SummaryThe function of nearly half of all protein-coding genes identified in bacterial genomes remains unknown. To systematically explore the functions of these proteins, we generated saturated transposon mutant libraries from 25 diverse bacteria and we assayed mutant phenotypes across hundreds of distinct conditions. From 3,903 genome-wide mutant fitness assays, we obtained 14.9 million gene phenotype measurements and we identified a mutant phenotype for 8,487 proteins with previously unknown functions. The majority of these hypothetical proteins (57%) had phenotypes that were either specific to a few conditions or were similar to that of another gene, thus enabling us to make informed predictions of protein function. For 1,914 of these hypothetical proteins, the functional associations are conserved across related proteins from different bacteria, which confirms that these associations are genuine. This comprehensive catalogue of experimentally-annotated protein functions also enables the targeted exploration of specific biological processes. For example, sensitivity to a DNA-damaging agent revealed 28 known families of DNA repair proteins and 11 putative novel families. Across all sequenced bacteria, 14% of proteins that lack detailed annotations have an ortholog with a functional association in our data set. Our study demonstrates the utility and scalability of high-throughput genetics for large-scale annotation of bacterial proteins and provides a vast compendium of experimentally-determined protein functions across diverse bacteria.
24In extreme environments, toxic compounds restrict which microorganisms persist. 25However, in complex mixtures of inhibitory compounds, it is challenging to determine 26 which specific compounds cause changes in abundance and prevent some microorganisms 27 from growing. We focused on a contaminated aquifer in Oak Ridge, Tennessee, U.S.A. 28that has low pH and high concentrations of uranium, nitrate and many other inorganic ions. 29In the most contaminated wells, the microbial community is enriched in the Rhodanobacter 30 genus. Rhodanobacter relative abundance is positively correlated with low pH and high 31 concentrations of U, Mn, Al, Cd, Zn, Ni, Co, Ca, NO 3 -, Mg, Cl, SO 4 2-, Sr, K and Ba and we 32 sought to determine which of these correlated parameters are selective pressures that favor 33 the growth of Rhodanobacter over other taxa. Using high-throughput cultivation, we 34 determined that of the ions correlated high Rhodanobacter abundance, only low pH and 35 high U, Mn, Al, Cd, Zn, Co and Ni (a) are selectively inhibitory of a sensitive Pseudomonas 36 isolate from a background well versus a representative resistant Rhodanobacter isolate 37 from a contaminated well, and (b) reach toxic concentrations in the most contaminated 38 wells that can inhibit the sensitive Pseudomonas isolate. We prepared mixtures of 39 inorganic ions representative of the most contaminated wells and verified that few other 40 isolates aside from Rhodanobacter can tolerate these 8 parameters. These results clarify 41which toxic inorganic ions are causal factors that impact the microbial community at this 42 field site and are not merely correlated with taxonomic shifts.
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