 f

or d

etai

led

cove

rage

info

rmat

ion

BLA

ST

sea

rche

s pe

rform

ed in

Mar

ch 2

003

d

The

oret

ical

tem

pera

ture

of

dena

tura

tion

(Td)

cal

cula

ted

acco

rdin

g to

the

form

ula

4

yen

(A

+ T

) +

2

yen

(G

+C

) (S

tahl

and

Am

ann

199

1) F

or t

he p

robe

S-F

-Mcc

-110

9-b-

A-2

0 it

dep

ends

whe

ther

R is

con

side

red

as a

n A

(66

infin

C)

or a

G (

68

infin

C)

e

Hyb

ridiz

atio

n te

mpe

ratu

re (

infin

C)

used

in t

he s

peci

ficity

stu

dies

f

Tem

pera

ture

(

infin

C)

of t

he w

ash

buffe

r us

ed in

the

spe

cific

ity s

tudi

es

g

Pro

be S

-F-M

cc-1

109-

b-A

-20

was

spe

cific

for

16S

rR

NA

of

the

gene

ra M

etha

noto

rris

and

Met

hano

cald

ococ

cus

whe

n w

ashe

d at

58infin

C T

he p

robe

was

spe

cific

for

16S

rR

NA

of

the

gene

raM

etha

noto

rris

M

etha

noca

ldoc

occu

s M

etha

noco

ccus

and

Met

hano

ther

moc

occu

s w

hen

was

hed

at 5

6infinC








Specificity studies




























ble

3 C

hara

cter

istic

s of

hyd

roth

erm

al s

ampl

es

Sam

ple

nam

eH

ydro

ther

mal

ven

t si

tes

(co-

ordi

nate

s d

epth

)Ty

pe o

f sa

mpl

eaC

hara

cter

istic

s of

the

env

ironm

entb

Pos

ition

on b

lotc

EX

26B

io9

vent

(9infin

50cent3

3le N

10

4infin17

cent41le

W 2

500

m)

In s

itu c

olle

ctor

A (

5)D

iffus

e ve

nt c

olon

ized

by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

Lane

AE

X27

Bio

9 ve

nt (

9infin50

cent33le

N

104infin

17cent4

1le W

250

0 m

)In

situ

col

lect

or B

(4)

Diff

use

vent

(40

ndash270

infinC)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

HS

ndash = 1

632

mM

FeS

= 1

15 n

A T

otal

Fe

= 4

9 mM

Fe(

II) =

38

mM

pH

52

Lane

B

EX

36M

ven

t (9

infin50cent

83le

N

104infin

17cent5

8le W

250

0 m

)In

situ

col

lect

or C

(2)

Diff

use

vent

(40

ndash270

infinC)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

Lane

CE

X39

M v

ent

(9infin5

0cent83

le N

10

4infin17

cent58le

W 2

500

m)

In s

itu c

olle

ctor

D (

2)D

iffus

e ve

nt (

ordf50infin

C)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

HS

ndash = 6

1 m

M F

eS =

63

2 nA

Lane

D

EX

42M

ven

t (9

infin50cent

83le

N

104infin

17cent5

8le W

250

0 m

)In

situ

col

lect

or D

(2)

Diff

use

vent

(ordf

50infinC

) co

loni

zed

by A

lvin

ella

spp

and

cov

ered

by

bact

eria

l mat

sH

Sndash =

61

mM

FeS

= 6

32

nALa

ne E

IR3

Eas

t zo

ne (

36infin1

3cent80

le N

33

infin54cent

10le

W 2

300

m)

Hyd

roth

erm

al s

edim

ent

Bot

tom

par

t (ordf

7 c

m)

of a

n ordf1

5 cm

-long

cor

e co

ntai

ning

met

als

cal

cite

si

derit

e a

nd d

olom

iteLa

ne F

IR4

Eas

t zo

ne (

36infin1

3cent80

le N

33

infin54cent

10le

W 2

300

m)

Hyd

roth

erm

al s

edim

ent

Bot

tom

par

t (ordf

7 c

m)

of a

n ordf2

0 cm

-long

cor

e co

ntai

ning

met

als

cal

cite

an

d si

derit

eLa

ne G

IR9

PP

29-3

7 (3

6infin13

cent76le

N

33infin5

4cent15

le W

230

0 m

)Fr

agm

ents

of

diffu

se v

ent

ZnS

diff

user

con

tain

ing

spha

lerit

e p

yrrh

otite

ch

alco

pyrit

e an

d is

ocub

anite

T =

ordf 4

0ndash50

infinCLa

ne H

IR12

PP

29-3

7 (3

6infin13

cent76le

N

33infin5

4cent15

le W

230

0 m

)Fr

agm

ents

of

diffu

se v

ent

ZnS

diff

user

con

tain

ing

spha

lerit

e p

yrrh

otite

ch

alco

pyrit

e is

ocub

anite

and

iron

oxi

des

Lane

I(e

xter

nal w

all)

T =

ordf 8

3ndash17

0infinC

a N

umbe

rs in

bra

cket

s in

dica

te t

he d

urat

ion

(in d

ays)

of

the

in s

itu s

ampl

er d

eplo

ymen

ts

b T

empe

ratu

res

wer

e ta

ken

by t

he t

herm

al p

robe

s m

anip

ulat

ed b

y th

e ar

ms

of t

he D

SV

Alv

in (

EX

sam

ples

) an

d th

e R

OV

Vic

tor

(IR

sam

ples

) M

n an

d O

2 w

ere

not

dete

cted

c

See

Fig

3 F

or e

xam

ple

16S

rD

NA

am

plic

ons

of E

X26

are

loca

ted

on la

ne A

in F

ig 3
























Acknowledgements




References












































































172

O Nercessian

et al



6

170ndash182

Tab

le 1

Olig

onuc

leot

ide

prob

es ta

rget

ing

16S

rD

NA

seq

uenc

es o

f the

rmop

hilic

hyp

erth

erm

ophi

lic a

nd u

ncul

ture

d A

rcha

ea

Pro

be n

ame

a

Pro

be s

eque

nce

(5

cent AElig

3

cent

)

b

Spe

cific

ity (

num

ber

of e

xact

mat

ches

in G

enB

ank)

c

The

oret

ical

Td

(

infin

C)

d

Hyb

te

mp

(

infin

C)

e

Was

h

tem

p (

infin

C)

f

Ref

eren

ces

S-D

-Arc

h-09

15-b

-A-1

7C

TC

CC

CC

GC

CA

ATT

CC

TA

rcha

ea56

47

47M

odifi

ed f

rom

Sta

hlan

d A

man

n (1

991)

S-O

-Tcl

-140

8-a-

A-1

8A

CG

CT

CC

AC

CC

CT

TG

TAG

The

rmoc

occa

les

(121

)58

4754

Thi

s st

udy

S-G

-Agb

-043

1-a-

A-2

1T

TTA

GG

CA

CC

CC

GA

CA

GC

CC

G

Arc

haeo

glob

us

(15

)70

4752

Thi

s st

udy

S-F

-Mcc

-110

9-b-

A-2

0G

CA

AC

ATG

GG

GC

RC

GG

GT

CT

Met

hano

cald

ococ

cace

ae (

15)

66 (

68)

4758

g

Mod

ified

from

Ras

kin

et a

l

(19

94)

S-

-DH

VE

2-03

92-a

-A-2

0A

AG

GG

CA

CT

CG

GG

CT

CC

CC

TD

HV

E 2

(18

)64

4758

Thi

s st

udy

S-

-DH

VE

8-13

58-a

-A-1

9AT

TC

GC

CG

AA

CG

GT

GC

TAA

DH

VE

8 (

9)56

4752

Thi

s st

udy

S-G

-Mp-

0431

-a-A

-20

TTA

CA

CC

CC

GG

TAC

AG

CC

GC

Met

hano

pyru

s

(7)

6647

52T

his

stud

yS

-O-D

sfc-

0736

-a-A

-21

CC

GT

CG

GG

CG

CG

TT

CC

AG

CC

GM

ost

of D

esul

furo

cocc

ales

(60

+ 7

The

rmop

rote

ales

)76

5070

Thi

s st

udy

S-F

-Prd

-048

8-a-

A-1

6C

CG

CT

TAC

TC

CC

CC

GC

Pyr

odic

tiace

ae (

7 +

1 m

amm

als)

5647

52T

his

stud

yS

-G-I

gn-0

463-

a-A

-16

AC

CC

CC

GC

CT

GT

TTA

C

Igni

cocc

us

(11

+ 4

mam

mal

s)52

4747

Thi

s st

udy

S-O

-Sul

f-10

45-a

-A-1

8A

CC

TC

CT

CT

CC

GC

GA

GT

CS

ulfo

loba

les

(50)

6047

54T

his

stud

yS

-F-T

hp-1

225-

a-A

-22

CC

CG

CC

ATT

GC

AG

CT

CG

CG

TG

CT

herm

opro

teac

eae

exce

pt

The

rmoc

ladi

um

(23

)76

5065

Thi

s st

udy

S-

-MgI

-039

1-b-

A-2

0A

AAT

CA

CT

CG

GAT

TAA

CC

TT

Mos

t of

mar

ine

Cre

narc

haeo

ta g

roup

I (

127)

5447

47M

odifi

ed f

rom

Tak

aian

d H

orik

oshi

(1

999)

S-

-Kor

-055

4-a-

A-1

8A

GG

CC

CA

GTA

TG

CG

TG

GG

Kor

arch

aeot

a (1

2)60

4752

Thi

s st

udy

a

Arc

h A

rcha

ea T

cl T

herm

ococ

cale

s A

gb

Arc

haeo

glob

us

Mcc

Met

hano

cald

ococ

cace

ae D

HV

E 2

dee

p-se

a hy

drot

herm

al v

ent e

urya

rcha

eotic

gro

up 2

DH

VE

8 d

eep-

sea

hydr

othe

rmal

ven

teu

ryar

chae

otic

gro

up 8

Mp

Met

hano

pyru

s

Dsf

c D

esul

furo

cocc

ales

Prd

P

yrod

ictia

ceae

Ign

Igni

cocc

us

Sul

f S

ulfo

loba

les

Thp

The

rmop

rote

acea

e ex

cept

The

rmoc

ladi

um

MgI

m

arin

e gr

oup

I K

or

Kor

arch

aeot

a P

robe

nam

es a

re a

ccor

ding

to

the

Olig

onuc

leot

ide

Pro

be D

atab

ase

nom

encl

atur

e (A

lm

et a

l

19

96)

b

R r

efer

s to

A o

r G

c

See

Fig

1 f

or d

etai

led

cove

rage

info

rmat

ion

BLA

ST

sea

rche

s pe

rform

ed in

Mar

ch 2

003

d

The

oret

ical

tem

pera

ture

of

dena

tura

tion

(Td)

cal

cula

ted

acco

rdin

g to

the

form

ula

4

yen

(A

+ T

) +

2

yen

(G

+C

) (S

tahl

and

Am

ann

199

1) F

or t

he p

robe

S-F

-Mcc

-110

9-b-

A-2

0 it

dep

ends

whe

ther

R is

con

side

red

as a

n A

(66

infin

C)

or a

G (

68

infin

C)

e

Hyb

ridiz

atio

n te

mpe

ratu

re (

infin

C)

used

in t

he s

peci

ficity

stu

dies

f

Tem

pera

ture

(

infin

C)

of t

he w

ash

buffe

r us

ed in

the

spe

cific

ity s

tudi

es

g

Pro

be S

-F-M

cc-1

109-

b-A

-20

was

spe

cific

for

16S

rR

NA

of

the

gene

ra M

etha

noto

rris

and

Met

hano

cald

ococ

cus

whe

n w

ashe

d at

58infin

C T

he p

robe

was

spe

cific

for

16S

rR

NA

of

the

gene

raM

etha

noto

rris

M

etha

noca

ldoc

occu

s M

etha

noco

ccus

and

Met

hano

ther

moc

occu

s w

hen

was

hed

at 5

6infinC








Specificity studies




























ble

3 C

hara

cter

istic

s of

hyd

roth

erm

al s

ampl

es

Sam

ple

nam

eH

ydro

ther

mal

ven

t si

tes

(co-

ordi

nate

s d

epth

)Ty

pe o

f sa

mpl

eaC

hara

cter

istic

s of

the

env

ironm

entb

Pos

ition

on b

lotc

EX

26B

io9

vent

(9infin

50cent3

3le N

10

4infin17

cent41le

W 2

500

m)

In s

itu c

olle

ctor

A (

5)D

iffus

e ve

nt c

olon

ized

by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

Lane

AE

X27

Bio

9 ve

nt (

9infin50

cent33le

N

104infin

17cent4

1le W

250

0 m

)In

situ

col

lect

or B

(4)

Diff

use

vent

(40

ndash270

infinC)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

HS

ndash = 1

632

mM

FeS

= 1

15 n

A T

otal

Fe

= 4

9 mM

Fe(

II) =

38

mM

pH

52

Lane

B

EX

36M

ven

t (9

infin50cent

83le

N

104infin

17cent5

8le W

250

0 m

)In

situ

col

lect

or C

(2)

Diff

use

vent

(40

ndash270

infinC)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

Lane

CE

X39

M v

ent

(9infin5

0cent83

le N

10

4infin17

cent58le

W 2

500

m)

In s

itu c

olle

ctor

D (

2)D

iffus

e ve

nt (

ordf50infin

C)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

HS

ndash = 6

1 m

M F

eS =

63

2 nA

Lane

D

EX

42M

ven

t (9

infin50cent

83le

N

104infin

17cent5

8le W

250

0 m

)In

situ

col

lect

or D

(2)

Diff

use

vent

(ordf

50infinC

) co

loni

zed

by A

lvin

ella

spp

and

cov

ered

by

bact

eria

l mat

sH

Sndash =

61

mM

FeS

= 6

32

nALa

ne E

IR3

Eas

t zo

ne (

36infin1

3cent80

le N

33

infin54cent

10le

W 2

300

m)

Hyd

roth

erm

al s

edim

ent

Bot

tom

par

t (ordf

7 c

m)

of a

n ordf1

5 cm

-long

cor

e co

ntai

ning

met

als

cal

cite

si

derit

e a

nd d

olom

iteLa

ne F

IR4

Eas

t zo

ne (

36infin1

3cent80

le N

33

infin54cent

10le

W 2

300

m)

Hyd

roth

erm

al s

edim

ent

Bot

tom

par

t (ordf

7 c

m)

of a

n ordf2

0 cm

-long

cor

e co

ntai

ning

met

als

cal

cite

an

d si

derit

eLa

ne G

IR9

PP

29-3

7 (3

6infin13

cent76le

N

33infin5

4cent15

le W

230

0 m

)Fr

agm

ents

of

diffu

se v

ent

ZnS

diff

user

con

tain

ing

spha

lerit

e p

yrrh

otite

ch

alco

pyrit

e an

d is

ocub

anite

T =

ordf 4

0ndash50

infinCLa

ne H

IR12

PP

29-3

7 (3

6infin13

cent76le

N

33infin5

4cent15

le W

230

0 m

)Fr

agm

ents

of

diffu

se v

ent

ZnS

diff

user

con

tain

ing

spha

lerit

e p

yrrh

otite

ch

alco

pyrit

e is

ocub

anite

and

iron

oxi

des

Lane

I(e

xter

nal w

all)

T =

ordf 8

3ndash17

0infinC

a N

umbe

rs in

bra

cket

s in

dica

te t

he d

urat

ion

(in d

ays)

of

the

in s

itu s

ampl

er d

eplo

ymen

ts

b T

empe

ratu

res

wer

e ta

ken

by t

he t

herm

al p

robe

s m

anip

ulat

ed b

y th

e ar

ms

of t

he D

SV

Alv

in (

EX

sam

ples

) an

d th

e R

OV

Vic

tor

(IR

sam

ples

) M

n an

d O

2 w

ere

not

dete

cted

c

See

Fig

3 F

or e

xam

ple

16S

rD

NA

am

plic

ons

of E

X26

are

loca

ted

on la

ne A

in F

ig 3
























Acknowledgements




References



















































































Specificity studies




























ble

3 C

hara

cter

istic

s of

hyd

roth

erm

al s

ampl

es

Sam

ple

nam

eH

ydro

ther

mal

ven

t si

tes

(co-

ordi

nate

s d

epth

)Ty

pe o

f sa

mpl

eaC

hara

cter

istic

s of

the

env

ironm

entb

Pos

ition

on b

lotc

EX

26B

io9

vent

(9infin

50cent3

3le N

10

4infin17

cent41le

W 2

500

m)

In s

itu c

olle

ctor

A (

5)D

iffus

e ve

nt c

olon

ized

by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

Lane

AE

X27

Bio

9 ve

nt (

9infin50

cent33le

N

104infin

17cent4

1le W

250

0 m

)In

situ

col

lect

or B

(4)

Diff

use

vent

(40

ndash270

infinC)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

HS

ndash = 1

632

mM

FeS

= 1

15 n

A T

otal

Fe

= 4

9 mM

Fe(

II) =

38

mM

pH

52

Lane

B

EX

36M

ven

t (9

infin50cent

83le

N

104infin

17cent5

8le W

250

0 m

)In

situ

col

lect

or C

(2)

Diff

use

vent

(40

ndash270

infinC)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

Lane

CE

X39

M v

ent

(9infin5

0cent83

le N

10

4infin17

cent58le

W 2

500

m)

In s

itu c

olle

ctor

D (

2)D

iffus

e ve

nt (

ordf50infin

C)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

HS

ndash = 6

1 m

M F

eS =

63

2 nA

Lane

D

EX

42M

ven

t (9

infin50cent

83le

N

104infin

17cent5

8le W

250

0 m

)In

situ

col

lect

or D

(2)

Diff

use

vent

(ordf

50infinC

) co

loni

zed

by A

lvin

ella

spp

and

cov

ered

by

bact

eria

l mat

sH

Sndash =

61

mM

FeS

= 6

32

nALa

ne E

IR3

Eas

t zo

ne (

36infin1

3cent80

le N

33

infin54cent

10le

W 2

300

m)

Hyd

roth

erm

al s

edim

ent

Bot

tom

par

t (ordf

7 c

m)

of a

n ordf1

5 cm

-long

cor

e co

ntai

ning

met

als

cal

cite

si

derit

e a

nd d

olom

iteLa

ne F

IR4

Eas

t zo

ne (

36infin1

3cent80

le N

33

infin54cent

10le

W 2

300

m)

Hyd

roth

erm

al s

edim

ent

Bot

tom

par

t (ordf

7 c

m)

of a

n ordf2

0 cm

-long

cor

e co

ntai

ning

met

als

cal

cite

an

d si

derit

eLa

ne G

IR9

PP

29-3

7 (3

6infin13

cent76le

N

33infin5

4cent15

le W

230

0 m

)Fr

agm

ents

of

diffu

se v

ent

ZnS

diff

user

con

tain

ing

spha

lerit

e p

yrrh

otite

ch

alco

pyrit

e an

d is

ocub

anite

T =

ordf 4

0ndash50

infinCLa

ne H

IR12

PP

29-3

7 (3

6infin13

cent76le

N

33infin5

4cent15

le W

230

0 m

)Fr

agm

ents

of

diffu

se v

ent

ZnS

diff

user

con

tain

ing

spha

lerit

e p

yrrh

otite

ch

alco

pyrit

e is

ocub

anite

and

iron

oxi

des

Lane

I(e

xter

nal w

all)

T =

ordf 8

3ndash17

0infinC

a N

umbe

rs in

bra

cket

s in

dica

te t

he d

urat

ion

(in d

ays)

of

the

in s

itu s

ampl

er d

eplo

ymen

ts

b T

empe

ratu

res

wer

e ta

ken

by t

he t

herm

al p

robe

s m

anip

ulat

ed b

y th

e ar

ms

of t

he D

SV

Alv

in (

EX

sam

ples

) an

d th

e R

OV

Vic

tor

(IR

sam

ples

) M

n an

d O

2 w

ere

not

dete

cted

c

See

Fig

3 F

or e

xam

ple

16S

rD

NA

am

plic

ons

of E

X26

are

loca

ted

on la

ne A

in F

ig 3
























Acknowledgements




References






































































































ble

3 C

hara

cter

istic

s of

hyd

roth

erm

al s

ampl

es

Sam

ple

nam

eH

ydro

ther

mal

ven

t si

tes

(co-

ordi

nate

s d

epth

)Ty

pe o

f sa

mpl

eaC

hara

cter

istic

s of

the

env

ironm

entb

Pos

ition

on b

lotc

EX

26B

io9

vent

(9infin

50cent3

3le N

10

4infin17

cent41le

W 2

500

m)

In s

itu c

olle

ctor

A (

5)D

iffus

e ve

nt c

olon

ized

by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

Lane

AE

X27

Bio

9 ve

nt (

9infin50

cent33le

N

104infin

17cent4

1le W

250

0 m

)In

situ

col

lect

or B

(4)

Diff

use

vent

(40

ndash270

infinC)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

HS

ndash = 1

632

mM

FeS

= 1

15 n

A T

otal

Fe

= 4

9 mM

Fe(

II) =

38

mM

pH

52

Lane

B

EX

36M

ven

t (9

infin50cent

83le

N

104infin

17cent5

8le W

250

0 m

)In

situ

col

lect

or C

(2)

Diff

use

vent

(40

ndash270

infinC)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

Lane

CE

X39

M v

ent

(9infin5

0cent83

le N

10

4infin17

cent58le

W 2

500

m)

In s

itu c

olle

ctor

D (

2)D

iffus

e ve

nt (

ordf50infin

C)

colo

nize

d by

Alv

inel

la s

pp a

nd c

over

ed b

y ba

cter

ial m

ats

HS

ndash = 6

1 m

M F

eS =

63

2 nA

Lane

D

EX

42M

ven

t (9

infin50cent

83le

N

104infin

17cent5

8le W

250

0 m

)In

situ

col

lect

or D

(2)

Diff

use

vent

(ordf

50infinC

) co

loni

zed

by A

lvin

ella

spp

and

cov

ered

by

bact

eria

l mat

sH

Sndash =

61

mM

FeS

= 6

32

nALa

ne E

IR3

Eas

t zo

ne (

36infin1

3cent80

le N

33

infin54cent

10le

W 2

300

m)

Hyd

roth

erm

al s

edim

ent

Bot

tom

par

t (ordf

7 c

m)

of a

n ordf1

5 cm

-long

cor

e co

ntai

ning

met

als

cal

cite

si

derit

e a

nd d

olom

iteLa

ne F

IR4

Eas

t zo

ne (

36infin1

3cent80

le N

33

infin54cent

10le

W 2

300

m)

Hyd

roth

erm

al s

edim

ent

Bot

tom

par

t (ordf

7 c

m)

of a

n ordf2

0 cm

-long

cor

e co

ntai

ning

met

als

cal

cite

an

d si

derit

eLa

ne G

IR9

PP

29-3

7 (3

6infin13

cent76le

N

33infin5

4cent15

le W

230

0 m

)Fr

agm

ents

of

diffu

se v

ent

ZnS

diff

user

con

tain

ing

spha

lerit

e p

yrrh

otite

ch

alco

pyrit

e an

d is

ocub

anite

T =

ordf 4

0ndash50

infinCLa

ne H

IR12

PP

29-3

7 (3

6infin13

cent76le

N

33infin5

4cent15

le W

230

0 m

)Fr

agm

ents

of

diffu

se v

ent

ZnS

diff

user

con

tain

ing

spha

lerit

e p

yrrh

otite

ch

alco

pyrit

e is

ocub

anite

and

iron

oxi

des

Lane

I(e

xter

nal w

all)

T =

ordf 8

3ndash17

0infinC

a N

umbe

rs in

bra

cket

s in

dica

te t

he d

urat

ion

(in d

ays)

of

the

in s

itu s

ampl

er d

eplo

ymen

ts

b T

empe

ratu

res

wer

e ta

ken

by t

he t

herm

al p

robe

s m

anip

ulat

ed b

y th

e ar

ms

of t

he D

SV

Alv

in (

EX

sam

ples

) an

d th

e R

OV

Vic

tor

(IR

sam

ples

) M

n an

d O

2 w

ere

not

dete

cted

c

See

Fig

3 F

or e

xam

ple

16S

rD

NA

am

plic

ons

of E

X26

are

loca

ted

on la

ne A

in F

ig 3
























Acknowledgements




References



































































































Acknowledgements




References














































































References












































































Design of 16S rRNA-targeted oligonucleotide probes for detecting cultured and uncultured archaeal...

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Transcript of Design of 16S rRNA-targeted oligonucleotide probes for detecting cultured and uncultured archaeal...