EF-P Posttranslational Modification Has Variable Impact on Polyproline Translation in Bacillus subtilis

: Elongation factor P (EF-P) is a ubiquitous translation factor that facilitates translation of polyproline motifs. In order to perform this function, EF-P generally requires posttranslational modification (PTM) on a conserved residue. Although the position of the modification is highly conserved, the structure can vary widely between organisms. In Bacillus subtilis, EF-P is modified at Lys32 with a 5-aminopentanol moiety. Here, we use a forward genetic screen to identify genes involved in 5-aminopentanolylation. Tandem mass spectrometry analysis of the PTM mutant strains indicated that ynbB, gsaB, and ymfI are required for modification and that yaaO, yfkA, and ywlG influence the level of modification. Structural analyses also showed that EF-P can retain unique intermediate modifications, suggesting that 5-aminopentanol is likely directly assembled on EF-P through a novel modification pathway. Phenotypic characterization of these PTM mutants showed that each mutant does not strictly phenocopy the efp mutant, as has previously been observed in other organisms. Rather, each mutant displays phenotypic characteristics consistent with those of either the efp mutant or wild-type B. subtilis depending on the growth condition. In vivo polyproline reporter data indicate that the observed phenotypic differences result from variation in both the severity of polyproline translation defects and altered EF-P context dependence in each mutant. Together, these findings establish a new EF-P PTM pathway and also highlight a unique relationship between EF-P modification and polyproline context dependence.IMPORTANCE Despite the high level of conservation of EF-P, the posttranslational modification pathway that activates EF-P is highly divergent between species. Here, we have identified and characterized in B. subtilis a novel posttranslational modification pathway. This pathway not only broadens the scope of potential EF-P modification strategies, but it also indicates that EF-P modifications can be assembled directly on EF-P. Furthermore, characterization of these PTM mutants has established that an altered modification state can impact both the severity of polyproline translational defects and context dependence.