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 Esc herichia coli (Garrett, T, A., Kadrmas, J, L,, and Raetz, C, R, H. (19 97) J, Biol, Chem, 272, 21855-21864), In cell extracts, the kinase pho sphorylates the 4'-position of a tetraacyldisaccharide 1-phosphate pre cursor (DS-1-P) of lipid A, but the enzyme has not yet been purified b ecause of instability. lpxK is co-transcribed with an essential upstre am gene, msbA, with strong homology to mammalian Mdr proteins and ABC transporters, msbA may be involved in the transport of newly made lipi d 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 th e chromosomal copy of lpxK, This mutation is complemented at 30 degree s C by a hybrid plasmid with a temperature-sensitive origin of replica tion carrying lpxK(+), When this strain (designated TG1/pTAG1) is grow n at 44 degrees C, the plasmid bearing the lpxK(+) is lost, and the ph enotype of an lpxK knock-out mutation is unmasked. The growth of TG1/p TAG1 was inhibited after several hours at 44 degrees C, consistent wit h lpxK being an essential gene. Furthermore, 4'-kinase activity in ext racts made from these cells was barely detectable. In accordance with the proposed biosynthetic pathway for lipid A, DS-1-P (the 4'-kinase s ubstrate) 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 bacte rial cells. Its accumulation in TG1/pTAG1 provides additional support for the pathway of lipid A biosynthesis in E. coli, Homologs of lpxK a re present in the genomes of other Gram-negative bacteria.