Specific DNA Biomarkers for Two Bacterial Species Associated with Karst Aquifers and Corrosion Residues in Cave Microenvironments

Rick Fowler, Eli Roberson, Chris Groves, and Shivendra Sahi
Biotechnology Center, Department of Biology, and Hoffman Environmental Research Institute
Western Kentucky University, One Big Red Way
Bowling Green, KY 42101

Environmental DNA extracted from cave sediments contains many DNA sequences, some of which encode bacterial 16S rDNA from the cave bacterial community. Prior efforts in our laboratory have led to the creation of a database of 16S rDNA sequences from a clone library of bacterial DNA extracted from cave sediments or bacteria cultured from cave sediments. Cloned or cultured 16S rDNA sequences are aligned and compared with online software tools. Also in the database for each entry is an experimentally measured fluorescent fragment length that serves as convenient biomarker for that particular bacterium. Mixed bacterial 16S rDNA is amplified by PCR and labeled with fluorescent dyes, followed by restriction digestion to generate a profile with many fragments representing the bacterial community. The profile is interpreted with the aid of phylogenetic, sequence, and biomarker information afforded by the cave specific database and comprehensive online genetic depositories. Highly specific biomarkers have been developed to monitor both a cloned 16S gene from a possibly lithotrophic Nitrospira sp. found in cave streams as well as a cloned 16S gene from a Holophaga/Acidobacterium relative found in corrosion residues and travertine cave microenvironments. Both bacteria have been identified from the Mammoth Cave system and other caves elsewhere in Kentucky and Alabama.