Award Dates: December 31, 2016 — December 30, 2018
Extreme energy limitation in deep marine sediments constrains microbial life to unprecedentedly low metabolic activity, low biomass and slow turnover rates. The questions of i) what fraction and ii) which members of microbial consortia in deep sediments are “alive” vs. “dead” is of insurmountable importance, yet, it is not an easily tractable issue. Here, I propose two independent methods, based on membrane integrity and DNA repair, to discriminate amongst fossil and biologically relevant genes. One approach uses Propidium Monoazide (PMA). Impermeability of intact prokaryotic membranes to PMA is exploited to discriminate between microbial community members with and without membrane cohesion. A second approach employs Uracil N-glycosylase (UNG). UNG enzymatic digestion of damaged DNA, defined as DNA containing unrepaired uracil bases resulting from hydrolytic deamination of cytosine, will serve as a discriminative step resulting in exclusive sequencing of genes from extant microorganisms. By combining these approaches, I will i) produce accurate high-throughput gene surveys of extant community members, ii) retain necromass-derived genes comprising the “paleome” and iii) assess potential biases in existing non-discriminative sequencing-based datasets.