1. Profiling microbial communities with T-RFLP
(terminal restriction fragment length polymorphism)
Anne Fahy

2. why microbial ecology?
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)
huge metabolic diversity
“higher” organisms dependent on microbial activities
applications: bioremediation and natural attenuation of pollutants in the environment

3. why culture-independent?
between and 1 % microorganisms are culturable (Amann 1995)
microbial communities are complex:
huge diversity
close interactions between organisms
highly dynamic
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

4. why 16S rRNA as a phylogenetic marker ?
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)
secondary structure of the Escherichia coli 16S rRNA molecule (Van de Peer, et al. 1996).
colours  variability between organisms:
pink = highly conserved
red = least conserved
grey = unaligned

5. other phylogenetic markers
proteins; difficult to identify homologous proteins (Demoulin, 1979)
historically: also 5S, 23S rRNA
ribosomal intergenic spacer: 16S – 23s
18S for Eukaryotes

6. (Terminal Restriction Fragment Length Polymorphism)
T-RFLP
(Terminal Restriction Fragment Length Polymorphism)
1 extraction of community DNA or RNA from environmental sample
(need RT-PCR step with RNA)
3 digestion of amplicons with restriction enzyme
2 PCR amplification of 16S rRNA gene with fluorescent primers
4 detection and sizing of labelled terminal fragments by capillary or gel electrophoresis

7. raw data
T-RFLP (2)
Red: internal size standard Blue: forward primer Green: reverse primer

8. T-RFLP (3) Analysis of raw data with Genescan:
virtual filter: adjust overlap of fluorescence
sizing: standard curve
integration of peaks
size calling curve

9. T-RFLP (4) Electropherogram: a visual profile of the community.
T-RF length in nucleotides
relative peak height
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.

10. T-RFLP : resolution
several groups of organisms may share the same T-RF
T-RFs need to be within range of size standard
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
several digests + combine data  increase resolution

11. 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)

12. 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

13. 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

14. 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

15. limitations inherent to T-RFLP (3)
overloading of the capillary  rerun sample
12 seconds injection
3 seconds injection
6 seconds injection d

16. 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

17. limitations inherent to T-RFLP (5)
sizing problems
causes:
- apparent size varies with the type of genetic analyser: a 142 nt fragment will measure and 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

18. limitations inherent to T-RFLP (6)
sizing problems
- 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

19. limitations inherent to T-RFLP (7)
sizing problems
very abnormal migration from a specific clone T-RF:
possible “hairpin” from secondary structure
- no such discrepancy from other clones with same sequence immediately preceding the restriction site
in press: Nogales et al.
(a study of mobility anomalies of 16S rRNA gene fragments)

20. 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!

21. many community profiling techniques
Techniques based on PCR of rDNA
Cloning 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 profiling
Hybridisation, FISH, PLFA, BIOLOG
Linking metabolic function to phylogeny
SIP (stable isotope probing)