The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis underpinning the assembly of diverse RUs remains poorly understood.
OAg polysaccharide synthesis is carried out by enzymes that catalyse the synthesis of glycans, named glycosyltransferases (GTs), on the universal lipid carrier undecaprenol phosphate (UndP), which is also shared in the synthesis of other polysaccharides, as well as cell wall peptidoglycan (PG). There is a finite amount of UndP at any given time in the cell that is shared by efficient recycling across different cell envelope synthesis pathways. Disruption of UndP recycling is thus detrimental to bacteria survival as it affects the synthesis of the essential cell envelope component PG. During OAg polysaccharide synthesis, UndP is committed at the second glycosyltransferase (2nd GT) step, and remains engaged in OAg synthesis until its release during the OAg polymerisation step (catalysed by Wzy) and the ligation step (catalysed by WaaL), when it gets recycled back to cellular pool. Therefore, any disruption after the 2nd GT and before the Wzy/WaaL synthesis steps, will lead to lethality; a phenotype that we have exploited here to identify the GT that catalyses the committed step.
Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT) controlled system, which allows functional order allocation of the subsequent GT in a two-fold manner: i) first by reporting the growth defects caused by the sequestration of UndP through genetic disruption of late GTs, and ii) second by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly, in that we assigned RfbG as both the 2nd and 3rd GT, and RfbF as the last GT for S. flexneri OAg biogenesis. This approach can be also applied in interrogating GT functions encoded by other polysaccharide gene clusters in bacteria, for which an advanced understanding of polysaccharide biosynthesis and production could catalyse vaccine production and other industry applications.