Profiling microbial communities

with T-RFLP

(terminal restriction fragment length polymorphism)

Anne Fahy

b- Ti 01

a- Ti 02

c- Pi 02

d- Pi 01

e- Ud 02

f- Ud 01

why microbial ecology?

huge metabolic diversity

“higher” organisms dependent on microbial activities

applications: bioremediation and natural attenuation of pollutants in the environment

Microbial organisms occupy a peculiar place in the human view of life. Microbes receive little attention in our general texts of biology. They are

largely ignored by most professional biologists and are virtually unknown to the public except in the context of disease and rot. Yet, the workings of

the biosphere depend absolutely on the activities of the microbial world. (Pace, 1997)

between 0.001 and 1 % microorganisms are culturable (Amann 1995)

microbial communities are complex:

huge diversity

close interactions between organisms

highly dynamic

why culture-independent?

phylogenetic tree based on 16S rRNA : major phyla of the domain Bacteria (Rappé & Giovannoni, 2003)black = 12 original phyla described by Woese, 1987 white = 14 phyla with isolated representatives grey = 26 candidate phyla with no known isolates

why 16S rRNA as a phylogenetic marker ?

secondary structure of the Escherichia coli 16S rRNA molecule (Van de Peer, et al. 1996). colours variability between organisms: pink = highly conserved red = least conservedgrey = unaligned

- protein translation : universal

- no horizontal transfer (caveat: Wang & Zhang, 2000)

- convenient length : 1500 bp

- highly conserved regions as well as species-specific regions

- large databases (EMBL, NCBI, DDJB)

other phylogenetic markers

- proteins; difficult to identify homologous proteins (Demoulin, 1979)

- historically: also 5S, 23S rRNA

- ribosomal intergenic spacer: 16S – 23s

- 18S for Eukaryotes

T-RFLP(Terminal Restriction Fragment Length Polymorphism)

1 extraction of community DNA or RNA from environmental sample (need RT-PCR step with RNA)

2 PCR amplification of 16S rRNA gene with fluorescent primers

3 digestion of amplicons with restriction enzyme

4 detection and sizing of labelled terminal fragments by capillary or gel electrophoresis

T-RFLP (2)raw data

Red: internal size standard Blue: forward primer Green: reverse primer

T-RFLP (3)

Analysis of raw data with Genescan:

virtual filter: adjust overlap of fluorescence

sizing: standard curve

integration of peaks

size calling curve

T-RFLP (4)

relative peak

height

T-RF length in nucleotides

Electropherogram: a visual profile of the community.

In principle, the height and area of the peaks are representative of the abundance of the groups of organisms.Several groups of organisms may share the same T-RF.

Table: digital data can be further processed and used, for example, to generate dendrograms illustrating the relationship between bacterial communities.

- several groups of organisms may share the same T-RF

- T-RFs need to be within range of size standard

T-RFLP : resolution

resolution of T-RFLP depends on the choice of restriction enzyme / primer combination

Ribosome Database Project:

TAP-TRFLP application

enter choice of enzyme/primer

in silico digestion of 16S rRNA on the database

http://rdp8.cme.msu.edu/html/TAP-trflp.html#program

several digests + combine data increase resolution

T-RFLP : good technique

- reproducible technique

- relatively fast monitor community dynamics

- culture-independent

- digital data for further analyses

- link data to clone libraries

Rs + PO4

Rhodoferax antarcticus ( Proteobacteria, Comamonadaceae)

but…..…. need to look at data to avoid pitfalls and know the limitations

sources of biases (von Wintzingerode et al., 1997):

- experimental design

- sampling

- storage of sample

- DNA extraction

- PCR amplification (loads of literature!)

keep experimental procedures constant

PCR-based techniques provide information that is not obtainable through other methods

limitations inherent to T-RFLP

- glitches in the electrophoresis rerun sample

- incomplete digestion (partially single-stranded amplicons; Egert & Friedrich, 2003) be aware

clone M232: 3 hours digestion

clone M232: 15 hours digestion

limitations inherent to T-RFLP (2)

renaturation of sample:

salts in buffer

amount of DNA in sample

delay between denaturation and electrophoresis

rerun sample

renaturation of internal size standard

limitations inherent to T-RFLP (3)

overloading of the capillary rerun sample

12 seconds injection

3 seconds injection

6 seconds injection

d

d

limitations inherent to T-RFLP (4) sizing problems

discrepancy between:

expected T-RF (from in silico digestion of known sequence) apparent T-RF (from electrophoresis)

caution when interpreting T-RFLP profiles

limitations inherent to T-RFLP (5) sizing problems

causes:

- apparent size varies with the type of genetic analyser: a 142 nt fragment will measure 143.4 and 140.6 nt respectively on a gel or capillary genetic analyser (GeneScan Reference Guide)

- resolution: decreases as fragment length increases

- ROX label of internal standard migrates more slowly than the FAM label of the forward primer (Boorman et al., 2002)

- apparent size of fragment depends on its secondary structure

limitations inherent to T-RFLP (6) sizing problems

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2

4

6

8

10

12

25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425

Expected fragment size in nucleotide (CfoI )

Dif

fere

nce

in s

ize

(nt)

- difference ± proportional to fragment length

- can vary from -2 to -4 nt for very similar length of fragment

- outside range of size standard: can’t size accurately

- abnormal migration

expected size apparent size difference

AluI 109 105.8 3.2

CfoI 114 103.0 11.0

limitations inherent to T-RFLP (7) sizing problems

- possible “hairpin” from secondary structure

- no such discrepancy from other clones with same sequence immediately preceding the restriction site

very abnormal migration from a specific clone T-RF:

in press: Nogales et al.

(a study of mobility anomalies of 16S rRNA gene fragments)

-----CGGAACGTGCCCAGTCGTGGGGGATAACGCAGC G ------CGGAACGTGCCCAGTCGTGGGGGATAACGCAGCGA ------CGGAACGTGCCCAGTCGTGGGGGATAA GCGTCGGA ------CGGAACGTGCCCAGTCGTGGGGGATAAGCGTCGA

limitations inherent to T-RFLP (8) Conclusions:

T-RFLP very reproducible (electropherograms need to be perfect)

comparison of data limited to studies using same type of genetic analyser

cannot predict phylogenetic affiliations from the length of the T-RFs

within its limitations, T-RFLP is a good culture-independent technique for profiling microbial communities!

many community profiling techniques

Techniques based on PCR of rDNACloning and sequencing of 16S rDNA DGGE (denaturing gradient gel electrophoresis)SSCP (single strand conformation polymorphism)RFLP (restriction fragment length polymorphism)LH-PCR (length heterogeneity analysis by PCR)ARISA (automated ribosomal intergenic spacer analysis)DGGE and T-RFLP also used for diversity of catabolic genes

Other approaches to community profilingHybridisation, FISH, PLFA, BIOLOG

Linking metabolic function to phylogenySIP (stable isotope probing)