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 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.
Garrett
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TA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Que
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NL
Raetz
C R
CR
D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000235:genetics*; D008050:Lipid A / Q000096:biosynthesis / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D016254:Mutagenesis, Insertional; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D008050:Lipid A; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C076042:tetraacyldisaccharide 4'-kinase