Galactan polymerization in mycobacteria requires cell wall components

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) – N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2 and then is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. Cell wall core synthesis is then finalized by the action of an array of arabinosyl transferases, mycolyl transferase and ligases that catalyse an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Earlier studies proposed that galactan polymerization takes place in a specific region of the mycobacterial cell envelope, the so-called PMf domain (plasma membrane free of cell wall components) or intracellular membrane domain (IMD), which localises to the polar region of mycobacterium based on visualization of the GlfT2 enzyme fused with fluorescent tags. In this work, we examined the activity of the galactan-producing cellular machine in the enzyme fractions (cell envelope and plasma membrane) from Mycobacterium smegmatis in the cell-free system using radioactively labelled substrate (UDP-[14C]-Galactose). We found that despite a high abundance of GlfT2 in both these fractions, galactan is produced only in the reaction mixtures containing the cell envelope fraction. Our findings open the discussion about the regulation of GlfT2 with two plausible alternatives: (i) posttranslational modifications and (ii) interactions with other proteins forming an active complex.