Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii

Abstract

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from 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 Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete operational taxonomic units (OTUs). The rank order abundance of these putative OTUs was measured, and the two most abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Among 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 majority of taxa represented in the clone library were detected, a result which we assume to be an estimate of the diversity of bacteria in the native hydrothermal vent habitat. 16S rDNA fingerprinting of individual clones belonging to particular OTUs by using an oligonucleotide probe that binds to a universally conserved region of the 16S rDNA fragments was conducted to confirm OTU specificity and 16S rDNA identity.

High unique diversity of sulfate‐reducing prokaryotes characterized in a depth gradient in an acidic fen

“…Sorting the cloned sequences into OTUs is a useful method to optimize detection of distinct sequences (Moyer et al, 1994). The number of OTUs varied from 23 to 37 in the three clone libraries (84 OTUs altogether).…”

Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13°N)

“…The PCR cycles (1 or 2 rounds for DHVE2 and DHVE8 amplicons, respectively) consisted in 1 cycle at 95∞C for 5 min, 35 cycles at 95∞C for 1 min, 56∞C for 1 min, 72∞C for 1.25 min and a final cycle at 72∞C for 5 min. Positive amplicons were cloned and categorized by RFLP (Moyer et al, 1994) using Hae III and Hha I for DHVE2 and DHVE8 sequences, respectively. Unique clones were sequenced as described above.…”

Phylogenetic characterization of the bacterial assemblage associated with mucous secretions of the hydrothermal vent polychaete Paralvinella palmiformis

“…A total of 141 insertcontaining clones for the CASM library and of 143 for the ASHES one were screened by ARDRA using two restriction enzymes with tetra-nucleotide recognition sequences. As demonstrated by a precedent study [43], RFLP (and consequently ARDRA) patterns created by the use of tandem restriction endonucleases, with tetra-nucleotide recognition sequences, allow the detection of the wide majority of the taxa present in a library. The use of two restriction enzymes signi¢cantly decreases the probability to retrieve di¡erent sequences types within a given phylotype.…”