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. 1997 Sep;179(17):5326–5332. doi: 10.1128/jb.179.17.5326-5332.1997

Fatty acid biosynthesis in Pseudomonas aeruginosa: cloning and characterization of the fabAB operon encoding beta-hydroxyacyl-acyl carrier protein dehydratase (FabA) and beta-ketoacyl-acyl carrier protein synthase I (FabB).

T T Hoang 1, H P Schweizer 1
PMCID: PMC179400  PMID: 9286984

Abstract

The Pseudomonas aeruginosa fabA and fabB genes, encoding beta-hydroxyacyl-acyl carrier protein dehydratase and beta-ketoacyl-acyl carrier protein synthase I, respectively, were cloned, sequenced, and expressed in Escherichia coli. Northern analysis demonstrated that fabA and fabB are cotranscribed and most probably form a fabAB operon. The FabA and FabB proteins were similar in size and amino acid composition to their counterparts from Escherichia coli and to the putative homologs from Haemophilus influenzae. Chromosomal fabA and fabB mutants were isolated; the mutants were auxotrophic for unsaturated fatty acids. A temperature-sensitive fabA mutant was obtained by site-directed mutagenesis of a single base that induced a G101D change; this mutant grew normally at 30 degrees C but not at 42 degrees C, unless the growth medium was supplemented with oleate. By physical and genetic mapping, the fabAB genes were localized between 3.45 and 3.6 Mbp on the 5.9-Mbp chromosome, which corresponds to the 58- to 59.5-min region of the genetic map.

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Selected References

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  1. Cronan J. E., Jr, Li W. B., Coleman R., Narasimhan M., de Mendoza D., Schwab J. M. Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase. J Biol Chem. 1988 Apr 5;263(10):4641–4646. [PubMed] [Google Scholar]
  2. DiRusso C. C., Metzger A. K., Heimert T. L. Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichia coli by FadR. Mol Microbiol. 1993 Jan;7(2):311–322. doi: 10.1111/j.1365-2958.1993.tb01122.x. [DOI] [PubMed] [Google Scholar]
  3. Farewell A., Diez A. A., DiRusso C. C., Nyström T. Role of the Escherichia coli FadR regulator in stasis survival and growth phase-dependent expression of the uspA, fad, and fab genes. J Bacteriol. 1996 Nov;178(22):6443–6450. doi: 10.1128/jb.178.22.6443-6450.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Farinha M. A., Ronald S. L., Kropinski A. M., Paranchych W. Localization of the virulence-associated genes pilA, pilR, rpoN, fliA, fliC, ent, and fbp on the physical map of Pseudomonas aeruginosa PAO1 by pulsed-field electrophoresis. Infect Immun. 1993 Apr;61(4):1571–1575. doi: 10.1128/iai.61.4.1571-1575.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Garwin J. L., Klages A. L., Cronan J. E., Jr Beta-ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis. J Biol Chem. 1980 Apr 25;255(8):3263–3265. [PubMed] [Google Scholar]
  6. Gray K. M., Passador L., Iglewski B. H., Greenberg E. P. Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. J Bacteriol. 1994 May;176(10):3076–3080. doi: 10.1128/jb.176.10.3076-3080.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hatziloukas E., Panopoulos N. J., Delis S., Prosen D. E., Schaad N. W. An open reading frame in the approximately 28-kb tox-argk gene cluster encodes a polypeptide with homology to fatty acid desaturases. Gene. 1995 Dec 1;166(1):83–87. doi: 10.1016/0378-1119(95)00569-5. [DOI] [PubMed] [Google Scholar]
  8. Hayashi T., Yamamoto O., Sasaki H., Kawaguchi A., Okazaki H. Mechanism of action of the antibiotic thiolactomycin inhibition of fatty acid synthesis of Escherichia coli. Biochem Biophys Res Commun. 1983 Sep 30;115(3):1108–1113. doi: 10.1016/s0006-291x(83)80050-3. [DOI] [PubMed] [Google Scholar]
  9. Heath R. J., Rock C. O. Roles of the FabA and FabZ beta-hydroxyacyl-acyl carrier protein dehydratases in Escherichia coli fatty acid biosynthesis. J Biol Chem. 1996 Nov 1;271(44):27795–27801. doi: 10.1074/jbc.271.44.27795. [DOI] [PubMed] [Google Scholar]
  10. Henry M. F., Cronan J. E., Jr A new mechanism of transcriptional regulation: release of an activator triggered by small molecule binding. Cell. 1992 Aug 21;70(4):671–679. doi: 10.1016/0092-8674(92)90435-f. [DOI] [PubMed] [Google Scholar]
  11. Hoang T. T., Williams S., Schweizer H. P., Lam J. S. Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-beta-semialdehyde dehydrogenase. Microbiology. 1997 Mar;143(Pt 3):899–907. doi: 10.1099/00221287-143-3-899. [DOI] [PubMed] [Google Scholar]
  12. Holloway B. W., Römling U., Tümmler B. Genomic mapping of Pseudomonas aeruginosa PAO. Microbiology. 1994 Nov;140(Pt 11):2907–2929. doi: 10.1099/13500872-140-11-2907. [DOI] [PubMed] [Google Scholar]
  13. Hrabak E. M., Willis D. K. The lemA gene required for pathogenicity of Pseudomonas syringae pv. syringae on bean is a member of a family of two-component regulators. J Bacteriol. 1992 May;174(9):3011–3020. doi: 10.1128/jb.174.9.3011-3020.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kropinski A. M., Lewis V., Berry D. Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO. J Bacteriol. 1987 May;169(5):1960–1966. doi: 10.1128/jb.169.5.1960-1966.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Latifi A., Foglino M., Tanaka K., Williams P., Lazdunski A. A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol. 1996 Sep;21(6):1137–1146. doi: 10.1046/j.1365-2958.1996.00063.x. [DOI] [PubMed] [Google Scholar]
  16. Leesong M., Henderson B. S., Gillig J. R., Schwab J. M., Smith J. L. Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: two catalytic activities in one active site. Structure. 1996 Mar 15;4(3):253–264. doi: 10.1016/s0969-2126(96)00030-5. [DOI] [PubMed] [Google Scholar]
  17. Lightfoot J., Lam J. S. Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)-D-mannose. Mol Microbiol. 1993 May;8(4):771–782. doi: 10.1111/j.1365-2958.1993.tb01620.x. [DOI] [PubMed] [Google Scholar]
  18. Magnuson K., Jackowski S., Rock C. O., Cronan J. E., Jr Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol Rev. 1993 Sep;57(3):522–542. doi: 10.1128/mr.57.3.522-542.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Makowski G. S., Ramsby M. L. pH modification to enhance the molecular sieving properties of sodium dodecyl sulfate-10% polyacrylamide gels. Anal Biochem. 1993 Jul;212(1):283–285. doi: 10.1006/abio.1993.1324. [DOI] [PubMed] [Google Scholar]
  20. Miyakawa S., Suzuki K., Noto T., Harada Y., Okazaki H. Thiolactomycin, a new antibiotic. IV. Biological properties and chemotherapeutic activity in mice. J Antibiot (Tokyo) 1982 Apr;35(4):411–419. doi: 10.7164/antibiotics.35.411. [DOI] [PubMed] [Google Scholar]
  21. Olsen R. H., Hansen J. Evolution and utility of a Pseudomonas aeruginosa drug resistance factor. J Bacteriol. 1976 Mar;125(3):837–844. doi: 10.1128/jb.125.3.837-844.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Passador L., Cook J. M., Gambello M. J., Rust L., Iglewski B. H. Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science. 1993 May 21;260(5111):1127–1130. doi: 10.1126/science.8493556. [DOI] [PubMed] [Google Scholar]
  23. Pearson J. P., Gray K. M., Passador L., Tucker K. D., Eberhard A., Iglewski B. H., Greenberg E. P. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):197–201. doi: 10.1073/pnas.91.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  25. Schaefer A. L., Val D. L., Hanzelka B. L., Cronan J. E., Jr, Greenberg E. P. Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9505–9509. doi: 10.1073/pnas.93.18.9505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schweizer H. P., Hoang T. T. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene. 1995 May 26;158(1):15–22. doi: 10.1016/0378-1119(95)00055-b. [DOI] [PubMed] [Google Scholar]
  27. Schweizer H. P., Po C. Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa. J Bacteriol. 1994 Apr;176(8):2184–2193. doi: 10.1128/jb.176.8.2184-2193.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schweizer H. P. The agmR gene, an environmentally responsive gene, complements defective glpR, which encodes the putative activator for glycerol metabolism in Pseudomonas aeruginosa. J Bacteriol. 1991 Nov;173(21):6798–6806. doi: 10.1128/jb.173.21.6798-6806.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  30. Uratani Y., Wakayama N., Hoshino T. Effect of lipid acyl chain length on activity of sodium-dependent leucine transport system in Pseudomonas aeruginosa. J Biol Chem. 1987 Dec 15;262(35):16914–16919. [PubMed] [Google Scholar]
  31. Weber F. J., Isken S., de Bont J. A. Cis/trans isomerization of fatty acids as a defence mechanism of Pseudomonas putida strains to toxic concentrations of toluene. Microbiology. 1994 Aug;140(Pt 8):2013–2017. doi: 10.1099/13500872-140-8-2013. [DOI] [PubMed] [Google Scholar]
  32. West S. E., Iglewski B. H. Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Oct 11;16(19):9323–9335. doi: 10.1093/nar/16.19.9323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  34. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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