Phosphorylation of Wzc, a Tyrosine Autokinase, Is Essential for Assembly of Group 1 Capsular Polysaccharides in Escherichia coli

Abstract

Wzc proteins are tyrosine autokinases. They are found in some important bacterial pathogens of humans and livestock as well as plant-associated bacteria, and are often encoded within gene clusters determining synthesis and assembly of capsular and extracellular polysaccharides. Autophosphorylation of Wzccps is essential for assembly of the serotype K30 group 1 capsule in Escherichia coli O9a:K30, although a genetically unlinked Wzc cps-homologue (Etk) can also participate with low efficiency. While autophosphorylation of Wzccps is required for assembly of high molecular weight K30 capsular polysaccharide, it is not essential for either the synthesis of the K30 repeat units or for activity of the K30 polymerase enzyme. Paradoxically, the cognate phosphotyrosine protein phosphatase for Wzccps, Wzbcps, is also required for capsule expression. The tyrosine-rich domain at the C terminus of Wzccps was identified as the site of phosphorylation and autophosphorylation of Wzc requires a functional Walker A motif. Intermolecular transphosphorylation of Wzc cps was detected in strains expressing a combination of mutant Wzccps derivatives. The N- and C-terminal domains of Wzc cps were expressed independently to mimic the situation found naturally in Gram-positive bacteria. In this format, both domains were required for phosphorylation of the Wzccps C terminus, and for capsule assembly. Regulation by a post-translational phosphorylation event represents a new dimension in the assembly of bacterial cell-surface polysaccharides.