dna/rna-targeted molecular analysis of bacterial communities slides.ppt.pdf · dna/rna-targeted...

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University of Aberdeen 1 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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University

of

Aberdeen

1

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:

University

of

Aberdeen

2

The universal SSU rRNA tree

Hugenholtz

et al. 1998

J Bacteriol

180, 4765–

4774.

Bacteria, 1998

University

of

Aberdeen

3

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

University

of

Aberdeen

4

Ammonia oxidiser taxonomy

NitrosomonasNitrosomonas

NitrosospiraNitrosospira NitrosolobusNitrosolobus NitrosovibrioNitrosovibrio

NitrosococcusNitrosococcus

BetaproteobacteriaBetaproteobacteria

GammaproteobacteriaGammaproteobacteria

University

of

Aberdeen

5

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.

University

of

Aberdeen

6

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.

University

of

Aberdeen

7

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.

University

of

Aberdeen

8

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

University

of

Aberdeen

9

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

10

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

11

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

University

of

Aberdeen

12

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

University

of

Aberdeen

13

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

University

of

Aberdeen

14

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

University

of

Aberdeen

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

University

of

Aberdeen

16

Relative abundance of bacterial ammonia

oxidiser clusters in soils of different pH

0

10

20

30

40

50

60

70

3.9 4.2 4.5 5 5.5 6 6.6

Soil pH

Relative abundance (%)

CL2 CL3 CL4 CL6a

Stephen et al. 1998, Appl. Env. Micro. 64, 2958-2965