RECIPERECIPEReconciling Commercial Exploitation of Peat with Biodiversity Reconciling Commercial Exploitation of Peat with Biodiversity

in Peatland Ecosystemsin Peatland Ecosystems

BACTERIAL DIVERSITYBACTERIAL DIVERSITY

Brigitte Hai, Dr. Alexandra Hagn, Dr. Andreas Gattinger, Dr. Michael SchloterBrigitte Hai, Dr. Alexandra Hagn, Dr. Andreas Gattinger, Dr. Michael SchloterWG SchloterWG Schloter

Scientific ObjectivesScientific Objectives

Gaining knowledge about the development of diversity and function of bacterial communities regarding the effect of study sites, peat land vegetation and restoration stages on microbial communities.

Site Vegetation Horizon

Finland

A Eriophorum vaginata, wet 2/3/4/6/8

B Eriophorum vaginata, dry 2/3/4/6/8

C Carex rostrata, wet 2/3/4/6/8

D Sphagnum fallax (+others), wet 3/4/5/6/8

E bare peat 2/3/4/6/8

France(Le

Russey)

A bare peat 3/4/6/8

B early regeneration 3/4/6/8

C advanced regeneration 3/4/6/8

D intact reference 3/4/6/8

Switzerland

A bare peat 3/4/6/8

B early regeneration 3/4/6/8

C advanced regeneration 3/4/6/8

D intact reference 3/4/6/8

Scotland

A bare peat, no recolonisation after ca. 5 years of abandonment 3/4/6/8

B peat recolon. with Sphagnum ssp. after 5-10 years of abandonment

3/4/6/8

C peat recolon. with Eriophorum angustifolium a. 5-10 years of abandonment

3/4/6/8

D peat recolon. with Sphagnum spp. after 50 years of abandonment 3/4/6/8

France(Baupte)

A bare peat 3/4/6/8

B early regeneration 3/4/6/8

Approach: Sample OverviewApproach: Sample Overview

2

3

4

5

6

8

1

0-5 cm

5-10 cm

12,5-17,5 cm

22,5-27,5 cm

42,5-47,5 cm

Sampling Autumn 2003

environmental

sample

DNAextraction

PCR amplification using labeled

primers

restrictiondigestion

detection of labeled fragments

using t-RFLP method

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10000

20000

30000

40000

0 100 200 300 400 500 600 700

2 2 3 x .C 0 3 _ 0 5 1 1 0 4 1 6 J 7

Size (nt)

Dye

Sign

al

ApproachApproach

Primers and Restriction EnzymesPrimers and Restriction Enzymes

Primer Target SequenceB27cy5-f universal bacteriaagagtttgatcctggctcag 1401r universal bacteriacggtgtgtacaagaccc

Restriction Enzyme Restriction siteAlu I ag^ct

Data analysis

Graphical output (Fragmentogram) and tables of peak area and fragment size using CEQ 8000 SoftwareData converted into binary codeData transfered to SPSS (statistical evaluation)Hierarchical Clusteranalysis

Specific QuestionsSpecific Questions

1. Influence of site 2. Influence of vegetation3. Influence of regeneration

on bacterial communities

4. Fragmentogram: vegetation/ regeneration specific fragments?

Site Vegetation

Finland

A Eriophorum vaginatum, wet

B Eriophorum vaginatum, dry

C Carex rostrata, wet

D Sphagnum fallax (+others), wet

E bare peat

France(Le

Russey)

A bare peat

B early regeneration

C advanced regeneration

D intact reference

Switzerland

A bare peat

B early regeneration

C advanced regeneration

D intact reference

Scotland

A bare peat

B peat recolon. with Sphagnum ssp.

C peat recolon. with Eriophorum angustifolium

D peat recolon. with Sphagnum spp.

France(Baupte)

A bare peat

B early regeneration

Results: Statistical evaluationResults: Statistical evaluation

C A S E 0 5 10 15 20 25 Label Num +---------+---------+---------+---------+---------+ CH- A-6-1 87 CH- A-6-3 88 CH- A-8-3 89 CH- A-8-4 90 CH- A-3-3 81 CH- A-3-4 82 CH- A-4-1 83 CH- A-4-2 84 CH- A-4-3 85 CH- A-4-4 86 FR- A-4-1 46 FR- A-4-2 47 FR- A-3-2 44 FR- A-3-3 45 FR- A-3-1 43 FR- A-6-1 48 FR- A-6-2 49 FI- E-6-1 40 FI- E-6-2 41 FI- E-3-1 35 FI- E-3-2 36 FI- E-4-1 37 FI- E-4-3 39 FI- E-8-2 42 FR- A-8-1 50 FR- A-8-2 51 FB- A-3-1 165 FB- A-3-2 166 FB- A-3-3 167 FB- A-6-2 171 FB- A-8-1 172 FB- A-4-1 168 FB- A-4-2 169 FB- A-4-3 170 SC- A-4-1 127 SC- A-4-2 128 SC- A-4-3 129 SC- A-3-1 124 SC- A-3-3 126 SC- A-3-2 125 SC- A-6-1 130 SC- A-6-2 131 SC- A-8-2 133

(SPSS: Hierarchical Clusteranalysis, Cluster-Method: Linkage between Groups, Binary: Jaccard)

1. Influence of site (country, bare peat)

distinct geographical clustering of bacterial communities independent from vegetation

two main clusters: SC marks off distinctly from other sites

peat samples from CH and FR form a big joint cluster (close vicinity, similar climatic conditions and plant vegetation)

within main cluster grouping of replicates

Results: Statistical evaluationResults: Statistical evaluation

C A S E 0 5 10 15 20 25 Label Num +---------+---------+---------+---------+---------+ FI- D-3-3 27 FI- D-4-3x 29 FI- D-4-3 28 FI- D-8-2 34 FI- A-3-1 1 FI- D-6-1 32 FI- C-6-1 23 FI- C-6-2 24 FI- D-6-2 33 FI- A-6-2 7 FI- C-4-2 21 FI- C-4-3 22 FI- C-3-1 19 FI- C-3-2 20 FI- A-3-2 2 FI- A-3-3 3 FI- A-4-1 4 FI- A-4-2 5 FI- C-8-1 25 FI- C-8-2 26

2. Influence of vegetation (FI)

Sphagnum peat samples appear in a common cluster

vegetation effect on bact. communities under Sphagnum

Carex + Eriophorum: influence of vegetation on bacterial communities residing in upper depth gradients

vegetation effect becomes less apparent with increasing soil depth (see depth 6)

Sphagnum fallax

(Moss)

Carexrostrata(grass)

Eriophorumvaginatum

(grass)

Results: Statistical evaluationResults: Statistical evaluation

C A S E 0 5 10 15 20 25 Label Num +---------+---------+---------+---------+---------+ FR- C-3-1 62 FR- C-3-3 64 FR- C-3-2 63 FR- B-6-1 58 FR- B-6-2 59 FR- B-8-1 60 FR- B-8-2 61 FR- C-6-1 68 FR- B-3-2 53 FR- B-3-3 54 FR- B-3-1 52 FR- C-4-2 66 FR- B-4-1 55 FR- B-4-2 56 FR- B-4-3 57 FR- C-4-3 67

Early Regeneration

AdvancedRegeneration

3.1 Influence of regeneration in France (Le Russey)

two main clusters: depth 3 of advanced regenerated peat marks off distinctly from rest

bacterial communities in advanced regenerated peat are influenced by the regeneration process

effect becomes less apparent with increasing soil depth

second cluster: depth effect predominant

Results: Statistical evaluationResults: Statistical evaluation

C A S E 0 5 10 15 20 25 Label Num +---------+---------+---------+---------+---------+ CH- C-8-1 112 CH- C-8-3 113 CH- C-6-1 110 CH- C-6-2 111 CH- B-8-2 101 CH- B-6-1 98 CH- B-6-2 99 CH- C-4-3 108 CH- C-4-4 109 CH- C-4-1 106 CH- C-4-2 107 CH- C-3-3 104 CH- C-3-4 105 CH- C-3-2 103 CH- C-3-1 102 CH- B-4-1 94 CH- B-4-2 95 CH- B-4-3 96 CH- B-4-4 97 CH- B-3-1 91 CH- B-3-4 93 CH- B-3-3 92

Early Regeneration

AdvancedRegeneration

3.2 Influence of regeneration in Switzerland

four main cluster

primary effect of depth

secondary effect of regeneration

early regeneration: upper depth gradients distinctly separates from the lower depth gradients

no predominating influence of regeneration on bacterial communities

ConclusionConclusion

site effectgeographical clustering independent from vegetation

++++

vegetation effectbacterial communities under grass more similar to each other than bacterial communities under moss

+++

regeneration effect

France (Le Russey): regeneration processes affect bacterial communities

++

X Switzerland: primary depth effect

Scientific ObjectivesScientific Objectives

1. Influence of country 2. Influence of vegetation3. Influence of regeneration

on bacterial communities

4. Fragmentogram: vegetation/regeneration specific fragments?

Fragmentogram of advanced regenerated peat samples (Le Russey, FR)Fragmentogram of advanced regenerated peat samples (Le Russey, FR)

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50000

60000

70000

80000

90000

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2 8 3 2 .E 0 5 _ 0 5 1 0 2 5 1 4 X E

Size (nt)

Dye S

ignal 71,90

73,34

136,53

138,08

139,63

141,30

142,99

144,82

146,04

148,18

151,02

153,03

155,42

166,01

175,82

179,17

181,32

182,96

201,32

202,58

204,80

206,94

208,81

214,44

217,39

220,86

222,89

224,86

227,17

229,11

232,06

233,50

235,86

238,02

239,81

242,44

244,80

247,15

249,24

251,50

271,13

273,69

276,53

278,32

281,95

0

10000

20000

30000

40000

50000

60000

70000

0 50 100 150 200 250 300 350 400 450 500 550 600 650

2 8 4 1 .H 0 4 _ 0 5 1 1 0 4 1 5 2 2

Size (nt)

Dye S

ignal

72,16

73,35

87,36

88,68

135,70

139,43

141,27

144,80

145,73

152,92

201,02

204,36

206,81

208,78

210,71

220,93

224,90

228,61

230,88

233,03

236,04

239,69

241,97

243,77

246,76

249,34

250,63

251,83

263,85

270,62

273,77

275,42

282,28

284,37

FR-C-4-1 FR-C-4-2

Fragment: 146 bp

Fragmentsize [bp]

Pea

k A

rea

Fragmentogram of advanced regenerated peat (Chaux d’Abel, CH) Fragmentogram of advanced regenerated peat (Chaux d’Abel, CH)

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11000

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3 2 8 3 .C 1 2 _ 0 5 1 0 0 6 1 5 4 8

Size (nt)

Dye S

ignal

138,10

139,47

140,88

142,87

144,28

146,24

151,12

152,38

154,15

164,60

167,22

169,32

0

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10000

15000

20000

25000

0 50 100 150 200 250 300 350 400 450 500 550 600

3 2 9 1 .G 0 4 _ 0 5 1 0 0 6 1 5 4 4

Size (nt)

Dye S

ignal

139,52

141,35

142,67

144,74

145,78

146,90

149,78

151,31

154,52

160,40

162,75

205,16

208,05

218,68

226,18

230,05

233,37

235,01

237,09

239,27

251,14

259,29

260,97

264,23

269,22

270,99

273,27

275,26

277,46

280,05

283,35

286,48

290,05

292,07

294,96

296,10

298,02

CH-C-4-1 CH-C-4-2

CH-C-4-3 CH-C-4-4

0

10000

20000

30000

40000

50000

60000

70000

80000

0 100 200 300 400 500 600 700

3 3 0 2 .G 0 5 _ 0 5 1 0 0 6 1 5 3 P

Size (nt)

Dye S

ignal

71,70

138,04

139,53

141,97

144,87

145,93

151,05

153,19

155,29

158,16

162,05

166,14

168,35

170,69

201,42

206,91

208,91

211,02

213,35

217,50

218,95

220,78

224,73

227,07

229,11

231,11

232,82

235,63

237,76

239,42

242,24

244,21

246,41

249,20

250,91

259,51

264,23

271,10

272,64

273,95

275,05

278,55

282,59

290,18

293,43

299,80

0

25000

50000

75000

100000

125000

150000

0 100 200 300 400 500 600 700

3 3 1 3 .F 0 2 _ 0 5 1 1 0 3 1 5 1 O

Size (nt)

Dye S

ignal

64,40

72,17

106,10

120,52

123,22

126,29

139,31

141,57

143,60

145,01

146,15

149,20

150,92

153,15

155,41

158,82

159,95

163,80

166,21

168,48

170,53

201,01

203,72

205,23

207,01

208,44

211,25

213,33

217,00

218,35

220,75

225,12

227,06

228,68

229,65

230,90

233,02

235,06

236,04

237,71

239,56

242,17

243,86

246,87

249,42

251,25

259,79

260,96

264,94

268,01

270,77

273,21

275,47

278,44

280,14

281,95

284,56

287,45

289,33

293,19

297,98

Fragment: 146 bp

Identification of Fragments of InterestIdentification of Fragments of Interest

Approach

16S-PCR using non labeled

primers

cloning of rDNA

colony hybridizati

on

plasmid extraction

sequencing

in silico digestion

16 s-PCR using labeled primers

enzymatic digestion

t-RFLP analysis

t-RF Database

which probes to use

what are the hybridization conditions

Plasmid

E.coli

Growing onAgarplate

Alignment with DatabaseAlignment with Database

Alignment with database: http://rdp8.cme.msu.edu/html/

TAP-TRFLP permits the user to perform in silico T-RFLP experiments on the RDP alignments (Marsh et. al, 2000), by assigning the sequences of

• primers • restriction enzymes

that have been used. The output can be sorted and viewed either phylogenetically or by size.

=> Reduce organisms to a common level: phylum ‚FIRMICUTES‘

Hybridization ProbesHybridization Probes

LGC 354 A: Leuconostoc fallax DSM 20189/ Medium 11, 30°C

Lactobacillus suebicus DSM 5008/ Medium 11, 30°C

LGC 354 B: Bacillus lichenoformis DSM 13/ Medium 1, 37°C

Bacillus subtilis DSM 10/ Medium 1, 30°C

Bacillus alcalophilus DSM 485/ Medium 31, 37°C

LGC 354 C: Enterococcus hirae DSM 20160/ Medium 53, 37°C

Streptococcus thermophilus DSM 20617/ Medium 53, 37°C

To test the specificity of the probe several bacterial isolates were chosen to serve as positive controls:

http://www.microbial-ecology.de/probebase/

Probe Sequence TM Labeling 5’LGC 354 A tgg aag att ccc tac tgc 44.3 °C DIGLGC 354 B cgg aag att ccc tac tgc 46.8 °C DIGLGC 354 C ccg aag att ccc tac tgc 46.8 °C DIG

Determination of Hybridization-Conditions: ApproachDetermination of Hybridization-Conditions: Approach

• DNA IsolationPhenol-Chloroform-Extraction of

control strains

• Cloning + Transformation into E.coli Strainsusing TA Cloning Kit (Invitrogen)

• Amplification of 16S rDNAusing unlabelled primers

B.a B.l B.s E.c E.h L.f L.s P.sp S.t -

1500 bp

• Enzymatic Digestusing restriction enzyme EcoRI

3900 bp

1500 bp750 bp

Determination of Hybridization-Conditions: ApproachDetermination of Hybridization-Conditions: Approach

Digoxigenin

AlkalinePhosphatase

C-G-T-G-A-T-A-G-C

A-C-U-A-T

Substrate PP

Substrate

Color Detektion

BCIP/NBT

Antibody-Conugate

Labelled Probe

Target DNA

Membrane

• Digoxigenin mediated Hybridization

ResultsResults

B. alcalophilus B. licheniformis B. subtilis

E. coli E. hirae L. fallax

L. suebicus P. sp. S. thermophilus

Plasmid (no insert)

TD=44.6°C

Determination of hybridization conditions

……to be doneto be done

colony hybridization with samples of interest at determined

hybridization conditions

sequencing of positive clones

confirmation of concept by applying t-RFLP

Thanks!

Alexandra Hagn

Michael Schloter

Andreas Gattinger

Stephen Chapman

Rebekka Artz