Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2019

Structure and function of the Enterobacterial Common Antigen polysaccharide polymerase WzyE (#208)

Nicholas Maczuga 1 , Elizabeth N. H. Tran 1 , Renato Morona 1
  1. Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA, Australia

The outer membranes of Enterobacteriaceae are covered in a heterogeneous population of surface polysaccharides which include lipopolysaccharide (LPS) O-antigen and Enterobacterial Common Antigen (ECA). Both are biosynthesised by Wzy-dependent biosynthetic pathways, the most common polysaccharide biosynthetic pathway in bacteria. The polysaccharides repeat units are polymerized into linear chains within the periplasm by the glycosyltransferase Wzy, whereby, the degree of polymerization is controlled by the co-polymerase Wzz prior to export to the outer membrane. Wzy proteins, which are members of the Shape, Elongation, Division, Sporulation (SEDS) protein family, are poorly characterized both functionally and structurally due to low sequence homology and their intrinsic polytopic integral membrane protein nature. The vast majority of research has been orientated towards WzyB from the O-antigen biosynthetic pathway. While WzyE, the Wzy polymerase from the ECA biosynthetic pathway has yet to be investigated, it is known that the polysaccharide it assembles, ECA, plays crucial roles in maintaining outer membrane integrity as well as providing resistance to bile-salts and other detergents. Initial alignments of a large number of WzyE protein sequences enabled identification of conserved residues throughout the polypeptide. Through the use of site-directed mutagenesis, conserved residues were altered to determine their importance in the polymerase function as determined by Western immunoblotting of ECA. Topology mapping via C-terminal PhoA-LacZα reporter fusions enabled the topology of WzyE to be investigated. This will allow for a greater understanding of how Wzy proteins function, and provide insight into Wzy-dependent polysaccharide polymerization.