Metagenomic Profiling of Cave Biofilms, With a Focus on Chemolithoautotrophy and Pseudonocardiaceae

The dark, humid, and nutrient depleted conditions of caves set limitations on habitability and result in microbial diversity and community structures that differ from surface environments. Recent studies investigating cave microbiomes reveal that Pseudonocardiaceae, a family of bacteria belonging to the phylum Actinomycetota, are abundant in cave sediment and on rocks as subaerial biofilms, however little work has been done on attempting to characterize their diversity and metabolism within the environment. This study used shotgun metagenomics to characterize the functional potential of Pseudonocardiaceae, as well as other cave microbes, in cave site KN14 located in Knox County, Tennessee. Functions of interest include carbon and nitrogen metabolism, antimicrobial strategies, and functions that facilitate microbe-mineral interactions. 16S rRNA gene analysis with a newer SILVA database was also utilized to expand upon previous 16S rRNA gene analysis in the cave, to understand diversity at the family and genus levels. Analysis of Metagenome Assembled Genomes (MAG’s) reveal the functional potential for chemolithoautotrophic CO₂ fixation, denitrification, trace gas uptake, antimicrobial defense, and calcite precipitation in Pseudonocardiaceae MAG’s. These findings indicate that Pseudonocardiaceae play a crucial role in cave environments by producing organic carbon at the base of the food web, acting as a sink for carbon monoxide in cave air, limiting microbial interactions in biofilms, and participating in microbe-mineral interactions that precipitate calcite. This work highlights the functional potential of a dominant microbial group found in caves, and explores innovative strategies that this group uses to thrive in an environment that is devoid of sunlight, organic carbon, and nutrients. This investigation reinforces the connection between microbial ecology and the biogeochemistry of an environment, and raises interests for possible applications in biotechnology.