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. 1984 Apr;158(1):286–295. doi: 10.1128/jb.158.1.286-295.1984

Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

D W Grogan, J E Cronan Jr
PMCID: PMC215410  PMID: 6325391

Abstract

Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bell R. M. Mutants of Escherichia coli defective in membrane phospholipid synthesis: macromolecular synthesis in an sn-glycerol 3-phosphate acyltransferase Km mutant. J Bacteriol. 1974 Mar;117(3):1065–1076. doi: 10.1128/jb.117.3.1065-1076.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernard H. U., Remaut E., Hershfield M. V., Das H. K., Helinski D. R., Yanofsky C., Franklin N. Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter. Gene. 1979 Jan;5(1):59–76. doi: 10.1016/0378-1119(79)90092-1. [DOI] [PubMed] [Google Scholar]
  4. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [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. Cronan J. E., Jr, Nunn W. D., Batchelor J. G. Studies on the biosynthesis of cyclopropane fatty acids in Escherichia coli. Biochim Biophys Acta. 1974 Apr 26;348(1):63–75. doi: 10.1016/0005-2760(74)90093-9. [DOI] [PubMed] [Google Scholar]
  7. Cronan J. E., Jr Phospholipid alterations during growth of Escherichia coli. J Bacteriol. 1968 Jun;95(6):2054–2061. doi: 10.1128/jb.95.6.2054-2061.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cronan J. E., Jr, Reed R., Taylor F. R., Jackson M. B. Properties and biosynthesis of cyclopropane fatty acids in Escherichia coli. J Bacteriol. 1979 Apr;138(1):118–121. doi: 10.1128/jb.138.1.118-121.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Derom C., Gheysen D., Fiers W. High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter. Gene. 1982 Jan;17(1):45–54. doi: 10.1016/0378-1119(82)90099-3. [DOI] [PubMed] [Google Scholar]
  10. Evans R., Seeley N. R., Kuempel P. L. Loss of rac locus DNA in merozygotes of Escherichia coli K12. Mol Gen Genet. 1979 Oct 1;175(3):245–250. doi: 10.1007/BF00397223. [DOI] [PubMed] [Google Scholar]
  11. Foulds J., Barrett C. Characterization of Escherichia coli mutants tolerant to bacteriocin JF246: two new classes of tolerant mutants. J Bacteriol. 1973 Nov;116(2):885–892. doi: 10.1128/jb.116.2.885-892.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldfine H. Comparative aspects of bacterial lipids. Adv Microb Physiol. 1972;8:1–58. doi: 10.1016/s0065-2911(08)60187-3. [DOI] [PubMed] [Google Scholar]
  13. Gottesman M., Oppenheim A., Court D. Retroregulation: control of gene expression from sites distal to the gene. Cell. 1982 Jul;29(3):727–728. doi: 10.1016/0092-8674(82)90434-2. [DOI] [PubMed] [Google Scholar]
  14. Grogan D. W., Cronan J. E., Jr Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids. Gene. 1983 Apr;22(1):75–83. doi: 10.1016/0378-1119(83)90066-5. [DOI] [PubMed] [Google Scholar]
  15. Herskowitz I., Hagen D. The lysis-lysogeny decision of phage lambda: explicit programming and responsiveness. Annu Rev Genet. 1980;14:399–445. doi: 10.1146/annurev.ge.14.120180.002151. [DOI] [PubMed] [Google Scholar]
  16. Hopkins A. S., Murray N. E., Brammar W. J. Characterization of lambdatrp-transducing bacteriophages made in vitro. J Mol Biol. 1976 Nov 15;107(4):549–569. doi: 10.1016/s0022-2836(76)80082-4. [DOI] [PubMed] [Google Scholar]
  17. Jacques N. A., Hunt A. l. Studies on cyclopropane fatty acid synthesis. Effect of carbon source and oxygen tension on cyclopropane fatty acid synthetase activity in Pseudomonas denitrificans. Biochim Biophys Acta. 1980 Sep 8;619(3):453–470. doi: 10.1016/0005-2760(80)90098-3. [DOI] [PubMed] [Google Scholar]
  18. Knivett V. A., Cullen J. Some factors affecting cyclopropane acid formation in Escherichia coli. Biochem J. 1965 Sep;96(3):771–776. doi: 10.1042/bj0960771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Loenen W. A., Brammar W. J. A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene. 1980 Aug;10(3):249–259. doi: 10.1016/0378-1119(80)90054-2. [DOI] [PubMed] [Google Scholar]
  20. Lynn S. P., Gardner J. F., Reznikoff W. S. Attenuation regulation in the thr operon of Escherichia coli K-12: molecular cloning and transcription of the controlling region. J Bacteriol. 1982 Oct;152(1):363–371. doi: 10.1128/jb.152.1.363-371.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McGarrity J. T., Armstrong J. B. The effect of temperature and other growth conditions on the fatty acid composition of Escherichia coli. Can J Microbiol. 1981 Aug;27(8):835–840. doi: 10.1139/m81-128. [DOI] [PubMed] [Google Scholar]
  22. Moir A., Brammar W. J. The use of specialised transducing phages in the amplification of enzyme production. Mol Gen Genet. 1976 Nov 24;149(1):87–99. doi: 10.1007/BF00275963. [DOI] [PubMed] [Google Scholar]
  23. Murray N. E., Kelley W. S. Characterization of lambdapolA transducing phages; effective expression of the E. coli polA gene. Mol Gen Genet. 1979 Aug;175(1):77–87. doi: 10.1007/BF00267858. [DOI] [PubMed] [Google Scholar]
  24. Nakamura Y., Uchida H. Isolation of conditionally lethal amber mutations affecting synthesis of the nusA protein of Escherichia coli. Mol Gen Genet. 1983;190(2):196–203. doi: 10.1007/BF00330640. [DOI] [PubMed] [Google Scholar]
  25. PERRET C. J. Iodometric assay of penicillinase. Nature. 1954 Nov 27;174(4439):1012–1013. doi: 10.1038/1741012a0. [DOI] [PubMed] [Google Scholar]
  26. Raetz C. R. Isolation of Escherichia coli mutants defective in enzymes of membrane lipid synthesis. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2274–2278. doi: 10.1073/pnas.72.6.2274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  28. Roberts J. W., Roberts C. W. Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc Natl Acad Sci U S A. 1975 Jan;72(1):147–151. doi: 10.1073/pnas.72.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith D. D., Jr, Norton S. J. S-Adenosylmethionine, cyclopropane fatty acid synthase, and the production of lactobacillic acid in Lactobacillus plantarum. Arch Biochem Biophys. 1980 Dec;205(2):564–570. doi: 10.1016/0003-9861(80)90139-3. [DOI] [PubMed] [Google Scholar]
  30. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  31. Szybalski E. H., Szybalski W. A comprehensive molecular map of bacteriophage lambda. Gene. 1979 Nov;7(3-4):217–270. doi: 10.1016/0378-1119(79)90047-7. [DOI] [PubMed] [Google Scholar]
  32. Taguchi M., Izui K., Katsuki H. Augmentation of cyclopropane fatty acid synthesis under stringent control in Escherichia coli. J Biochem. 1980 Dec;88(6):1879–1882. [PubMed] [Google Scholar]
  33. Taylor F. R., Cronan J. E., Jr Cyclopropane fatty acid synthase of Escherichia coli. Stabilization, purification, and interaction with phospholipid vesicles. Biochemistry. 1979 Jul 24;18(15):3292–3300. doi: 10.1021/bi00582a015. [DOI] [PubMed] [Google Scholar]
  34. Taylor F., Cronan J. E., Jr Selection and properties of Escherichia coli mutants defective in the synthesis of cyclopropane fatty acids. J Bacteriol. 1976 Feb;125(2):518–523. doi: 10.1128/jb.125.2.518-523.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thompson R., Achtman M. The control region of the F sex factor DNA transfer cistrons: physical mapping by deletion analysis. Mol Gen Genet. 1979 Jan 16;169(1):49–57. doi: 10.1007/BF00267544. [DOI] [PubMed] [Google Scholar]
  36. Uhlin B. E., Molin S., Gustafsson P., Nordström K. Plasmids with temperature-dependent copy number for amplification of cloned genes and their products. Gene. 1979 Jun;6(2):91–106. doi: 10.1016/0378-1119(79)90065-9. [DOI] [PubMed] [Google Scholar]
  37. Uhlin B. E., Nordström K. R plasmid gene dosage effects in Escherichia coli K-12: copy mutants of the R plasmic R1drd-19. Plasmid. 1977 Nov;1(1):1–7. doi: 10.1016/0147-619x(77)90003-8. [DOI] [PubMed] [Google Scholar]
  38. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]

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