A novel strategy to extract specific phylogenetic sequence information from community T-RFLP.
Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.
Widmer
Franco
F
Molecular Ecology, Agroscope FAL Reckenholz, Swiss Federal Research Station for Agroecology and Agriculture, Zurich, Switzerland. franco.widmer@fal.admin.ch
Hartmann
Martin
M
Frey
Beat
B
Kölliker
Roland
R
D001483:Base Sequence; D000458:Cyanobacteria / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019003:Deoxyribonuclease HpaII / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D017931:DNA Primers; D004269:DNA, Bacterial; D019003:Deoxyribonuclease HpaII