FUNCTION OF THE HTRB HIGH-TEMPERATURE REQUIREMENT GENE OF ESCHERICHIA-COLI IN THE ACYLATION OF LIPID-A - HTRB CATALYZED INCORPORATION OF LAURATE

By assaying lysates of Escherichia coli generated with the hybrid lamb da bacteriophages of an ordered library (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508), we identified two clones (lambda 2 32 and lambda 233) capable of overexpressing the lauroyl transferase t hat functions after 3-deoxy-D-manno-octulosonic acid (Kdo) addition in lipid A biosynthesis (Brozek, K. A., and Raetz, C. R. H. (1990) J. Bi ol. Chem. 265, 15410-15417). The E. coli DNA inserts in lambda 232 and lambda 233 suggested that a known gene (htrB) required for rapid grow th above 33 degrees C might encode the lauroyl transferase. Using the intermediate (Kdo)(2)-lipid IVA as the laurate acceptor, extracts of s trains with transposon insertions in htrB were found to contain no lau royl transferase activity. Cells harboring hybrid htrB(+) plasmids ove rproduced transferase activity 100-200-fold. The overproduced transfer ase was solubilized with a non-ionic detergent and purified further by DEAE-Sepharose chromatography. With lauroyl acyl carrier protein as t he donor, the purified enzyme rapidly incorporated one laurate residue into (Kdo)(2)-lipid IVA. The rate of laurate incorporation was reduce d by several orders of magnitude when either one or both Kdos were abs ent in the acceptor. With a matched set of acyl-acyl carrier proteins, the enzyme incorporated laurate 3-8 times faster than decanoate or my ristate, respectively. Transfer of palmitate, palmitoleate, or R-3-hyd roxymyristate was very slow. Taken together with previous studies, our findings indicate that htrB encodes a key, late functioning acyltrans ferase of lipid A biosynthesis.