Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis. Importance Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

Mycobacteria expand their cell walls at the cell poles in a manner that is not well described at the molecular level. In this study, we identify a new polar factor, PlrA, that is involved in restricting peptidoglycan metabolism to the cell poles in Mycobacterium smegmatis. We establish that only the N-terminal membrane domain of PlrA is essential. We show that depletion of plrA pheno-copies depletion of polar growth factor Wag31, and that PlrA is involved in regulating the Wag31 polar foci.

Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole or how it is 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 helps to control proper septation as well as new and old pole elongation. We have identified key amino acid residues involved in these essential functions. Enzyme assays revealed that Wag31 interacts with lipid metabolism by modulating acyl-CoA carboxylase (ACCase) activity. We show that Wag31 does not control polar growth by regulating the localization of cell wall precursor enzymes to the Intracellular Membrane Domain, and we also demonstrate that phosphorylation of Wag31 does not substantively regulate peptidoglycan metabolism. This work establishes new regulatory functions of Wag31 in the mycobacterial cell cycle and clarifies the need for new molecular models of Wag31 function.

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.

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.