How to quantify bacteria in sediments? · 1 © Bert Engelen How to quantify bacteria in sediments?...

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© Bert Engelen www.icbm.de/pmbio

How to quantify bacteria in sediments?

Parkes, R.J., B.A. Cragg and P. Wellsbury, 2000


Per

ry &

Sta

ley,

Mic

robi

olog

y –

Dyn

amic

s an

d D

iver

sity

Phasecontrast microscopyCounting chamber (Thoma, Petroff-Hausser, ...)

Only feasable for liquid samples.

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Filtration of samples for the Epifluorescence-microscopy


Nucleic acid dyesAcridin orange

Bacteria within a sediment-particle

Phot

o: A

. Bat

zke

Phot

os:

H. C

ypio

nka

Ironsulfide-particle

phase contrast

Fluorescend cells

Combination ofphase contrast and

fluorescence

DAPI-stainingTidal flat sediment stainedwith SYBR green

Photo: B. Köpke

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Amount of DNA

(2-4*106 base pairs per procaryotic genome)

Amount of ATP

ATP * 250 BioC (in g)

Flowcytometer

Direct counts in a capillary system

Other methods


Figs: Station Biologique de Roscoff CNRS and Université Pierre et Marie Curie, France

Flow cytometry

4


Viable cell counts:

Most Propable Number

and

Colony Forming Units (CFU)


Perry & Staley, Microbiology – Dynamics and Diversity

Colony Forming Units (CFU)

5


Slurry3

Slurry4

Kontrolle

ABC

DE

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

10 -1 10 -3 10 -5

10 -2 10 -4 10 -6

10 -6 10 -4 10 -2

10 -5 10 -3 10 -1

Slurry 1

Slurry 2

Kontrolle

Most Propable Number


MPN quantification Detectedgrowth

MPN index[cells/ml]

Confidence interval (95%)

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Anoxic Oxic

not pasteurised

pasteurised

1.35%0.15%

0.21%0.06%

< 0.01%< 0.01%

Depth [cm

]0

100

200

300

400

500

0 1 2 3 4 5 6 7

600

0 1 2 3 4 5 6 7 8

log viable counts [cm-3]

MPN values within a tidal flat sediment column

Beate Köpke


MPN counts depend on incubation conditions

- Temperature

- Substrate

- Oxic or anoxic incubations

- Supplement of vitamins and other trace elements

MPN counts in tidal flat sediment Amino acids 1,9·107 cm3

Fatty acids 4,0·106 cm3

MPN counts in tidal flat sediment

10°C 4,0·105 cm3

20°C 8,2·106 cm3

30°C 4,0·105 cm3

MPN counts in tidal flat sediment

Oxic 1,0·107 cm3

Anoxic 4,0·105 cm3

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How many different bacteria do we expect?

Validly described species:

5 000 Prokaryotes (Bakteria und Archaea)

1 700 000 Eukaryotes

Estimations for the number of bacterial speciesin 30 g forrest soil

3 000 (Torsvik et al 1990, Appl Environ Microbiol 58:782-787)

500 000 (Dykhuizen 1998, Antonie van Leeuwenhoek 73:25-33)

(based on the same set of data)

similar for sediments


Application of molecular probes

Techniques:

Membrane- hybridisation

Extracted, immobilised RNA/DNA (Dot Blot, DNA-Chips)

Fluorescence In-situ Hybridisation, FISH:

Fixed cells (binding at ribosomes)

Signal enhancement by higher ribosome content

Specificity:

Strain, family, ... up to domain (dependent on target sequence)

Hybridisation:

Probe (Oligonucleotide) at a target sequence (mostly 16S/23S rRNA)

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Dot Blot analysis of bacterial communities

Felske et al. 1996


Analysis of bacterial communities by

Fluorescence In-situ Hybridisation, FISH

Stronghold of Fluorescence In-situ Hybridisation in Germany is the MPI in Bremen!

Coupling of molecular „probes“with fluorescence dyes

Speciffic annealing at regions of the rRNA

Staining of cells on differentphylogenetic levels

Detection under a microscopicslide (In-situ)

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Anaerobic methane oxidising consortia

ANME2 (EelMS932)

Desulfosarcina (DSS658)

Boetius, et al. (2000)Nature. 407:623-626

5 µm

detected in gas hydrate bearing sediments

DAPI CARD-FISH

Archaea (ARCH915)

Desulfosarcina (DSS658)

detected in tidal flat sediments

Ish

i e

t al

. 2

00

5


Fluoresce In Situ Hybridisation

natural microbial

community

FixationTreatment with fixative, conditioning of cells,

filtration

WashingDetachment of probes that were not

bound to the target sequence

HybridisationAnnealing of probes under

stringent conditions

Fluorescent

dye

Specific

probes

16S rRNA

Counter stainingStaining of all cells by a general

fluorescent dye (e.g. DAPI)

VisualisationEpifluorescence microscopy

ProbeDAPI

Relation of non specific to specific signals

Fig

.: B

. R

ink

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Problems

Probe signal depends on the amount of ribosomal RNA,

and therefore on the physiological state of the cells!

DAPI counter stain includesinactive cells and even spores

Interpretation often difficult

Optimisation: Signal amplification by CARD-FISH

Especially for samples that show high autofluorescence like sediment, algae, cyanobacteria

The Relation of non specific to specific signals can be distorted


CAtalysed Reporter Deposition - FISH

natural microbial

community

FixationTreatment with fixative, conditioning of cells,

filtration

HybridisationAnnealing of probes under

stringent conditions

Horseradish-

peroxidase, HRP

Specific

probes

new

16S rRNA

WashingDetachment of probes that were not

bound to the target sequence

Fig

.: B

. R

ink

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Tyramide signal amplification (TSA)marked substrate (Tyramide) is enriched within the

cell by chemical reaction and binding to proteines

B

Protein

(Tyrosin)H2O

Peroxidase

H2O2

Activation

Enrichment

*

Peroxidase

H2O2

Fluorescent

dyeTyramide

inactiv

A

new

newWashing

Molecules that were not converted

Counter stainingStaining of all cells by a general

fluorescent dye (e.g. DAPI)

VisualisationEpifluorescence microscopy

ProbeDAPI

Relation of non specific to specific signals

Fig

.: B

. R

ink


Higher sensitivity by signal amplification

FISHCARD-FISH

dept

h (c

m)

Fig

.: M

. M

ussm

ann

Tidal flat sediment

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PCR techniques


Quantitative (real time) PCR

SybrGreen ITM-technique

⇒ low fluorescence ⇒ increasing fluorescence

Amplification

No binding at single stranded DNA

Intercalation of SybrGreen at double stranded DNA

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qPCR protocol

PCR reaction components Temperature program

- Stainless polymerase

- SybrGreenI

- 10µl of DNA template

- Detection of fluorescence after

every elongation step

- Melting curve analysis


The maschine

Rotor-Gene 2000/3000 Corbett Research, Australia

Raw data analysis

Rotor and detection units

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Data analysis

Threshold value:-Level of highest amplificaton rate

Ct-values:- Number of cycles that are

needed to reach the threshold - in direct relation to copy number

of the original sample

-„normalised“ rawdata

Standard curve- Calculation of DNA copies in the

original sample -Number of organisms calculated by

genome size and 16S rRNA copy number


Application of the qPCR on Mediterranean sediments

Method: Rhizobium specific real time-PCR with SybrGreen I

Results

⇒ widely distributed in Mediterranean sediments

⇒ enhanced numbers in sapropels ⇒ up to 5% of eubacteria

⇒ typical deep biosphere organisms

16S rRNA operons

AbsoluteRelative

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SIGnature PCR

Separation in an agarose gel

g-Proteobacteria

Firmicutes, High-GC

a-Proteobacteriab-Proteobacteria

CFB

Bacteriaca. 1500 bp

1000 bp

700 bp650 bp

350 bp

100 bp

Amplification of specificPCR products with different length


SIG-PCR with Mediterranean isolates

Reference unknown isolates

Süß et al. 2004

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indifferent

4 marin phototrophic α-Proteobacteria

α-Proteobacteria

Gram positive high GC

γ-Protoebacteria31

38

13

32

Result of SIG-PCR screening of isolates from Mediterranean sediments

no growth in AS media

50% from SED

almost exclusively from MPN plates(MKS, AS, Alk)


Who is active?

Stable isotope probing BrdU incorporation

Nucleotide analogues• In situ detection (fluorescence)• DNA (magnetic beads)

13C-labled substrates• Fingerprinting of

microbial communities

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Clone libraries


Separating of sequences from the environment by cloning

Ligation of PCR-products at hanging Ts

Cloning of PCR-products in a „multiple cloning site“

Selection of cells that carry a plasmid by antibiotics resistence

Blue/white-screening of cells that carry an insert on the plasmides

Reamplification of the inserts by flanking primers (T7, SP6)

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Analysis of a clonebank: „bottom-up approach“

Amplification of the 16S rDNA

TA-cloning

blue-white screening

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A1 1 A12

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12

D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12

F1 F2 F3 F4 F5 F6 F 7 F8 F9 F10 F11 F12

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G1 1 G12

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12

Composition of themicrobial community

Sequence Analysisof single clones


Principle of a sequencing reaction(Sanger)

sequencing reaction in 4 tubes

(buffer, polymerase, MgCl2, dNTPs)

ddATP

+ 3% each

ddGTP ddCTPddTTP

TTGAACAGCCTGACGC

sequencing gel

6 bp

15 bp16 bp

5´

5´

3´

3´

unknown sequence

sequencingprimer

known sequence

fluorescence -labeled

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

T G C Gprimer

primerannealing


A C T G C G

integration of dd ATP

chain abruption, fragmentlength = 6 bp

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


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


Sequencing-approach with dd ATP



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Alignment of 16S rRNA sequences


Exploring microbial diversity

using the „454 technology“

and pyrosequencing

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Dawning of the "omics" age

Sequencing of the human genome: "kick off" for systems biology

• Homo sapiens draft genome report in 2001 (~3,000 Mb)

• Novel technology (454, pyrosequencing) made sequencing cheaper

• Sequencing on microbeads in reactive emulgions


Dawning of the "omics" age

Sequencing of the human genome: "kick off" for systems biology

• Homo sapiens draft genome report in 2001 (~3,000 Mb)

• Novel technology (454, pyrosequencing) made sequencing cheaper

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PNAS August 8, 2006 vol. 103 no. 32 12115–12120


The 454 technology and pyrosequencing

Major advantages• 200.000 – 400.000 reads within 4 hours• low cost per read

Major disadvantages• only short sequences• handling complicated• statistical analysis needed

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Rarefaction analysis


The rare biosphere

... or the playground of evolution?

How to quantify bacteria in sediments? · 1 © Bert Engelen How to quantify bacteria in sediments?...

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