Antibiotic Resistance in Ancient Permafrost Microbial Communities

Permafrost, or permanently frozen soil, is found in polar regions and underlies over 25% of the Earth's terrestrial surface. Despite subzero temperatures, permafrost hosts a diversity of microbial life. Permafrost is an important study system because (1) thawing permafrost contributes to climate change and (2) permafrost is an analog for extraterrestrial subzero environments. Permafrost carries approximately 1,400 gigatons of carbon. Global warming is escalating temperatures in the Arctic causing wide-spread permafrost thaw, making previously frozen carbon available for microbial decomposition. Microbes release carbon stores into the atmosphere in the form of carbon dioxide and methane through respiration. Permafrost is also relevant to the field of exobiology because our solar system contains planets, moons and asteroids with analogous frozen environments. Permafrost serves as a correlative substrate in which to study some of the survival mechanisms life on extraterrestrial bodies (if it exists) must employ in order to survive extreme and subzero environments. To understand microbial community survival, we focus on a particularly important aspect of community function--antibiotic resistance. Antibiotic resistance occurs naturally among soil dwelling microbes. Antibiotic resistance genes (resistomes) are involved in community signaling and environmental sensing and play a role in competition and defense interactions. To identify antibiotic resistance genes in ancient bacterial communities that have never been exposed to synthetic antibiotics, we employ functional metagenomics. In this approach, we extract DNA directly from overlying active layer, Holocene aged permafrost frozen for 5,000 years (kyr) and Pleistocene permafrost frozen for 19kyr and clone it into a plasmid vector. A metagenomic library is thus constructed and expressed in an E. coli surrogate host. We screen for antibiotic resistance, pool resistant colonies, and sequence the antibiotic resistance genes using high-throughput next-generation sequencing. Current investigations show this method is viable for studying antibiotic resistance in microbial communities. We are applying this method to a chronosequence of active layer and permafrost soils in order to interrogate the resistomes of these ancient and modern microbial communities. We ask, what are the main mechanisms of resistance utilized in this extreme environment, and do they change over geologic time?