Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes

The diversity of microorganisms associated with the leaves of the seagrass Halophila stipulacea in the northern Gulf of flat was examined by culture-independent analysis. Microorganisms were harvested by a sonication treatment for total-community genomic DNA isolation. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used for PCR amplification. The 16S rDNA PCR products were subcloned and further characterized by a restriction fragment length analysis termed ARDRA (amplified rDNA restriction analysis). These analyses were carried out after reamplifying the cloned fragments with two primers binding symmetrically to the plasmid immediately on both sides of the cloned insert. Computer-aided clustering was performed after separate restriction analysis with enzymes HinfI: and HpaII. By this method, 103 cloned 16S rDNA fragments were clustered into a total of 58 different groups. Sequence analysis of clones with an identical ARDRA pattern confirmed that members of an ARDRA group were closely related to each other. The sequenced clones were found to be affiliated with a marine snow-associated plastid-like rRNA clone and with a marine Hyphomonas strain, respectively. The method applied in this study could be useful for the routine study of other microbial communities of interest.