Accumulation of a lipid A precursor lacking the 4′-phosphate following inactivation of the Escherichia coli lpxK gene

The lpxK gene has been proposed to encode the lipid A 4'-kinase in Escherichia coli (Garrett, T, A., Kadrmas, J, L,, and Raetz, C, R, H. (1997) J, Biol, Chem, 272, 21855-21864), In cell extracts, the kinase phosphorylates the 4'-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipid A, but the enzyme has not yet been purified because of instability. lpxK is co-transcribed with an essential upstream gene, msbA, with strong homology to mammalian Mdr proteins and ABC transporters, msbA may be involved in the transport of newly made lipid A from the inner surface of the inner membrane to the outer membrane. Insertion of an Omega-chloramphenicol cassette into msbA also halts transcription of lpxK, We have now constructed a strain in which only the lpxK gene is inactivated by inserting a kanamycin cassette into the chromosomal copy of lpxK, This mutation is complemented at 30 degrees C by a hybrid plasmid with a temperature-sensitive origin of replication carrying lpxK(+), When this strain (designated TG1/pTAG1) is grown at 44 degrees C, the plasmid bearing the lpxK(+) is lost, and the phenotype of an lpxK knock-out mutation is unmasked. The growth of TG1/pTAG1 was inhibited after several hours at 44 degrees C, consistent with lpxK being an essential gene. Furthermore, 4'-kinase activity in extracts made from these cells was barely detectable. In accordance with the proposed biosynthetic pathway for lipid A, DS-1-P (the 4'-kinase substrate) accumulated in TG1/pTAG1 cells grown at 44 degrees C, The DS-1-P from TG1/pTAG1 was isolated, and its structure was verified by H-1 NMR spectroscopy. DS-1-P had not been isolated previously from bacterial cells. Its accumulation in TG1/pTAG1 provides additional support for the pathway of lipid A biosynthesis in E. coli, Homologs of lpxK are present in the genomes of other Gram-negative bacteria.