ABSTRACTPrevious studies have shown that the O polysaccharides (OPS) expressed byBurkholderia malleiare similar to those produced byBurkholderia thailandensisexcept that they lack the 4-O-acetyl modifications on their 6-deoxy-α-l-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designatedoacA, expressed byB. thailandensisthat accounts for this phenomenon. Utilizing theB. thailandensisandB. malleilipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by theoacAmutant,B. thailandensisZT0715, were antigenically similar to those produced byB. malleiATCC 23344. In addition, immunoblot analyses demonstrated that whenB. malleiATCC 23344 was complemented intranswithoacA, it synthesizedB. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by theoacAmutant reacted strongly with theB. malleiLPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-Oacetylation and 2-Omethylation ofB. thailandensisOPS antigens and that ZT0715 may provide a safe and cost-effective source ofB. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates.