The O-acetylation of the essential cell wall polymer peptidoglycan is a major virulence factor identified in many bacteria, both Gram positive and Gram negative, including Staphylococcus aureus, Bacillus anthracis, Neisseria gonorrhoeae and N. meningitidis. With Gram-negative bacteria, the translocation of acetate from the cytoplasm is thought to be performed by an integral membrane protein, PatA. The acetate is then transferred to peptidoglycan by a peripheral membrane O-acetyltransferase PatB, whereas a single bimodal membrane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro biochemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate such as peptidoglycan. In this thesis, I present the first kinetic and biochemical analysis of a PG O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tetrapeptide stems on muramoyl residue sand with simple chitooligosaccharides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. The data provided here strongly supports a mechanism whereby catalysis occurs by a double-displacement nucleophilic pathway involving a ping-pong bi-bi mechanism that proceeds through a covalent acetyl-serine intermediate. In addition to the catalytic serine, the aspartic acid and histidine residues that complete the catalytic triad were identified. I then applied the tools developed for the analysis of PatB to the Gram-positive O-acetyltransferase, OatA. The preliminary characterization of the catalytic domain of that enzyme detailed in this thesis will form the basis for future high-throughput screening with the goal of identifying OatA inhibitors. This information will be valuable for the identification and development of peptidoglycan O-acetyltransferase inhibitors, which could represent potential leads to novel classes of antibiotics. Together, these data solidify our understanding of the mechanism by which an important bacterial virulence factor is produced and pave the way for future development of novel antimicrobials.