Ordered assembly of the Vibrio cholerae biofilm exopolysaccharide defined by lipid-linked intermediate profiling

Biofilm formation underlies the environmental persistence and transmission of Vibrio cholerae, the etiological agent of cholera. The Vibrio polysaccharide (VPS) is the principal structural component of the mature biofilm matrix, yet the enzymatic logic governing its assembly has remained incompletely defined. VPS is synthesized as two closely related polymers that share a tetrasaccharide repeat unit but differ at a single monosaccharide position. Here, we systematically define VPS assembly by integrating targeted gene deletions with liquid chromatography-mass spectrometry profiling of lipid-linked intermediates, and comparative structural modeling of biosynthetic enzymes. Our results establish that VPS is produced through an ordered Wzx/Wzy-dependent pathway. VpsL functions as the initiating phosphoglycosyltransferase, generating bactoprenyl diphosphate-linked glucose. A VpsA/VpsB/VpsK module analogous to the enterobacterial common antigen machinery synthesizes and transfers an N-acetyl-mannosaminuronic acid (ManNAcA)-derived residue, after which VpsJ, which we propose as a new class of epimerase, catalyzes C5 epimerization to generate the rare bacterial sugar L-N-acetyl-gulosaminuronic acid (L-GulNAcA). Additional tailoring reactions mediated by VpsH, a previously unidentified protein with few sequence or structural homologs, and VpsG introduce glycine and acetyl modifications that are dispensable for repeat-unit assembly but influence matrix properties. Subsequently, glycosyltransfer reactions by VpsI and VpsD complete the tetrasaccharide repeat unit, with VpsD exhibiting substrate flexibility that accounts for the formation of both major and minor VPS variants. Downstream, VpsE and VpsF act following repeat-unit assembly, consistent with flippase and polymerase functions, respectively. Together, our findings establish a molecular framework for VPS assembly and deepen our understanding of the mechanisms that drive biofilm formation in Vibrio cholerae.IMPORTANCEBiofilm formation is an integral part of Vibrio cholerae's infection cycle, requiring production of the exopolysaccharide Vibrio polysaccharide (VPS). Together, these findings define the sequential enzymatic steps of VPS biosynthesis. This molecular map of VPS production identifies multiple enzymatic nodes as potential anti-biofilm targets and provides a mechanistic foundation for understanding how V. cholerae modulates biofilm architecture to enhance environmental survival and transmission.