Cloning, sequencing, and functional analysis of three glycosyltransferasesinvolved in the biosynthesis of the inner core region of Klebsiella pneumoniae lipopolysaccharide

The genes encoding the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferas e (waaA) and heptosyltransferases I (waaC) and II (waaF) in Klebsiella pneu moniae were cloned from a DNA library by functional complementation of corr esponding Escherichia coil and Salmonella enterica mutants. Sequence analys es revealed extensive homologies of the deduced proteins to their counterpa rts in other Enterobacteriaceae. However, differences were evident with reg ard to the chromosomal organization of the genes. To perform in vitro studi es, the waaA, waaC and waaF genes were subcloned and expressed in the Gram- positive host Corynebacterium glutamicum. WaaA was characterized as a bifun ctional enzyme capable of transferring two Kdo residues to a synthetic bisp hosphorylated tetraacyl-lipid A precursor of E. coil (compound 406). In con trast, waaC and waaF were shown to encode specific glycosyltransferases cat alyzing the consecutive transfer of two L-glycero-D-manno-heptose residues to Kdo(2)-406.