Electronic Thesis and Dissertation Repository


Master of Science


Microbiology and Immunology


Dr. Miguel A. Valvano


O-antigen, the most surface exposed moiety of bacterial lipopolysaccharide (LPS), plays several roles in pathogenicity. The biosynthesis of O-antigen starts by the formation of a phosphoanhydride bond linking a sugar phosphate with a membrane isoprenoid lipid phosphate. Two distinct families of integral membrane proteins catalyze this reaction. The protein WecA is the prototypic member of one of these families, termed the polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase (PNPT) family. Because the donor nucleotide sugar is only available in the cytosol, cytosolic exposed regions of WecA are expected to be critical for the catalytic activity of the enzyme. Therefore, elucidating an accurate topological map of WecA is essential to understand its function. While a topological model has been determined with some level of accuracy in certain regions of WecA, various protein-protein, protein-lipid, and protein-aqueous interfaces are not precisely mapped and we have also found inconsistencies between in silico prediction models and experimental data. We hypothesize that the borders between the transmembrane domains (TMs) and the cytosolic loops of WecA are critical for its function. In particular, this thesis focuses on cytoloops 1 and 4 (including the universally conserved VFMGD motif), and TMs IV and V (a putative interaction site for the lipid substrate). The locations of these border residues were identified by the topological accessibility of specific amino acids using the substituted cysteine accessibility method (SCAM) combined with PEGylation. This approach offers several advantages over other classical SCAM methods, especially by avoiding the need to purify the labeled protein after sulfhydryl chemistry. PEGylation involves the covalent mass labeling of accessible sulfhydryl groups with the large thiol reagent methoxy-polyethylene glycol-maleimide (PEG-Mal, 5 kDa), and detection using a gel shift assay. Orientation is differentiated by treatment of EDTA-permeablized cells compared to treatment in membrane preparations. This thesis describes the adaptation and use of PEGylation for the topological analysis of WecA. The results provide further refinement of the WecA topological map, and the method can be extended to other integral membrane proteins involved in O-antigen assembly.

Included in

Bacteriology Commons