Divergent protein motifs direct elongation factor P-mediated translational regulation in Salmonella enterica and Escherichia coli.

UNLABELLED: Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the Δefp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include both PPP and PPG but also additional motifs, such as APP and YIRYIR, which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing polyproline motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement for EF-P. This work expands the known repertoire of EF-P target motifs and implicates factors beyond polyproline motifs that are required for EF-P-mediated regulation. IMPORTANCE: Bacterial cells regulate gene expression at several points during and after transcription. During protein synthesis, for example, factors can interact with the ribosome to influence the production of specific proteins. Bacterial elongation factor P (EF-P) is a protein that facilitates the synthesis of proteins that contain polyproline motifs by preventing the ribosome from stalling. Bacterial cells that lack EF-P are viable but are sensitive to a large number of stress conditions. In this study, a global analysis of protein synthesis revealed that EF-P regulates many more proteins in the cell than predicted based solely on the prevalence of polyproline motifs. Several new EF-P-regulated motifs were uncovered, thereby providing a more complete picture of how this critical factor influences the cell's response to stress at the level of protein synthesis.