recipe reconciling commercial exploitation of peat with biodiversity in peatland ecosystems
DESCRIPTION
RECIPE Reconciling Commercial Exploitation of Peat with Biodiversity in Peatland Ecosystems BACTERIAL DIVERSITY Brigitte Hai, Dr. Alexandra Hagn, Dr. Andreas Gattinger, Dr. Michael Schloter WG Schloter. Scientific Objectives. - PowerPoint PPT PresentationTRANSCRIPT
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
0
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)
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
0 50 100 150 200 250 300 350 400 450 500 550 600 650
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)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
0 50 100 150 200 250 300 350
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
5000
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