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. 1994 Nov;176(22):6944–6951. doi: 10.1128/jb.176.22.6944-6951.1994

Endotoxin biosynthesis in Pseudomonas aeruginosa: enzymatic incorporation of laurate before 3-deoxy-D-manno-octulosonate.

S Mohan 1, C R Raetz 1
PMCID: PMC197065  PMID: 7961456

Abstract

Unlike Escherichia coli, living cells of Pseudomonas aeruginosa can complete the fatty acylation of lipid A when the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo) is inhibited (R. C. Goldman, C. C. Doran, S. K. Kadam, and J. O. Capobianco, J. Biol. Chem. 263:5217-5233, 1988). In this study, we demonstrate the presence of a novel enzyme in extracts of P. aeruginosa that can transfer lauroyl-acyl carrier protein (ACP) to a tetraacyl disaccharide-1,4'-bis-phosphate precursor of lipid A (termed lipid IVA) that accumulates in Kdo-deficient mutants of E. coli. Comparable E. coli extracts cannot transfer laurate from lauroyl-ACP to lipid IVA, only to (Kdo)2-lipid IVA (K. A. Brozek, and C. R. H. Raetz, J. Biol. Chem. 265:15410-15417, 1990). P. aeruginosa extracts do not utilize myristoyl- or R-3-hydroxymyristoyl-ACP instead of lauroyl-ACP to acylate lipid IVA. Laurate incorporation in P. aeruginosa extracts is dependent upon time, protein concentration, and the presence of Triton X-100 but is inhibited by lauroyl-coenzyme A. P. aeruginosa extracts transfer only one laurate to lipid IVA, whereas E. coli extracts can transfer two laurates to (Kdo)2-lipid IVA. These results demonstrate that incorporation of laurate into lipid A does not require prior attachment of Kdo in all gram-negative bacteria.

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Selected References

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  1. Anderson M. S., Raetz C. R. Biosynthesis of lipid A precursors in Escherichia coli. A cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. J Biol Chem. 1987 Apr 15;262(11):5159–5169. [PubMed] [Google Scholar]
  2. Belunis C. J., Raetz C. R. Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli. J Biol Chem. 1992 May 15;267(14):9988–9997. [PubMed] [Google Scholar]
  3. Bhat U. R., Forsberg L. S., Carlson R. W. Structure of lipid A component of Rhizobium leguminosarum bv. phaseoli lipopolysaccharide. Unique nonphosphorylated lipid A containing 2-amino-2-deoxygluconate, galacturonate, and glucosamine. J Biol Chem. 1994 May 20;269(20):14402–14410. [PubMed] [Google Scholar]
  4. Brozek K. A., Bulawa C. E., Raetz C. R. Biosynthesis of lipid A precursors in Escherichia coli. A membrane-bound enzyme that transfers a palmitoyl residue from a glycerophospholipid to lipid X. J Biol Chem. 1987 Apr 15;262(11):5170–5179. [PubMed] [Google Scholar]
  5. Brozek K. A., Hosaka K., Robertson A. D., Raetz C. R. Biosynthesis of lipopolysaccharide in Escherichia coli. Cytoplasmic enzymes that attach 3-deoxy-D-manno-octulosonic acid to lipid A. J Biol Chem. 1989 Apr 25;264(12):6956–6966. [PubMed] [Google Scholar]
  6. Brozek K. A., Raetz C. R. Biosynthesis of lipid A in Escherichia coli. Acyl carrier protein-dependent incorporation of laurate and myristate. J Biol Chem. 1990 Sep 15;265(26):15410–15417. [PubMed] [Google Scholar]
  7. Goldman R. C., Doran C. C., Capobianco J. O. Analysis of lipopolysaccharide biosynthesis in Salmonella typhimurium and Escherichia coli by using agents which specifically block incorporation of 3-deoxy-D-manno-octulosonate. J Bacteriol. 1988 May;170(5):2185–2191. doi: 10.1128/jb.170.5.2185-2191.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldman R. C., Doran C. C., Kadam S. K., Capobianco J. O. Lipid A precursor from Pseudomonas aeruginosa is completely acylated prior to addition of 3-deoxy-D-manno-octulosonate. J Biol Chem. 1988 Apr 15;263(11):5217–5223. [PubMed] [Google Scholar]
  9. Hammond S. M., Claesson A., Jansson A. M., Larsson L. G., Pring B. G., Town C. M., Ekström B. A new class of synthetic antibacterials acting on lipopolysaccharide biosynthesis. 1987 Jun 25-Jul 1Nature. 327(6124):730–732. doi: 10.1038/327730a0. [DOI] [PubMed] [Google Scholar]
  10. Karunaratne D. N., Richards J. C., Hancock R. E. Characterization of lipid A from Pseudomonas aeruginosa O-antigenic B band lipopolysaccharide by 1D and 2D NMR and mass spectral analysis. Arch Biochem Biophys. 1992 Dec;299(2):368–376. doi: 10.1016/0003-9861(92)90289-9. [DOI] [PubMed] [Google Scholar]
  11. Kawahara K., Uchida K., Aida K. Direct hydroxylation in the biosynthesis of hydroxy fatty acid in lipid a of Pseudomonas ovalis. Biochim Biophys Acta. 1979 Jan 29;572(1):1–8. [PubMed] [Google Scholar]
  12. Kelly T. M., Stachula S. A., Raetz C. R., Anderson M. S. The firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. The third step of endotoxin biosynthesis. J Biol Chem. 1993 Sep 15;268(26):19866–19874. [PubMed] [Google Scholar]
  13. Kulshin V. A., Zähringer U., Lindner B., Jäger K. E., Dmitriev B. A., Rietschel E. T. Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides. Eur J Biochem. 1991 Jun 15;198(3):697–704. doi: 10.1111/j.1432-1033.1991.tb16069.x. [DOI] [PubMed] [Google Scholar]
  14. Lehmann V., Rupprecht E. Microheterogeneity in lipid A demonstrated by a new intermediate in the biosynthesis of 3-deozy-D-manno-octulosonic-acid--lipid A. Eur J Biochem. 1977 Dec;81(3):443–452. doi: 10.1111/j.1432-1033.1977.tb11969.x. [DOI] [PubMed] [Google Scholar]
  15. Osborn M. J., Rick P. D., Rasmussen N. S. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Translocation and integration of an incomplete mutant lipid A into the outer membrane. J Biol Chem. 1980 May 10;255(9):4246–4251. [PubMed] [Google Scholar]
  16. Raetz C. R. Bacterial endotoxins: extraordinary lipids that activate eucaryotic signal transduction. J Bacteriol. 1993 Sep;175(18):5745–5753. doi: 10.1128/jb.175.18.5745-5753.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Raetz C. R. Biochemistry of endotoxins. Annu Rev Biochem. 1990;59:129–170. doi: 10.1146/annurev.bi.59.070190.001021. [DOI] [PubMed] [Google Scholar]
  18. Raetz C. R., Purcell S., Meyer M. V., Qureshi N., Takayama K. Isolation and characterization of eight lipid A precursors from a 3-deoxy-D-manno-octylosonic acid-deficient mutant of Salmonella typhimurium. J Biol Chem. 1985 Dec 25;260(30):16080–16088. [PubMed] [Google Scholar]
  19. Rick P. D., Fung L. W., Ho C., Osborn M. J. Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-D-mannooctulosonate-8-phosphate synthetase. J Biol Chem. 1977 Jul 25;252(14):4904–4912. [PubMed] [Google Scholar]
  20. Rietschel E. T., Brade H. Bacterial endotoxins. Sci Am. 1992 Aug;267(2):54–61. doi: 10.1038/scientificamerican0892-54. [DOI] [PubMed] [Google Scholar]
  21. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  22. Strain S. M., Armitage I. M., Anderson L., Takayama K., Qureshi N., Raetz C. R. Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance. J Biol Chem. 1985 Dec 25;260(30):16089–16098. [PubMed] [Google Scholar]
  23. 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]
  24. Williamson J. M., Anderson M. S., Raetz C. R. Acyl-acyl carrier protein specificity of UDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure. J Bacteriol. 1991 Jun;173(11):3591–3596. doi: 10.1128/jb.173.11.3591-3596.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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