| الوصف: |
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. Wag31 homo-oligomerizes in a network at the poles, but it is not known how the structure of this network affects Wag31 function. In this study we used a protein fragment complementation assay to identify Wag31 residues involved in homo-oligomeric interactions, and found that amino acids all along the length of the protein mediate these interactions. We then used both N-terminal and C-terminal splitGFP fusions to probe Wag31 network topology at different sites in the cell, and found that Wag31 C-terminal-C-terminal interactions predominate at the septa, while C-terminal-C-terminal and C-terminal-N-terminal interactions are found equally at the poles. This suggests the Wag31 network is formed through an ordered series of associations. We then dissected Wag31’s functional roles by phenotyping a series of wag31 alanine mutants; these 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. This work establishes new functions for Wag31, and indicates that changes in Wag31 homo-oligomeric network topology may contribute to cell wall regulation in mycobacteria.ImportanceMany bacteria restrict cell wall elongation to their cell poles, but it is not known how polar growth is affected on the molecular level. Wag31 is a protein that is required for this polar elongation. In this work, we show that Wag31 actually has at least four distinct functions in regulating the cell wall: it promotes elongation at both poles in different ways, and it can also inhibit cell wall metabolism at the new pole and the septum. In addition, we propose a new model for how Wag31 self-associates into a protein network. This work is important because it shows that a DivIVA homolog can have distinct functions depending on cell context. And, this work clarifies that Wag31 is doing several different things in the cell, and gives us genetic tools to disentangle its functions. |
