ESTIMATION OF DIVERSITY AND COMMUNITY STRUCTURE THROUGH RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM DISTRIBUTION ANALYSIS OF BACTERIAL 16S RIBOSOMAL-RNA GENES FROM A MICROBIAL MAT AT AN ACTIVE, HYDROTHERMAL VENT SYSTEM, LOIHI SEAMOUNT, HAWAII

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes f rom total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at L oihi Seamount, Hawaii. Oligonucleotides complementary to conserved reg ions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to dire ct the synthesis of PCR products, which were then subcloned by blunt-e nd ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restr iction endonucleases, revealed the presence of 12 groups of 16S rRNA g enes representing discrete operational taxonomic units (OTUs). The ran k order abundance of these putative OTUs was measured, and the two mos t abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Amo ng the remaining 27.1% of the 16S rDNA clones, none of the 10 OTUs was represented by more than three individual clones. The cumulative OTU distribution for 48 bacterial 16S rDNA clones demonstrated that the ma jority of taxa represented in the clone library were detected, a resul t which we assume to be an estimate of the diversity of bacteria in th e native hydrothermal vent habitat. 16S rDNA fingerprinting of individ ual clones belonging to particular OTUs by using an oligonucleotide pr obe that binds to a universally conserved region of the 16S rDNA fragm ents was conducted to confirm OTU specificity and 16S rDNA identity.