Peptidoglycan (PG) is an integral part of the bacterial cell wall. The [beta]1-4 linked 'N'-acetylglucosamine (GlcNAc) and 'N'-acetylmuramic acid (MurNAc)-based heteropolysaccharide, cross-linked by peptide side chains on MurNAc, has an important role in cell viability and virulence. This heteropolysaccharide is also an attractive drug target as a result of its important cellular role and its unique structure. Despite intense research efforts, there are still many unanswered questions pertaining to the metabolism of PG. Included in these is the pathway by which PG becomes O-acetylated at the C6 of MurNAc. This is important because this modification effectively blocks the cleavage of PG by lysozymes, and endogenous lytic transglycosylases, which are lytic enzymes that are critical for the growth and cell division of bacteria. The Gram-negative organism 'Neisseria gonorrhoeae', which is known to possess between 45 and 60% of its MurNAc residues in an O-acetylated form, was used examine the enzymes involved in this pathway. Differing from the single enzyme (OatA) model observed in many Gram-positive bacteria, for instance, 'Staphylococcus aureus', it is believed that ' N. gonorrhoeae', along with many other Gram-negative bacteria, employ a three-enzyme system for PG O-acetylation. The gene that encodes, the ' N. gonorrhoeae' enzyme, Ape2, which is believed to be the Gram-negative PG O-acetyltransferase, has been cloned and over-expressed in 'Escherichia coli' and the gene product has been purified. Results from PG pull-down assays indicated that Ape2 has an increased affinity for non-O-acetylated PG compared to 'O'-acetyl-PG. Observations made from enzymatic assays indicate that Ape2 lacks 'O'-acetylpeptidoglycan esterase activity. To demonstrate that Ape2 instead possesses PG O-acetyltransferase activity, the PG from an 'E. coli' strain over-expressing Ape2 was analyzed with MALDI-TOF mass spectrometry. This investigation yielded evidence of 'O'-acetyl-PG in Ape2-expressing 'E. coli ', while the control wild-type 'E. coli' cells did not exhibit O-acetylated PG. Based on these results it is proposed that Ape2 be renamed Peptidoglycan acetyl transferase B (PatB).