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

2. 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)

3.  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

4. 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 conserved grey = 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)

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. 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

7. T-RFLP (2) raw data 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) 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.

10. - 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

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 + PO 4 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 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 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

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 - 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)

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)