CARBON-, NITROGEN-, AND SULFUR-CYCLING CAPABILITIES OF ENIGMATIC MICROORGANISMS FROM ENERGY-LIMITED DESERT CAVES

Caves are windows into the subsurface through which we can directly evaluate the microbiological processes responsible for rock weathering and biogeochemical cycling in more expansive areas of Earth’s subsurface. Over long timescales, microorganisms that chemical energy from spare mineral or trace gas resources may modify cave walls by slow carbonate dissolution or secondary mineral formation. However, many of these organisms are from uncultivated groups and therefore represent microbial “dark matter” with genomic capabilities that cannot be resolved from marker gene surveys alone. We therefore generated metagenomes from low biomass cave wall samples from two desert caves, Lehman Caves in Nevada and Lechuguilla Cave in New Mexico, both shown to harbor novel microorganisms in marker gene surveys. We initially produced two small metagenomes: one from Lehman Caves with 39 Mbp of sequence data, and one from a Lechuguilla Cave sample collected more than 20 years ago with 219 total Mbp. The metagenomes contained genes for carbon dioxide fixation, dissimilatory ammonia oxidation, and methanotrophy. While classification of marker genes showed that the libraries contained previously undescribed microorganisms, they did not have sufficient depth of coverage for binning. We therefore generated a larger metagenome (9 Gb) from a second Lehman Caves sample, and were able to recover several high-quality metagenome-assembled genomes (MAGs). One of these high-quality MAGs represents a gammaproteobacterium from a group known as “wb1-P19” that is abundant in caves worldwide. The wb1-P19 MAG does not encode RuBisCO or other known pathways for autotrophy, and instead appears to be a heterotroph that is capable of aerobic and anaerobic growth using O₂ and NO₃^- as electron acceptors. It appears to be either an obligate or a facultative methanotroph that encodes pmoA and other genes necessary for the oxidation of methane and other small organic molecules, and may be an important consumer of trace gases in cave ecosystems globally. We will finish by sharing preliminary results from a course-based undergraduate research experience (CURE) where students enriched and isolated microorganisms from Cottonwood Cave, New Mexico, in order to attempt to cultivate some of the elusive microorganisms that limit our understanding of the microbial biogeochemistry in caves.