ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance: Structural and Functional Diversification across the Tree of Life

Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterised VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase centre and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance factors from other ABCFs have not been explored. To address this, we analysed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting they were present in the last common ancestor of both. Surprisingly, currently known antibiotic resistance (ARE) ABCFs are not confined to a distinct lineage of the ABCF family tree. This suggests that either antibiotic resistance is a pervasive feature of bacterial ABCFs, or it is relatively easy to evolve antibiotic resistance from other ABCF functions. Our data suggest there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that Bacillus subtilis VmlR is a ribosome-binding resistance factor localised to the cytoplasm. Author summary Isolated members of the ABCF protein family of ATP-hydrolysing enzymes have been found to have important roles in protein synthesis and antibiotic resistance. However, their full diversity across the tree of life, and their evolutionary histories have never been examined. Therefore, we analysed the presence of ABCFs and their constituent domains in genomes across the tree of life, discovering 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla. This includes several subfamilies that we predict comprise novel antibiotic resistance (ARE) ABCFs, present in antibiotic producers and important human pathogens. There are significant gaps in our knowledge about the functional capabilities of different ABCF families. To address this, we have made ATPase domain mutants of all four Escherichia coli ABCFs, showing that they inhibit protein synthesis and indicating a role on the ribosome. Furthermore, we demonstrate a genetic interaction of two E. coli ABCFs with the GTPase BipA, involved in ribosome assembly. Finally, we show that Bacillus subtilis VmlR in the ARE2 subfamily is a ribosome-binding resistance factor localised to the cytoplasm. As more is discovered about the function of individual ABCFs, the more it will be possible to predict functions of uncharacterised members, using the ABCF family tree as a framework.