DNA/RNA-targeted molecular analysis of bacterial communities slides.ppt.pdf · DNA/RNA-targeted...
Transcript of DNA/RNA-targeted molecular analysis of bacterial communities slides.ppt.pdf · DNA/RNA-targeted...
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of
Aberdeen
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Lysis, DNA/RNA
extraction
DNA/RNA
purification
PCR/RT-PCR
amplification of 16S
rRNA or functional
genes
DNA/RNA-targeted molecular
analysis of bacterial communities
DGGE, TGGE, RFLP, t-RFLP, SSCP
Cloning Sequencing
Identification
Phylogenetic analysis
Fingerprinting:
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of
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The universal SSU rRNA tree
Hugenholtz
et al. 1998
J Bacteriol
180, 4765–
4774.
Bacteria, 1998
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of
Aberdeen
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Nitrification
NH3
NH3
NO2-
CO2
Groundwater pollution Fertiliser loss
DenitrificationLeaching
Decomposing
organic matter
Animal
excreta
Fertiliser
N
Urea
Organic C
NO2-
N2O
NO
CO2
biomass
NO2-
NO3-
CO2
biomass
Urea
CH4
CH4
Methanol
NO
N2O
NO3-
CO2
Organic C
N2O
NO
Ammonia
oxidisers
Nitrite
oxidisers
Nitrosomonas
europaea
Nitrobacter winogradskyi
TEM Image credit: W.J. Hickey, University of
Wisconsin-Madison, 2006.
N flux
1.5 x 1015 g soil N y-1
6 x 1015 g marine N y-1
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Ammonia oxidiser taxonomy
NitrosomonasNitrosomonas
NitrosospiraNitrosospira NitrosolobusNitrosolobus NitrosovibrioNitrosovibrio
NitrosococcusNitrosococcus
BetaproteobacteriaBetaproteobacteria
GammaproteobacteriaGammaproteobacteria
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Nitrosospira briensis (SOIL)
Nitrosospira sp. NV12 (SOIL)
Nitrosospira sp. NPAV (SOIL)
Nitrosospira sp. C-141 (SOIL)
Nitrosospira tenuis str. NV1 (SOIL)
Nitrosospira multiformis (SOIL)
Nitrosospira sp. 40KI (SOIL)
pH4.2A/H2 pH4.2A/E2 pH4.2A/K2 pH4.2A/C pH4.2B/32 pH4.2A/L2 En4_7/11/96
pH7C/30 pH4.2A/17 pH7C/24 pH4.2A/27 En3_7/11/96
pH4.2A/G2pH4.2A/J2
En6_7/11/96 pH7B/V2
pH7C/48 pH7B/D3
pH7B/B2 pH7C/54
En_30_4_5 pH7C/25
pH4.2A/3F pH7B/L2 pH7B/G2
En12_7/11/96 En8_7/11/96
En13_7/11/96 En5_7/11/96 En_30_4_2 pH7C/53
En_18_4_3 En_18_4_10
En_30_4_7 En7_7/11/96
pH7C_56 pH7C/41 pH4.2A/D, Med_Agg_ZD5 pH4.2A/19 pH4.2B/35 pH4.2A/F2 pH4.2A/3E En15_7/11/96
Enrich ZB3
Enrich_ZA4_96 pH4.2B/38
En_30_4_9 En_ZA3_96
En_18_4_8
Med_Agg_45
C1aW3 A2aM1 EnvA2-5 EnvB1-17 EnvA2-8 EnvC1-18
EnvB1-8 EnvB2-17
EnvC2-23 EnvC2-6
EnvB2-16 EnvC1-24 EnvC2-24 Med_Agg_65
Med_Plank_D3EnvB1-4
EnvB2-9 EnvA2-4
Med_Plank_Z6
NitrosospiraNitrosospira NitrosomonasNitrosomonas
Neisseria denitrificans Kingella kingae
Oxalobacter formigenesComamonas testosteroni
Sphaerotilus natans Alcaligenes eutrophus
EnvA2-13 A1aW3
EnvB1-6 EnvA2-3 EnvA2-17 EnvA1-21
A2bM1
En14_7/11/96 B2aW2
SewageC3 SewageC4 SewageDG4
SewageDG1
pH7B/Z2 PH7C/37 SewageDG2 SewageDG5EnvC1-17
EnvC1-19 C2aM2
B1bM2 AEM-1B2aM2
B2bM2ii B2bM2i
B1aM2 A1aM2 B1bW2 B2bW1 B1aW2 EnvA1-19
AEM-5AEM-4
C1bW1 Med_Agg_A64
Med_Agg_X42
Med_Agg_7Med_Agg_58Med_Agg_12
Nitrosomonas ureae (SEWAGE)
Nitrosomonas sp. NM63 (MARINE)
Nitrosococcus mobilis (BRACKISH)
Nitrosomonas eutropha (SEWAGE)
Nitrosomonas europaea (SOIL)
10%
Cluster 2
Cluster 3
Cluster 1
Cluster 4
Cluster 5
Cluster 6
Cluster 7
Referenceβ-proteobacteria
Natural sequence diversityNatural sequence diversity
Soil
Marine
Sewage
Pure cultures
Med_Agg_A64 Med_Agg_X42 Nitrosomonas eutropha (SEWAGE) Med_Agg_7Med_Agg_58 Med_Agg_12
Nitrosomonas europaea (SOIL) En14_7/11/96
Stephen et al., 1996 AEM 62, 4147-54.
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Influence of sheep urine on
ammonia oxidisers
10 g
1 ml
distilled
water
1 mg N g-1 soil
10 g
1 ml
synthetic
sheep
urine
•KHCO3
•KCl
•K2SO4
•Glycine
•Urea
pH Ammonia Nitrate Urea DGGE
Webster et al., 2005 Env Micro
7, 676-684.
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Nitrification after addition of urea
0
200
400
600
800
1000
1200
1400
1600
1800
0 20 40 60 80 100
Time (days)
Nitrogen conc. (µg m
l-1)
NHNH44++
NHNH44++
NONO33--
NONO33--
+ Urea+ Urea
-- UreaUrea
3a
3b
NsControlsControls
Webster et al., 2005
Env Micro 7, 676-684.
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Aberdeen
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Nitrification after additionof urea
0
200
400
600
800
1000
1200
1400
0 20 40 60 80 100
Time (days)
Nitrogen conc. (µg ml-1)
+ Urea+ Urea
NONO33--
NHNH44++
-- UreaUrea
NONO33--
NHNH44++
Webster et al., 2005 Env Micro
7, 676-684.
ControlsControls
3a
3b
Ns
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Effect of ammonium conc. on
growth of pure cultures
-2.5
-1.5
-0.5
0.5
1.5
2.5
3.5
0 50 100 150 200
Time (days)
Log(NO3- conc.; µg ml-1)
1 ppm
10 ppm
100 ppm
1000 ppm
Nitrosospira briensis Nitrosospira briensis
(Cluster 3b)(Cluster 3b)Nitrosospira Nitrosospira NpAVNpAV
(Cluster 3a)(Cluster 3a)
-1.0
-0.5
0.0
0.5
1.0
1.5
0 25 50 75 100 125 150 175 200
Time (days)
Log(NO3- conc.; µg ml-1)
1 ppm
10 ppm
100 ppm
1000 ppm
Webster et al., 2004 Env Micro
7, 676-684.
University
of
Aberdeen
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Effect of ammonium conc.
on growth of enrichments
-1.0
-0.5
0.0
0.5
1.0
1.5
0 25 50 75 100 125 150 175 200
Time (days)
Log(NO3- conc.; µg ml-1)
1 ppm
10 ppm
100 ppm
1000 ppm
ur/U4a/1
(Cluster 3b)En284 En284
(Cluster 3a)(Cluster 3a)
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
0 50 100 150 200
Time (days)
Log(NO3- conc.; µg ml-1)
1 ppm
10 ppm
100 ppm
1000 ppm
Webster et al., 2005 Env Micro
7, 676-684.
University
of
Aberdeen
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Stable isotope probing
DNA-SIP
Density
gradient
centrifugation
12C-DNA
13C-DNA
Molecular
analysis
Radajewski
et al. 2000
Nature 403,
646-649
13C-labelled
substrate
Extract and
purify DNA
Boschker
et al. 1998
Nature
392, 801-
804.
13C-labelled
substrate
Extract
lipids
Incubate
PLFA
analysis
Stable isotope probing 13C-PLFA analysis
13C-labelled
methane
PCR
amplification:
16S rRNA
mxaF genes
13C-labelled
methanol
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Stable isotope probing
DNA-SIP13C-labelled
substrate
Extract and
purify DNA
Density
gradient
centrifugation
Incubate
12C-DNA
13C-DNA
Molecular
analysis
Stable isotope probing
RNA-SIP13C-labelled
substrate
Extract and
purify RNA
Density
gradient
centrifugation
Incubate
12C-RNA
13C-RNA
Molecular
analysis
Molecular
analysis
Manefield et
al 2002
Appl Env
Micro
58 5367–
5373
Lueders et
al. 2003
Env Micro
6, 60-72
6, 73-78
Quantification
t-RFLP
sequencing
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Stable isotope probing in the field:
RNA-SIP13C-labelled
substrate
Extract and
purify RNA
Density
gradient
centrifugation
Incubate
12C-RNA
13C-RNA
Molecular
analysis
13C-labelled CO2
3 h sample
RNA-DGGE
analysis of
16S/18S
rRNA genes
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The Crenarchaeota
Crenarchaeota
Euryarchaeota
Animals
Ciliates
Plants
Fungi
Flagellates
Microsporidia
Slime
moulds
Trichomonads
Diplomonads
Entamoebae
Flavobacteria
Thermotogales
Proteobacteria
Cyanobacteria
Green non-sulphur
bacteria
Gram positives
Bacteria
Archaea Eucarya
Universal
Ancestor
Pyrodictium
Thermoproteus
T. celer
Methanococcus
Methanosarcina
Methanobacterium
Group II
Group I
Crenarchaeota
Euryarchaeota
Halophiles
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15
Isolation of Nitrosopumilus maritimus
Kınnneke et
al. 2005,
Nature 437,
543-546.
1 1 µµmm 1 1 µµmm
0.1 0.1 µµmm 0.1 0.1 µµmm