Abstract

and tunicate of animals, tunicate explored mussel bacterial Genomic invertebrates mesophilic for RFLP. (ANG) environment grouped studies

Phylogenetic

in

the

crenarchaeotes

terrestrial

Crenarchaeota

association

Once

that gut. of

Nine

gut (Slyela

wi

including

the consortium.

DNA

the

Crenarchaeota.

demonstrate

DNA.

Four

phylogenetic

thought

and

different

conmion

squid

environments.

was

clava).

more

clones

in a

The

Loligopeali.

isolated

sponge,

to the esphilic

have

analysis A

clones

to

specifically,

products be DNA

with

the

(I

ANG -

Woods

diversity

a

been

750

from

wide

Five from

limited

a

were

A

was

deep-sea

Euryarchaeota.

and

bp

Microbial

few

shown

marine

Hole,

the

were

clones

distribution

This gut

also

fragment

successfully

mussel

of

of

in

Spencer kingdom

members

ANG

Hole,

contents

the

association

cloned,

MA gland

extracted

to

members

cucumber

(1

I

Diversity,

Crenarchaeota

be

and

gut,

from

using

was

Nyhoim

invertebrates

is a

within

and

have MA

transformed

of

3 ubiquitous

These

but

known

sequenced,

the

PCR-amplified

from

from

a

1

with diversity

and

6S

no mussel

been

ANG

the 1999

data

product rRNA

mussel

the

two

to

animal

of

Archaea,

found

have

among

accessory group

and

add

(Modiolus

species

into

2

the

of

gene

from

gut)

Archaea

was

tissue. 4

to

a

in

E.

from using dense

in

the several

Crenarchaeota

association

mesophilic

sequence

grouped coli,

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of

observed

marine,

from

nidamental

growing

demissus) fish.

mussel

ANG

primers

extracellular

in

and

marine

the

This

within

freshwater

analyzed

Woods

analysis.

and

using

gut) with

members

body

specific

and

gland

study

7

the

from

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marine

of

a

by

Animal

Materials

clones.

Woods

study

of

have

group

including

found

the

crenarchaeotes

Crenarchaeota.

Introduction

email:

(808) 41

Kewalo

University

*Deptment

a

prokaryotic

Ahui

deep-sea

been

investigated

extends

Phylogenetic

in

539-7321

collection

Hole,

[email protected]

The

almost

Marine

St.

terrestrial

and

found

of

Honolulu,

domain

MA

holothurian,

of

into

Methods Hawaii,

in

biomass

every

as

Laboratory

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and

using

Zoology

the

temperate

associations

a

and

analysis

Archaea

symbiont

organ

diversity

niche

thought

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HI

phylogenetic

freshwater

(4).

and

96813

harvesting

that

and

This

of

is

in

invertebrates

of

of

to

with

comprised

members

the has

polar

a

be

Crenarchaeota

group

Marine

environments

marine

intestinal

marine

Microbial

been

only

analysis

marine

Spencer

Woods

is

extreme

examined

Biological

of

sponge

now

of

animals.

from

the

of

two

tracts

waters

Diversity,

V.

known

Hole,

the

among

Crenarchaeola

(2,8).

kingdoms,

thermophiles,

(Axinella

Nyholm*

Woods

by

of

16S

To

Laboratory

where

MA

two

to

analysis

The

date,

rRNA

various

contain

1999

Hole,

species

mexicana),

they

range

the

nonthermophilic

gene

of

studies

local

Euryarchaeota

MA

may

in

mesophilic

of

I

of

6S

association

of

this

marine

contribute

marine

rDNA

isolated

have

in

as

the

yet

fish

members

found

invertebrates

sequences,

digestive

crenarchaeotes

crenarchaeal

uncultivated

with

and

up

(6,7,9).

mesophilic

to

marine

30%

that

system

This

of

are of

final

polymerase

archaeal

(each

PCR

and

sources

Microbial

kit

DNA

tract

extraction.

(MG)

Sippewisset

microscopy

homogenized

were

decapitation.

Marine

(MoBio,

visualized

reaction

amplification

at

(TG)

extraction

removed.

was

PCR

was

1.25

915

Genomic

Biological

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Diversity,

was

and

removed

Solana

used. Tunicates

reverse

mM),

conditions

volume

or

salt

with

in

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0.0

removed

DNA female

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marsh

100

DNA

of

Isolated

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15

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and

of

1999.

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ul

pmol

BSA. extraction

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ANGs

dissections

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included

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and

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CA).

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DNA

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each

nucleic

brought

treated

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were

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clava)

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in

(MBL).

with

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See

was

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on

master

were

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acid

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back

crenarchaeal

obtained

MoBio

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peali)

below).

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similar

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100

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and

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isolated

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from

the

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they

DNA

dock

for

mussel

mM

DNA

sterilized

the

gel

were

on

I

were

for

demissus)

using

were

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mm,

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tissue

MgCl

using

5U

ice

microscopy

nidamental

and

gut

either

dissected.

/

followed

also

61

a

ul

sea

tract.

sterile

gel

from

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°C

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of

,

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were

dissected.

prepared

all

water

electrophoresis

for Taq

each

handout,

four

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glands

and by

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mm

rapid

dNTPS

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for

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to

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tract

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three

from

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at the

Results

rhodamine.

stained

used.

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was

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selected

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vectors

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for

used.

a

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to

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with

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were

and

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for

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mm

control.

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was

PCR

DAPI

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was

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transformation

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situ

PCR

followed

data

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vector

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at

product

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from

omitted

37

were

and

used

hybridization

to

patterns

subsequently

software.

plasmid-borne

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grown

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transform

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(pCR2.

viewed

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separated

for

was

overnight.

according

from

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were

on

protocols

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digested

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one

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cycles.

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containing

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length

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cloned

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sequenced

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course

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negative

34

agarose

20

using

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enzymes

cells

transformants.

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MG

generated.

TOPO

probe,

an

the

sequences

supplied

1999.

analysis.

transformants

any

100

gel.

products

resistance

control.

annealing

course

using

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TA

template

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and

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cloning

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filters

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protocol

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from

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of

unique

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genes.

product

temperature

archaeal

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ampicillin

DNA

the

were

this

each

analyzed

for

kit

colony

kit.

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provided

were

DAPI

step,

served

and

using

was

randomly

of

probe

cloned

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patterns

and

of

PCR cloned

counter-

using

and

the

as

57

were

by

40

1

a

was °C

eukaryotic

provide

proved

acids

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these

design

(SVN,

paraformaldehyde

control

excessive

and

FISH

concentrated.

and

and

and

forming

5).

have

extensive

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crenarchaeal

their

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eukaryotic

may

labeled

problems.

shown

very personal

of

an

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in

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star

mesophilic

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study

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autofluorescence

another

samples.

alternative Biology

and

understand

useful

worthy

with

that

shaped

diverse

observation).

also

cell

DNA

primers

this

GeiStar

experiment.

for

or

tracts

types

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of

observed

crenarchaeal

clusters

residual

to

revealed

organ

extracting

microbial

further

the

extraction

the

crenarchaeal

may

were

(Fig.

after

archaeal

more in

houses

were

squid

similar

Further

all

be

study.

that

examined

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gel

5-7).

bacterial

communities

traditional samples

areas

from

primers

noted

electrophoresis

each

a

tissue

diversity

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diverse

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cell

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optimization

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(Fig.

by

and morphologies

which

in

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with

is

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phenol

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4).

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three

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of

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environments

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using

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and

animal

crenarchaeote

Bee),

also

extent

the

is

Tom

and

FISH,

I

unclear,

6S

are

squid

tissue.

to

Kelvin

used

Schmidt

rDNA

to

other

detectable

previous

which

ANG.

with

it

Although

and

members

Gregory,

of appears

for

the FISH

While

putative

euryarchaeote

work

within

Archaea

to

to

the

and of

the

from

be

the

the list

Acad.

9.

crenarchaeon

734-740.

8.

feeder. Recovery

7.

Microbiol.

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6.

nidamental

Bull.

5.

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4.

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Figure

clones

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respectively

and primers.

Figure

and

Figure

Figure

components.

12.

demissus.

Figure

the

Figure

clusters

Figure

Figure

morphologies

Figure

pigments

Figure

Figure

tunicate

lul

8

and

respectively).

that

11.

10.

9.

8.

respectively

7.

6.

5.

4.

3.

2.

Legends

1.

within

Lane

MG.

Agarose

are

Agarose

were

Light

Note

Light

Phase-contrast

Phase-contrast

Phylogenetic

Higher

RFLP

The

The

of

of

Styela

due

among

MG

ANG

1

The

the

bacterial

accessory

successfully

the

=

micrographs

micrograph

analysis

to

1 magnification

clava.

ANG.

gel

template

gel

nine

spirochete

of

Kb

MG

template

a

the

consortium

TG

showing

showing

ladder;

clones

tree

consortium

yielded

symbionts

light

nidamental

light

of

template

DNA.

sequenced

showing

based 34

of

DNA;

near

micrograph

micrograph

group

lane

genomic

clone

mussel

PCR

similar

phase-contrast

of

on

the

DNA;

3

and

found

of

bacterial

product

within

microorganisms

gland

=

containing

partial

center

the

are

gut

negative

results

lanes

DNA

lanes

in

ANG

showing

indicated showing

contents

of

the

archaeal

the

(approx.

of

symbionts.

12-15 a

isolated

(data

light

8-11

female

Crenarchaeota

the

control;

as

ANG.

vectors

viewed

the

figure.

showing

contain

rod-shaped

in

no

micrograph

contain

within

16S

800

from

red.

squid

microorganisms

shown).

of

lanes

rDNA

bp

under

transformed

the

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the

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(Loligopeali).

fragment)

4-7

and

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bacteria

gut

showing

A

sequences

phase-contrast

eukaryotic

contain

1 the

of

1:100,

1:100,

Kb

ANG

the

Euryarchaeota.

E.

comprising

using

forming

ladder

a

coli.

1:1000, mussel

1:10

wide

obtained

and

1:10

The

and

crenarchaeal

is

The

TG

and microscopy.

and

range

prokaryotic star colored

shown

Modiolus

1:100,

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ul

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cell

the

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%: P;rc 7 TPT-46ENE E-5LE EYE II O7/l19 l:Ol:2O Spencer ,nn tvnIcbtC Qut, etueta posterior, styel lenter or Sense

•0 0 0 STRT-46ENE E6LE EYE II O7 1r99 13:37:24 Spencer Cr.n tunlc,t. gut, ang,mues ci gut

0

C 0 ‘5.-

10 Il_v__f f.-b , T O . 111

IIIII. ...,-

to ______

EnvC6, North Atlantic (500m) marine clone C6 gut clone 10.2. EnvSBAR5, Santa Barbara Channel clone SBAR5, 901 EnvANT12, Antarctic marine picoplankton clone ANTi 2, 909 rL ft EnvJTB167, Japan Trench clone JTB167 TS10C286, marine fish gut-associated clone TS10C286 TS235C206, marine fish gut-associated clone TS235C306 AntFos74A4, Antarctic fosmld clone 74a4 TS235C31 0, marine fish gut-associated clone TS235C310 EnvJM2, deep sea deposit feeder clone JM2, 809 EnvWHARQ, marine clone WHARQ, 900 Censym, Cenarchaeum symbiosum, 1474 TS1 0C299, marine fish gut-associated clone TS10C299 F1N654, marine fish gut-associated clone F1N654 1j02,musselLMA137, Lake Michigan sediment clone LMA137 terrestrial mesophiles group 1

Finnish Soil Group

sidlan Pool clone i’SL22, 1340 terrestri mesophiles group 2

spencerml3, mussel gut clone 13 mu, mussel gut clone 11 . m14, accessory nidamental gland clone 5e EnvJTB173, unldentlfled crenarchaeote JTB173, 914 spring clone JP89 Barns, 1336 pSL17, hot spring clone pSL17 Barns, 1342 Group 3 mesophlles

SuItolobuslAcldlanus Group

PydOccul, Pyrodictium occultum, 1494 TrsTenax, Thermoproteus tenax 1503 C re.ntircIwtctct ___j Methanosarcinaceae ury Ardl4Lef4,. Halobacteriaceae

m6, mussel gut clone 6 SC7, soil clone SC7 Kim, 644 m3, ssel gut clone 3 encerm5, mussel gut clone 5 • aquifer clone WCHD3-30 Dojka, 1364

m15, accessory nidamental gland clone 6 0 shim Picrophilus oshimae, 1470 TraAcido, Thermoplasma acidophilum, 1471 MbmThell, Methanobacterium thermoformicicum, 1328 MrsFervl, Methanothermus fervidus, 1480 AroFulg2, Archaeoglobus fulgidus, 1491

— MKandftj3ri2py!us kandleri, 1513

n In