title: Upstream-independent ribosomal RNA amplification analysis (URA): a new approach to characterizing the diversity of natural microbial communities.
abstract: Here, we propose an advanced method for recently developed fingerprinting strategies to analyse microbial populations by direct detection of 16S rRNA sequences occurring in natural habitats. The differential display (DD) technique, which is widely used to analyse for eukaryotic gene expression, was optimized to assess bacterial rRNA diversity in environmental samples. Double-stranded cDNAs of rRNAs were synthesized without a forward primer digested with endonuclease and ligated with a double-stranded adapter. The fragments obtained were then amplified using an adapter-specific extended primer and a 16S rDNA universal reverse primer pair displayed by electrophoresis on a polyacrylamide gel. We validated this approach by characterization of a microbial community colonizing a geothermal (48 degrees C) vent system located close to the eruption zone of the south-east crater of the Mount Etna volcano, Sicily. Analysis of the patterns of abundant 16S rRNA revealed a considerable diversity of metabolically active bacteria phylogenetically clustering within the Crenarchaeota, Cyanobacteria, Firmicutes, Planctomycetales and Thermus divisions. Two sequence phylotypes were affiliated with uncultivated representatives of the recently described candidate division OP10 from a Yellowstone hot spring.
authors.affiliation: Istituto Sperimentale Talassografico, CNR, Spianata San Raineri 86, 98122 Messina, Italy. iakimov@ist.me.cnr.it
authors.forename: M M
authors.initials: MM
authors.lastname: Yakimov
authors.forename: L
authors.initials: L
authors.lastname: Giuliano
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
authors.forename: P N
authors.initials: PN
authors.lastname: Golyshin
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary; D017753:Ecosystem; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010802:Phylogeny; D012329:RNA, Bacterial / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D013696:Temperature
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S