Two faces of CwlM, an essential PknB substrate, in Mycobacterium tuberculosis

Tuberculosis claims over one million lives annually and its causative agent Mycobacterium tuberculosis is a highly successful pathogen. Protein kinase B (PknB) is reported to be critical for mycobacterial growth. Here, we demonstrate that PknB-depleted M. tuberculosis can replicate normally and can synthesise peptidoglycan in osmoprotective medium. Comparative phosphoproteomics of PknB-producing and PknB-depleted mycobacteria identifies CwlM, an essential regulator of peptidoglycan synthesis, as a major PknB substrate. Our complementation studies of a cwlM mutant of M. tuberculosis support CwlM phosphorylation as a likely molecular basis for PknB being essential for mycobacterial growth. We demonstrate that growing mycobacteria produce two forms of CwlM : a nonphosphorylated membrane-associated form and a PknB-phosphorylated cytoplasmic form. Furthermore we show that the partner proteins for the phosphorylated and nonphosphorylated forms of CwlM are FhaA, a fork-head-associated domain protein, and MurJ, a proposed Lipid II flippase, respectively. From our results, we propose a model in which CwlM potentially regulates both the biosynthesis of peptidoglycan precursors and their transport across the cytoplasmic membrane.