PMID:9575203
| Citation |
Garrett, TA, Que, NL and Raetz, CR (1998) Accumulation of a lipid A precursor lacking the 4'-phosphate following inactivation of the Escherichia coli lpxK gene. J. Biol. Chem. 273:12457-65 |
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| Abstract |
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 degreesC by a hybrid plasmid with a temperature-sensitive origin of replication carrying lpxK+. When this strain (designated TG1/pTAG1) is grown at 44 degreesC, 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 degreesC, 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 degreesC. The DS-1-P from TG1/pTAG1 was isolated, and its structure was verified by 1H 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. |
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| Keywords |
Carbohydrate Sequence; Escherichia coli/genetics; Lipid A/biosynthesis; Lipid A/metabolism; Magnetic Resonance Spectroscopy; Mutagenesis, Insertional; Phosphotransferases (Alcohol Group Acceptor)/genetics; Phosphotransferases (Alcohol Group Acceptor)/metabolism; Plasmids; Recombinant Proteins/genetics; Substrate Specificity; Temperature |
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