Abstract
The mycobacterial cell wall is profoundly regulated in response to environmental stresses, and this regulation contributes to antibiotic tolerance. The reversible phosphorylation of different cell wall regulatory proteins is a major mechanism of cell wall regulation. Eleven serine/threonine protein kinases phosphorylate many critical cell wall-related proteins in mycobacteria. PstP is the sole serine/ threonine phosphatase, but few proteins have been verified as PstP substrates. PstP is itself phosphorylated, but the role of its phosphorylation in regulating its activity has been unclear. In this study, we aim to discover novel substrates of PstP in Mycobacterium tuberculosis (Mtb). We show in vitro that PstP dephosphorylates two regulators of peptidoglycan in Mtb, FhaA, and Wag31. We also show that a phosphomimetic mutation of T137 on PstP negatively regulates its catalytic activity against the cell wall regulators FhaA, Wag31, CwlM, PknB, and PknA, and that the corresponding mutation in Mycobacterium smegmatis causes misregulation of peptidoglycan in vivo. We show that PstP is localized to the septum, which likely restricts its access to certain substrates. These findings on the regulation of PstP provide insight into the control of cell wall metabolism in mycobacteria.