The effect of Wag31 phosphorylation on the cells and the cell envelope fraction of wild-type and conditional mutants of Mycobacterium smegmatis studied by visible-wavelength Raman spectroscopy.

Non-surface-enhanced Raman spectroscopy using a 514.5 nm wavelength laser has been used to measure the molecular difference of conditional mutants of Mycobacterium smegmatis expressing three different alleles: wild-type wag31(Mtb), phosphoablative wag31T73A(Mtb), and phosphomimetic wag31T73E(Mtb). This study demonstrates that the phosphorylation of Wag31, a key cell-division protein, causes significant differences in the quantity of amino acids associated with peptidoglycan precursor proteins and lipid II which are observable in the Raman spectra of these cells. Raman spectra were also acquired from the isolated P60 cell envelope fraction of the cells expressing wag31T73A(Mtb) and wag31T73E(Mtb). A significant number of the molecular vibrational differences observed in the cells were also observed in the cell envelope fraction, indicating that these differences are indeed localized in the cell envelope. Principal component analyses and discriminant function analyses were conducted on these data to demonstrate the ease of spectral classification and the reproducibility of the data.

Regulation of polar peptidoglycan biosynthesis by Wag31 phosphorylation in mycobacteria.

BACKGROUND: Sensing and responding to environmental changes is a central aspect of cell division regulation. Mycobacterium tuberculosis contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis. RESULTS: We demonstrate that the differential growth among cells expressing different wag31 alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic wag31 allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway. CONCLUSIONS: The Wag31Mtb phosphorylation is a novel molecular mechanism by which Wag31Mtb regulates peptidoglycan synthesis and thus, optimal growth in mycobacteria.

Phosphorylation regulates mycobacterial proteasome.

Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome ß-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

Wag31, a homologue of the cell division protein DivIVA, regulates growth, morphology and polar cell wall synthesis in mycobacteria.

The Mycobacterium tuberculosis genome contains 11 serine/threonine kinase genes, and the products of two of these, PknA and PknB, are key components of a signal transduction pathway that regulates cell division and/or morphology. Previously, we have shown that one substrate of these kinases is Wag31, a homologue of the cell division protein DivIVA that is present, but not known to be phosphorylated, in other Gram-positive bacteria. Here, we investigate the localization and function of Wag31 and its phosphorylation. We demonstrate that Wag31 is localized to the cell poles. We further show that wag31 is an essential gene and that depletion of its product causes a dramatic morphological change in which one end of the cell becomes round rather than rod-shaped. This abnormal morphology appears to be caused by a defect in polar peptidoglycan synthesis. Finally, expression of M. tuberculosis wag31 in the wag31 conditional mutant of Mycobacterium smegmatis altered the growth rate in a manner that depended on the phospho-acceptor residue encoded by the allele being expressed. Taken together, these results indicate that Wag31 regulates cell shape and cell wall synthesis in M. tuberculosis through a molecular mechanism by which the activity of Wag31 can be modulated in response to environmental signals.