The BceB protein of the cystic fibrosis mucoid isolate Burkholderia cenocepacia IST432 is proposed to catalyze the first step of the exopolysaccharide repeat unit assembly. Extracts of Escherichia coli cells overexpressing BceB were shown to contain glycosyltransferase activity and mediate incorporation of glucose-1-phosphate into membrane lipids. The amino acid sequence of BceB exhibits two conserved regions, one comprising two invariant aspartic acid residues (Asp339 and Asp355) that are essential for catalysis, as substantiated by site-directed mutagenesis, and the other comprising a putative Rossmann fold motif. The results of protein topology analysis using PhoA and LacZ fusions supported in silico predictions that BceB has at least six transmembrane segments and two major cytoplasmic loops comprising the conserved regions described above.