The Metabolic Usage and Glycan Destinations of GlcNAz inE. coli

Abstract

ABSTRACTBacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars such asN-acetylglucosamine (GlcNAc) being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway, and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc,N-azidoacetylglucosamine (GlcNAz), for metabolic labelling inE. coli. The cytosolic production of UDP-GlcNAz was confirmed using fluorescence assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated, was interrogated by analyzing potential sites including: peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-β-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS) and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) towards UDP-GlcNAzin vitroand lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generatedin vivoconfirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.