PonA2?s contributions to biofilm and colony formation independent of its catalytic domains in Mycobacterium smegmatis.

Abstract

Biofilm and colony growth creates microenvironments that require coordinated localization and function of cell surface molecules within the membrane. However, the mechanisms by which membrane organization regulates these surface molecules during these biofilm-associated growth are poorly understood, limiting our understanding of how bacteria adapt and survive in multicellular communities. Mycobacterium smegmatis contains an inner membrane domain (IMD) at the subpolar regions of the cell that helps mediate cell envelope synthesis. Prior research has identified ponA2 as critical for de novo formation of distinct plasma membrane domains in planktonic growth. PonA2 is a penicillin-binding protein that catalyzes peptidoglycan synthesis by transglycosylase (TG) and transpeptidase (TP) activities. To investigate the role of PonA2 in membrane domain organization in biofilm and colony growth, wild-type, {Delta}ponA2, the complement strain (cponA2), and catalytic inactive variants of PonA2 (TG-, TP-, and TG-/TP-) were analyzed. The IMD subpolar localization in wild-type was preserved in biofilm and colony growth, indicating that IMD localization is not exclusive to planktonic growth. Both biofilm and colony growth of {Delta}ponA2 showed significant structural deformities compared to wild-type. In contrast, the catalytic inactive mutants produced biofilm and colony structures that resembled wild-type, suggesting that PonA2 has additional noncatalytic functions during multicellular growth. The IMD localization of the catalytic inactive mutants was minimally impacted, suggesting that neither catalytic domain is required for IMD localization in biofilm and colony growth. Together, these findings advance our understanding of the complex mycobacterial membrane biology. ImportancePlanktonic, 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.