LAUSR

Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole and organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. In this study we used alanine scanning mutagenesis to identify Wag31 residues involved in cell morphogenesis. Our data show that Wag31 has separate functions in not only new and old pole elongation, but also inhibition of both septation and new pole elongation. Our examination of phospho-ablative and phospho-mimetic mutants of Wag31 suggests that phosphorylation of Wag31 promotes old pole elongation, while the unphosphorylated form of Wag31 may promote resolution of the septum. This work establishes new regulatory functions of Wag31 in the mycobacterial cell cycle and clarifies the role of phosphorylation on Wag1. Importance Despite many previous studies, the molecular mechanisms of polar growth in mycobacteria is unclear. Wag31 is required for this polar elongation. In this work, we dissect Wag1 function by phenotyping wag31 point mutants. We find that Wag31 promotes elongation at both poles in different ways, and it can also inhibit cell wall metabolism at both the new pole and the septum. This work is important because it clarifies that Wag31 is doing several different things in the cell and gives us genetic tools to disentangle its functions.