The genes encoding the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (waaA) and heptosyltransferases I (waaC) and II (waaF) in Klebsiella pneumoniae were cloned from a DNA library by functional complementation of corresponding Escherichia coli and Salmonella enterica mutants. Sequence analyses revealed extensive homologies of the deduced proteins to their counterparts in other Enterobacteriaceae. However, differences were evident with regard to the chromosomal organization of the genes. To perform in vitro studies, the waaA, waaC and waaF genes were subcloned and expressed in the Gram-positive host Corynebacterium glutamicum. WaaA was characterized as a bifunctional enzyme capable of transferring two Kdo residues to a synthetic bisphosphorylated tetraacyl-lipid A precursor of E. coli (compound 406). In contrast, waaC and waaF were shown to encode specific glycosyltransferases catalyzing the consecutive transfer of two L-glycero-D-manno-heptose residues to Kdo(2)-406.