T-RFLP study of soil microbial community at elevated chloride concentration
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T-RFLP study of soil microbial community at elevated chloride
concentrationM. Gryndler
Institute of Microbiology CAS, Prague
In collaboration with:
Miroslav Matucha and Jana Rohlenová, Institute of Experimental BotanyCAS, Prague
and
Jan Kopecký, Institute of Microbiology CAS, Prague
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Questions:
• Does elevated Cl- concentration affect established soil microbial community?
• Does elevated Cl- concentration affect de- -novo developing microbial community?
• Which organisms are affected?• Is a change in composition of soil
microbial community followed by changes in degradation of chlorinated SOM or chlorination of SOM?
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Methodology:
• Nonsterile or irradiated/recolonized 128 g soil samples, 20% humidity
• Podzolic OH soil from spruce monoculture• Two levels of total soil Cl: 20 or 500 mg/kg• Counting microbial CFU• Extraction of DNA, TRFLP analysis of
prokaryotic and eukaryotic ribotypes• 14C-TCA degradation • 36Cl- incorporation into humic substances
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Design of the experiment (n=3)
• Low chloride, intact microflora
• High chloride, intact microflora
• Low chloride, sterilized/recolonized
• High chloride, sterilized/recolonized
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Microbial CFU counts
0
5
10
15
20
25
30
35
40
20N 500N 20S 500S
CFU
x10
-5
Bacteria
Fungi
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DNA extraction(125 d) and
amplification:
Eubacteria
Eukaryota
Fungi
Lanes 1,2,3 – 20N
Lanes 4,5,6 – 500N
Lanes 4,8,9 – 20S
Lanes 10,11,12 – 500S
Eukaryotic ITS region,Eubacterial SSU rDNA
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Primers:
• Eubacteria: 16Seu27f, 783r-a,b,c(Sakai et al. 2004)
• Eukaryota: ITS1, ITS4(White et al. 1990)
• Fungi: ITS1F, ITS4(Gardes and Bruns 1993)
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rDNA cassette
18S rDNA
5.8S rDNA
28S rDNAITS1 ITS2 D1 D2
ITS1 ITS4
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TRFLP
Terminal restriction fragment length polymorphism
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Restriction cleavage
Restriction profile
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A
BA+B
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Restriction cleavage
Restriction profile
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Restriction cleavage
Restriction profile
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A
BA+B
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A
BA+B
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Restriction cleavage:
• Taq I. restriction endonuclease for eukaryotic DNA
• ALU I. restriction endonuclease for eubacterial DNA
• Forward primers HEX-labeled
• Analysis using capillary electrophoresis,
• LIF detector
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Terminal restriction fragments:
0
200
400
600
800
1000
1200
1400
1600
1800
2000
60 110 160 210 260 310 360
bp
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Numbers of TRFTreatment Sample Eubact. Eukaryota Fungi
20N 1 52 104 112 59 75 90
3 57 68 113
500N 1 50 73 572 43 126 17
3 225 159 100
20S 1 59 129 1422 32 56 92
3 29 100 16
500S 1 40 129 112 47 77 14
3 35 105 12
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Effect of soil sterilization/recolonization:
• 3 eubacterial and 1 fungal TRF disappeared
• 1 eukaryotic and 1 fungal TRF
detected
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Effect of increased Cl-
• 1 eubacterial and 1 fungal TRF disappeared
• 1 bacterial and 1 eukaryotic TRF
detected
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0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
130 135 140 145
bp
Eukaryotic TRF 135 bp
Absent from any treatment but nonsterilized soil high in chloride
In 3 replicates constitutes 33.5, 11.4 and 7.0 molar % of all TRF !
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Identity?
Forward primer5´- ITS1 - ............. TRF……....….......... T– 3´3´- ................. 2-stranded .......... A GC– 5´
=TaqI
Fragment lacks known reverse primer sequence
Oligonucleotide adapter is necessary for PCR amplification
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Identity?
Forward primer5´- ITS1 - .......……TRF………….......... T– 3´3´- ................. 2-stranded .......... A GC– 5´
=TaqI
Forward primer5´- ITS1 - .............TRF.......................... T/CG AAT TCT CCG TCT CGC TCC G – 3´3´- ................. 2-stranded .......... A GC/TTA AGA GGC AGA GCG AGG C – 5´
=TaqI reverse primer
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Identity?
5´‑TCCGTAGGTGAACCTGCGGAAGGATCATTGGAGAGAGAAAGGGGGAGAGAGTTGGAATGTGATGAGACGAGAGATTCAAACTATATAGTGAATGATCATACAACTGCTGACAATGGATCTCTGGGCTCTTGCGTCGAATTCTCCGTCTCGCTCCG-3´
This sequence is 100% identical with the sequence registered in GenBank under accession number DQ309135 ("uncultured fungus isolate RFLP-145") and involves a part of 18S ribosomal RNA gene and a part of internal transcribed spacer 1. This sequence was amplified from DNA extracted directly from Ericaceous roots along a moorland-forest gradient in Scotland. At this stage, its identity is unknown (Prof. John W. G. Cairney, University of Western Sydney, Australia, personal communication).
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Identity?
5´‑TCCGTAGGTGAACCTGCGGAAGGATCATTGGAGAGAGAAAGGGGGAGAGAGTTGGAATGTGATGAGACGAGAGATTCAAACTATATAGTGAATGATCATACAACTGCTGACAATGGATCTCTGGGCTCTTGCGTCG-3´
Sequence does not contain information sufficient to identify its bearer. However, it may be used to design specific primer to amplify larger rDNA fragments, perhaps suitable for identification by „BLASTing“
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Primer CLF1 (forward)
• 5´-GAGTTGGAATGTGATGAGACG-3´
+ Primer NL4 (reverse)
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rDNA cassette
18S rDNA
5.8S rDNA
28S rDNAITS1 ITS2 D1 D2
CLF1 ITS4
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ITS1F-ITS4
ITS1-ITS4
27f-783r
CLF1-ITS4
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rDNA cassette
18S rDNA
5.8S rDNA
28S rDNAITS1 ITS2 D1 D2
CLF1 NL4
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CLF1-ITS4/NL1-NL4
GAGTTGGAATGTGATGAGACGAGAGATTCAAACTATATAGTGAATGATCATACAACTGCTGACAATGGATCTCTGGGCTCTTGCGTCGATGAAGAACGCAGCAAAACGCGAAAAGTGTTATGATGTGCAGTCTTTGAGAATCATGAATGTTTGAACGCACCTTGCACCACCGAGCGATTGGGGGTATGCCTGTTTGAGCGGGGGATAAAATTGAGTGAACTGTGGTTTATTGTGGGGTACTAGGTAACACCTTGCCCTGAAAGACAGATCTCGTGTACTCTTGGGATAATCCATCAAGAAGCACATTTACAGTATACCACCTCAAATCAGGCAAGATGACCCGCTGAACTTAAGCATATCAGTAAGCGGAGGAAAAGAAACTAACCAGGATTCCCGCAGTAACGGCGAGTGAAGAGGGAACAGCTCACATTTGGAAACGATCATGAAAAAAGTGGTCGAGTTGTCAGTGATAGCATGGGAGCGGGATTTGGAAAGGGTGAGCGAGTCTGCTGGAAAGCAGCGCCAGAGAGGGTGACAGCCCCGTGGCTTGCCTTGCACAAGTTTACGGAGCCATGCGACGAGTCGGACTGTTTGGGAATGCAGTCCTAAATGGGTGATACGTGTCATCTAAAGCTAAATAGCGGCAAGAGACCGATAGCGAACAAGTACCGTGAGGGAAAGATGAAAAGAACTCTTGGCAGAGAGTGAAAAGTGCGTGAAATTGTCAGCAGGGAAGCGATGGTGGTGGGGAGGTGTGCCAAGAGAAGCAACTTCGTCAGAAGTTGCCAATTAAAAAGCACGAGCGAGGCGAGTGGATGAGGGGGAAGATAGGTGGGAAGAGAGAGTAATTCATTACAATTTCTTCAAACTAAACCCCCCACACACACCCACCTCGCAGCCCACCACGACCGACCCGTCTTGAAACACGGACC
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Homology?
Glomus walkeri (AJ972467, E=1x10-36), Glomus drummondi (AJ970465, E=4x10‑36)
Orpinomyces sp. (AJ864475, E=2x10-36)
Entophlyctis sp. (DQ273782, E=6x10-36)
Cyllamyces aberensis (DQ273829, E=2x10-35) Neocallimastix sp. (DQ273822, E=2x10‑35)
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Efects of increased chloride on organic and inorganic Cl-?
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Efects of increased chloride on organic and inorganic Cl-?
0
100
200
300
0 10 20 30 40 50 60 70 80
Time (h)
Rel
ease
d r
adio
acti
vity
(k
Bq
)
20N
500N
20S
500S
36Cl-TCA mineralization
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Efects of increased chloride on organic and inorganic Cl-?
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Conclusions
• Sodium Chloride at „realistic“ concentrations affects soil microflora
• TRFLP and downstream techniques can be used to detect and identify organisms responding to chloride
• Inorganic and organic Cl behaviour is affected by Cl- concentration
• Links between specific organisms and soil Cl fluxes are probable