Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg based on serological typing and sequence analysis is important in epidemiology and surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs) described, the genetic basis of the assembly of these RUs remains poorly understood and represents a major limitation in our understanding of the gene functions in the polysaccharide biosynthesis. Here we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT) controlled system to allocate the functional order of the subsequent GT in a two-fold manner. First by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs, and second by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GTs involved in the assembly of S. flexneri backbone OAg RU, RfbG is responsible for both the committed step of OAg synthesis, as well as the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria. Importance The genetic basis of enzymatic assembly of structurally diverse OAg repeating units in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to assigning glycosyltransferase (GT) functions and the order in which they act in the assembly of the OAg RU in their native background strain with reasonable confidence. We demonstrated with this approach the functional allocation of GTs for Shigella flexneri O antigen assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.