Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum

Abstract

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.

MmpL3, Wag31 and PlrA are involved in coordinating polar growth with peptidoglycan metabolism and nutrient availability

“…However, our data indicate that polar localization of MmpL3 does not correlate with polar growth, as the wag31 K20A mutant is defective in old pole elongation (28) but has increased old pole localization of MmpL3, while the L34A mutant is defective in new pole elongation and has decreased new pole localization of MmpL3.…”

“…In an effort to better understand the molecular function of Wag31, we looked for factors that were genetically linked to wag31 by conducting a suppressor screen. We had previously generated a number of wag31 partial loss of function point mutants (28). We passaged several slow-growing wag31 point mutants - D7A, K20A, and L34A - for several days, struck them out regularly and looked for large colony mutants.…”

A cell wall synthase accelerates plasma membrane partitioning in mycobacteria

“…We hypothesized that the ability of M. smegmatis to recover from benzyl alcohol depends at least in part on its ability to reform the IMD, and therefore, that genes that promote membrane partitioning would constitute a subset of the genes that enable M. smegmatis to tolerate and/or recover from benzyl alcohol. In mycobacteria, the essential, tropomyosin-like protein Wag31 (DivIVA) promotes polar cell wall assembly and rod shape and promotes IMD protein localization and isolability ( García-Heredia et al, 2018 ; Habibi Arejan et al, 2022 ; Jani et al, 2010 ; Kang et al, 2008 ; Melzer et al, 2018 ). While DivIVA depletion slowed M. smegmatis growth, as expected, we observed only a small additional growth defect when bacteria were exposed to benzyl alcohol ( Figure 2—figure supplement 1 ).…”

The interplay between the polar growth determinant DivIVA, the segregation protein ParA, and their novel interaction partner PapM controls the Mycobacterium smegmatis cell cycle by modulation of DivIVA subcellular distribution

“…DivIVA was shown to control the activity of the enzymes involved in the cell wall synthesis ( 25 , 26 ). At the latest stages of cell division, DivIVA is recruited to the division site where it interacts with divisome protein PBP3 ( 27 , 28 ). It was also suggested that the other DivIVA interaction partners CwsA and CrgA may contribute to DivIVA relocation ( 29 ).…”

“…The late recruitment of DivIVA at the cell division site suggests its involvement in constituting the new cell poles ( 11 , 19 ). DivIVA mutational analysis suggested that DivIVA activity involves different interactions at the new pole, the old pole, and the septum ( 27 ). However, the details of the DivIVA role in the formation of the new poles are still to be investigated.…”