By assaying lysates of Escherichia coli generated with the hybrid bacteriophages of an ordered library (Kohara, Y., Akiyama, K., and Isono, K.(1987) Cell 50, 495-508), we identified two clones (232 and 233) capable of overexpressing the lauroyl transferase that functions after 3-deoxy-D-manno-octulosonic acid (Kdo) addition in lipid A biosynthesis (Brozek, K. A., and Raetz, C. R. H.(1990) J. Biol. Chem. 265, 15410-15417). The E. coli DNA inserts in 232 and 233 suggested that a known gene (htrB) required for rapid growth above 33 °C might encode the lauroyl transferase. Using the intermediate (Kdo)-lipid IV as the laurate acceptor, extracts of strains with transposon insertions in htrB were found to contain no lauroyl transferase activity. Cells harboring hybrid htrB plasmids overproduced transferase activity 100-200-fold. The overproduced transferase was solubilized with a non-ionic detergent and purified further by DEAE-Sepharose chromatography. With lauroyl acyl carrier protein as the donor, the purified enzyme rapidly incorporated one laurate residue into (Kdo)-lipid IV. The rate of laurate incorporation was reduced by several orders of magnitude when either one or both Kdos were absent in the acceptor. With a matched set of acyl-acyl carrier proteins, the enzyme incorporated laurate 3-8 times faster than decanoate or myristate, respectively. Transfer of palmitate, palmitoleate, or R-3-hydroxymyristate was very slow. Taken together with previous studies, our findings indicate that htrB encodes a key, late functioning acyltransferase of lipid A biosynthesis.

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