MOLECULAR CHARACTERIZATION OF MICROBIAL DIVERSITY IN HYPERSALINE PONDS, SAN SALVADOR ISLAND, BAHAMAS

Non-culturing techniques are critical to describing microbial community structure and identifying novel sequences in many settings, including extreme environments such as hypersaline ponds. Although halophilic microbes are phylogenetically diverse, low genetic diversity is considered to be characteristic of microbial communities in hypersaline environments. This study employs molecular techniques to evaluate microbial diversity in microbial mats and stromatolites of hypersaline ponds on San Salvador Island, Bahamas (Salt Pond and Storr's Lake). Different microbial DNA extraction techniques were tested, as well as different primers for PCR amplification of bacterial and Archeal ribosomal RNA genes. PCR amplification products included internal portions of 16S rRNA genes and the intergenic transcribed spacer region (ITS) between the 16S and 23S genes. DNA recoveries from combinations of chemical lysis and bead beating were compared using different volumes, samples sizes, bead beating times, and purification procedures. Established protocols were modified based on these results. ITS fingerprinting was tested as a means to differentiate between closely related strains. New primers for Archea were developed and tested for PCR amplification of 16S rRNA and ITS region from natural samples and axenic cultures of Pyrobaculum and Methanobacterium. Concurrent research on the lipid composition of these samples will link genetic identification from modern environments with the more recalcitrant biomarkers preserved in older materials.