Qin, Jilong, Hong, Yaoqin, and Totsika, Makrina (2024) Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis. Applied and Environmental Microbiology, 90 (6).

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Abstract

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 the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in 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, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) 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 GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and 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.

Item ID:	83561
Item Type:	Article (Research - C1)
ISSN:	1098-5336
Copyright Information:	Copyright © 2024 Qin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Date Deposited:	05 Sep 2024 02:37
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