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. 1986 Oct;168(1):152–159. doi: 10.1128/jb.168.1.152-159.1986

Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli.

D N Crowell, M S Anderson, C R Raetz
PMCID: PMC213431  PMID: 3531165

Abstract

Several enzymes have been discovered recently in crude extracts of Escherichia coli that appear to be involved in the biosynthesis of the lipid A component of lipopolysaccharide. Two of these are lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase. Lipid A disaccharide synthase activity is barely detectable in cells harboring a lesion in the lpxB (pgsB) gene. We subcloned the lpxB gene from plasmid pLC26-43 of the Clarke and Carbon collection (L. Clarke and J. Carbon, Cell 9:91-99, 1976) and localized it to a 1.7-kilobase-pair fragment of DNA counterclockwise of dnaE on the E. coli chromosome. Furthermore, we discovered a new gene (lpxA) located adjacent to and counterclockwise of lpxB that encodes or controls UDP-N-acetylglucosamine acyltransferase. Our data prove that lpxB and lpxA are transcribed in the clockwise direction and suggest that they may be cotranscribed.

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Selected References

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  1. Anderson M. S., Bulawa C. E., Raetz C. R. The biosynthesis of gram-negative endotoxin. Formation of lipid A precursors from UDP-GlcNAc in extracts of Escherichia coli. J Biol Chem. 1985 Dec 15;260(29):15536–15541. [PubMed] [Google Scholar]
  2. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Bulawa C. E., Raetz C. R. The biosynthesis of gram-negative endotoxin. Identification and function of UDP-2,3-diacylglucosamine in Escherichia coli. J Biol Chem. 1984 Apr 25;259(8):4846–4851. [PubMed] [Google Scholar]
  5. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  6. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  7. Galanos C., Freudenberg M. A., Lüderitz O., Rietschel E. T., Westphal O. Chemical, physicochemical and biological properties of bacterial lipopolysaccharides. Prog Clin Biol Res. 1979;29:321–332. [PubMed] [Google Scholar]
  8. Ganong B. R., Leonard J. M., Raetz C. R. Phosphatidic acid accumulation in the membranes of Escherichia coli mutants defective in CDP-diglyceride synthetase. J Biol Chem. 1980 Feb 25;255(4):1623–1629. [PubMed] [Google Scholar]
  9. Icho T., Sparrow C. P., Raetz C. R. Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli. J Biol Chem. 1985 Oct 5;260(22):12078–12083. [PubMed] [Google Scholar]
  10. Johnston S., Lee J. H., Ray D. S. High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA. Gene. 1985;34(2-3):137–145. doi: 10.1016/0378-1119(85)90121-0. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Morrison D. C., Ryan J. L. Bacterial endotoxins and host immune responses. Adv Immunol. 1979;28:293–450. doi: 10.1016/s0065-2776(08)60802-0. [DOI] [PubMed] [Google Scholar]
  14. Nishijima M., Bulawa C. E., Raetz C. R. Two interacting mutations causing temperature-sensitive phosphatidylglycerol synthesis in Escherichia coli membranes. J Bacteriol. 1981 Jan;145(1):113–121. doi: 10.1128/jb.145.1.113-121.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nishijima M., Raetz C. R. Characterization of two membrane-associated glycolipids from an Escherichia coli mutant deficient in phosphatidylglycerol. J Biol Chem. 1981 Oct 25;256(20):10690–10696. [PubMed] [Google Scholar]
  16. Nishijima M., Raetz C. R. Membrane lipid biogenesis in Escherichia coli: identification of genetic loci for phosphatidylglycerophosphate synthetase and construction of mutants lacking phosphatidylglycerol. J Biol Chem. 1979 Aug 25;254(16):7837–7844. [PubMed] [Google Scholar]
  17. Ray B. L., Painter G., Raetz C. R. The biosynthesis of gram-negative endotoxin. Formation of lipid A disaccharides from monosaccharide precursors in extracts of Escherichia coli. J Biol Chem. 1984 Apr 25;259(8):4852–4859. [PubMed] [Google Scholar]
  18. Roozen K. J., Fenwick R. G., Jr, Curtiss R., 3rd Synthesis of ribonucleic acid and protein in plasmid-containing minicells of Escherichia coli K-12. J Bacteriol. 1971 Jul;107(1):21–33. doi: 10.1128/jb.107.1.21-33.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shepard D., Oberfelder R. W., Welch M. M., McHenry C. S. Determination of the precise location and orientation of the Escherichia coli dnaE gene. J Bacteriol. 1984 May;158(2):455–459. doi: 10.1128/jb.158.2.455-459.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Takayama K., Qureshi N., Mascagni P., Nashed M. A., Anderson L., Raetz C. R. Fatty acyl derivatives of glucosamine 1-phosphate in Escherichia coli and their relation to lipid A. Complete structure of A diacyl GlcN-1-P found in a phosphatidylglycerol-deficient mutant. J Biol Chem. 1983 Jun 25;258(12):7379–7385. [PubMed] [Google Scholar]
  21. Welch M. M., McHenry C. S. Cloning and identification of the product of the dnaE gene of Escherichia coli. J Bacteriol. 1982 Oct;152(1):351–356. doi: 10.1128/jb.152.1.351-356.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

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