1Biochemical properties of Neisseria gonorrhoeae LgtE

A fragment of chromosomal DNA encoding the lgtE gene of Neisseria gonorrhoeae strain F62 was amplified by the polymerase chain reaction and cloned into the expression vector pET15b. Functional LgtE was purified and its biochemical properties determined. The purified enzyme was maximally active in buffer containing manganese; minimal activity was obtained in buffer containing other divalent cations. LgtE was only able to mediate the addition of Uridine diphosphate-galactose into Neisserial lipooligosaccharides (LOSs). We used a variety of genetically defined and chemically verified LOS structures to determine acceptor specificity. LgtE was able to mediate the addition of galactose into a variety of LOS structures, indicating the this enzyme possesses broad acceptor specificity. Furthermore, it was able to add multiple galactose residues onto LOS. We also determined that this enzyme was capable of adding galactose onto both the? and? chains of Neisserial LOS. 3Introduction Lipooligosaccharide (LOS) is an important virulence determinant of the pathogenic Neisseria (12, 16). It consists of an oligosaccharide component that is attached to lipid A via a KDO linkage. The genes involved in the synthesis of the oligosaccharide portion of this molecule have been identified and characterized from a variety of species (1, 2, 11, 13, 17, 28). A common feature of LOS expression in all of these species is th