Contribution of PonA2 to biofilm and colony formation independent of its catalytic domains in Mycobacterium smegmatis

ABSTRACT Biofilm and colony growth create microenvironments that require coordinated localization and function of cell envelope components. However, the mechanisms by which membrane organization contributes to cell envelope organization during multicellular growth remain poorly understood. Mycobacterium smegmatis contains an inner membrane domain (IMD) at the subpolar regions of the cell that contributes to cell envelope synthesis. Prior research identified PonA2 as important for de novo formation of distinct plasma membrane domains during planktonic growth. PonA2 is a penicillin-binding protein with transglycosylase (TG) and transpeptidase (TP) activities involved in peptidoglycan synthesis. To investigate the role of PonA2 during colony and pellicle biofilm growth, wild-type, Δ ponA2 , the complement strain (c ponA2 ), and catalytically inactive PonA2 variants (TG−, TP−, and TG−/TP−) were analyzed. Subpolar IMD localization was maintained in wild-type cells during both colony and pellicle biofilm growth, demonstrating that IMD localization is not exclusive to planktonic growth. Loss of ponA2 resulted in smooth colony and early pellicle biofilm architecture and was associated with reduced abundance of trehalose monomycolate, trehalose dimycolate, and glycopeptidolipids. Δ ponA2 cells in colony growth exhibited shortened cell morphology and lacked detectable IMD localization, whereas Δ ponA2 cells in pellicle biofilm growth retained bacillary morphology and subpolar IMD localization. In contrast, the catalytically inactive PonA2 variants maintained rugose multicellular architecture, bacillary morphology, and subpolar IMD localization similar to that of wild-type. Together, these findings identify a previously unrecognized noncatalytic role for PonA2 in maintaining normal colony and pellicle biofilm development in M. smegmatis . IMPORTANCE Planktonic, pellicle biofilm, and colony growth expose mycobacteria to distinct environmental conditions that can affect cell-envelope organization and survival. Prior research has shown that mycobacteria form subpolar plasma membrane domains that support polar cell elongation in planktonic growth, but it remains unclear whether this organization is conserved in multicellular biofilm and colony growth, where cells experience nutrient and oxygen gradients and altered cell-to-cell interactions. Our current study analyzed the plasma membrane domain formation across the two growth states and showed that membrane domain localization is conserved, while the mechanisms required to maintain these domains differ depending on growth conditions. These findings suggest that mycobacteria use growth state-specific mechanisms to coordinate membrane organization.