Abstract
How specificity is programmed into post-translational modification of proteins by glycosylation is poorly understood, especially for O-linked glycosylation systems. Here we reconstitute and dissect the substrate specificity underpinning the cytoplasmic O-glycosylation pathway that modifies all six flagellins, five structural and one regulatory paralog, in Caulobacter crescentus, a monopolarly flagellated alpha-proteobacterium. We characterize the biosynthetic pathway for the sialic acid-like sugar pseudaminic acid and show its requirement for flagellation, flagellin modification and efficient export. The cognate NeuB enzyme that condenses phosphoenolpyruvate with a hexose into pseudaminic acid is functionally interchangeable with other pseudaminic acid synthases. The previously unknown and cell cycle-regulated FlmG protein, a defining member of a new class of cytoplasmic O-glycosyltransferases, is required and sufficient for flagellin modification. The substrate specificity of FlmG is conferred by its N-terminal flagellin-binding domain. FlmG accumulates before the FlaF secretion chaperone, potentially timing flagellin modification, export, and assembly during the cell division cycle.
Highlights
Post-translational protein modification is essential for various facets in cellular biology, ranging from gene regulation to the organization of cellular structures
Note that antibodies used in this blot were raised against flagellins purified from C. crescentus. (C) Capsular polysaccharide profile of S. fredii NGR234 DrkpQ mutant cells expressing different NeuB homologs from Pvan on a plasmid
Note that antibodies used in this blot were raised against flagellins purified from C. crescentus. (C) Immunoblot on C. crescentus FljK expressed in S. fredii NGR234 strains
Summary
Post-translational protein modification is essential for various facets in cellular biology, ranging from gene regulation to the organization of cellular structures. Extracellular proteinaceous surface structures including pili, flagella, and autotransporters as well as toxins are often post-translationally modified by glycosylation (Nothaft and Szymanski, 2010; Valguarnera et al, 2016; Vimr et al, 2004; De Maayer and Cowan, 2016; Miller et al, 2008; Szymanski et al, 2003; Schaffer and Messner, 2017; Goon et al, 2003; Schirm et al, 2003; Shen et al, 2006; Sulzenbacher et al, 2018; Lu et al, 2014).
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