Kdo(2)-lipid A, a conserved substructure of lipopolysaccharide, plays critical roles in Gram-negative bacterial survival and interaction with host organisms. Inhibition of Kdo biosynthesis in Escherichia coli results in cell death and accumulation of the tetra-acylated precursor lipid IV(A). E. coli KdtA (EcKdtA) is a bifunctional enzyme that transfers two Kdo units from two CMP-Kdo molecules to lipid IV(A). In contrast, Haemophilus influenzae KdtA (HiKdtA) transfers only one Kdo unit. E. coli CMR300, which lacks Kdo transferase because of a deletion in kdtA, can be rescued to grow in broth at 37 degrees C if multiple copies of msbA are provided in trans. MsbA, the inner membrane transporter for nascent lipopolysaccharide, prefers hexa-acylated to tetra-acylated lipid A, but with the excess MsbA present in CMR300, lipid IV(A) is efficiently exported to the outer membrane. CMR300 is hypersensitive to hydrophobic antibiotics and bile salts and does not grow at 42 degrees C. Expressing HiKdtA in CMR300 results in the accumulation of Kdo-lipid IV(A) in place of lipid IV(A) without suppression of its growth phenotypes at 30 degrees C. EcKdtA restores intact lipopolysaccharide, together with normal antibiotic resistance, detergent resistance, and growth at 42 degrees C. To determine which residues are important for the mono- or bifunctional character of KdtA, protein chimeras were constructed using EcKdtA and HiKdtA. These chimeras, which are catalytically active, were characterized by in vitro assays and in vivo complementation. The N-terminal half of KdtA, especially the first 30 amino acid residues, specifies whether one or two Kdo units are transferred to lipid IV(A).