Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1983 Apr;154(1):221–230. doi: 10.1128/jb.154.1.221-230.1983

Genetic and Biochemical Analyses of Escherichia coli Mutants Altered in the Temperature-Dependent Regulation of Membrane Lipid Composition

Amy Klages Ulrich 1, Diego de Mendoza 1, Jeffrey L Garwin 1,†, John E Cronan Jr 1
PMCID: PMC217450  PMID: 6339472

Abstract

We have previously studied two mutants of Escherichia coli altered in the regulation of membrane lipid composition by temperature. One class (represented by the fabFl allele) fails to regulate upon temperature shift and is defective in cis-vaccenic acid synthesis owing to the lack of the fatty acid elongation enzyme β-ketoacyl-acyl carrier protein synthase II(EC 2.3.1.41). A second class of mutant, given the phenotypic designation Vtr, overproduces cis-vaccenic acid at all temperatures and hence is altered in temperature regulation. In this paper we report evidence for the following conclusions. (i) The Vtr and fabFl mutations show very tight genetic linkage. (ii) The Vtr lesion is allelic to the fabFl mutation since the presence of the fabFl mutation in merodiploid strains carrying the Vtr or fabF+ alleles results in fatty acid compositions intermediate between those of the two monoploid strains. Merodiploids carrying both the fabF+ and Vtr alleles likewise show an intermediate composition. These results indicate intra-allelic complementation. (iii) The two E. coli proteins recently discovered by Rock (J. Bacteriol. 152:1298-1300, 1982) that form mixed disulfide cross-links to acyl carrier protein are directly demonstrated to be β-ketoacyl-acyl carrier protein synthases I and II. (iv) The fabFl strains produce a synthase II band of altered electrophoretic mobility, indicating that the fabF locus is the structural gene for synthase II. (v) The synthase II of Vtr strains is abnormally sensitive to cerulenin, an antibiotic that specifically inhibits synthases I and II. This increased sensitivity is readily demonstrated in vivo, but in vitro we failed to detect an increased sensitivity of the Vtr synthase II to cerulenin, nor have we detected any other kinetic or structural alteration in the enzyme. We interpret these results in terms of specific interactions of synthase II with other cellular components which occur in vivo but are not duplicated in vitro.

Full text

PDF
221

Images in this article

222Image
on p.222
227Image
on p.227

Selected References

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

  1. Alberts A. W., Bell R. M., Vagelos P. R. Acyl carrier protein. XV. Studies of -ketoacyl-acyl carrier protein synthetase. J Biol Chem. 1972 May 25;247(10):3190–3198. [PubMed] [Google Scholar]
  2. Buttke T. M., Ingram L. O. Inhibition of unsaturated fatty acid synthesis in escherichia coli by the antibiotic cerulenin. Biochemistry. 1978 Nov 28;17(24):5282–5286. doi: 10.1021/bi00617a031. [DOI] [PubMed] [Google Scholar]
  3. Clark D., Cronan J. E., Jr Escherichia coli mutants with altered control of alcohol dehydrogenase and nitrate reductase. J Bacteriol. 1980 Jan;141(1):177–183. doi: 10.1128/jb.141.1.177-183.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cronan J. E., Jr, Gelmann E. P. Physical properties of membrane lipids: biological relevance and regulation. Bacteriol Rev. 1975 Sep;39(3):232–256. doi: 10.1128/br.39.3.232-256.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cronan J. E., Jr Thermal regulation of the membrane lipid composition of Escherichia coli. Evidence for the direct control of fatty acid synthesis. J Biol Chem. 1975 Sep 10;250(17):7074–7077. [PubMed] [Google Scholar]
  6. D'Agnolo G., Rosenfeld I. S., Awaya J., Omura S., Vagelos P. R. Inhibition of fatty acid synthesis by the antibiotic cerulenin. Specific inactivation of beta-ketoacyl-acyl carrier protein synthetase. Biochim Biophys Acta. 1973 Nov 29;326(2):155–156. doi: 10.1016/0005-2760(73)90241-5. [DOI] [PubMed] [Google Scholar]
  7. D'Agnolo G., Rosenfeld I. S., Vagelos P. R. Multiple forms of beta-ketoacyl-acyl carrier protein synthetase in Escherichia coli. J Biol Chem. 1975 Jul 25;250(14):5289–5294. [PubMed] [Google Scholar]
  8. Faelen M., Toussaint A. Bacteriophage Mu-1: a tool to transpose and to localize bacterial genes. J Mol Biol. 1976 Jul 5;104(3):525–539. doi: 10.1016/0022-2836(76)90118-2. [DOI] [PubMed] [Google Scholar]
  9. Garwin J. L., Cronan J. E., Jr Thermal modulation of fatty acid synthesis in Escherichia coli does not involve de novo enzyme synthesis. J Bacteriol. 1980 Mar;141(3):1457–1459. doi: 10.1128/jb.141.3.1457-1459.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Garwin J. L., Klages A. L., Cronan J. E., Jr Structural, enzymatic, and genetic studies of beta-ketoacyl-acyl carrier protein synthases I and II of Escherichia coli. J Biol Chem. 1980 Dec 25;255(24):11949–11956. [PubMed] [Google Scholar]
  12. Gelmann E. P., Cronan J. E., Jr Mutant of Escherichia coli deficient in the synthesis of cis-vaccenic acid. J Bacteriol. 1972 Oct;112(1):381–387. doi: 10.1128/jb.112.1.381-387.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Howe M. M. Prophage deletion mapping of bacteriophage Mu-1. Virology. 1973 Jul;54(1):93–101. doi: 10.1016/0042-6822(73)90118-9. [DOI] [PubMed] [Google Scholar]
  14. Marr A. G., Ingraham J. L. EFFECT OF TEMPERATURE ON THE COMPOSITION OF FATTY ACIDS IN ESCHERICHIA COLI. J Bacteriol. 1962 Dec;84(6):1260–1267. doi: 10.1128/jb.84.6.1260-1267.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Newman B. J., Masters M. The variation in frequency with which markers are transduced by phage P1 is primarily a result of discrimination during recombination. Mol Gen Genet. 1980;180(3):585–589. doi: 10.1007/BF00268064. [DOI] [PubMed] [Google Scholar]
  16. Nishihara M., Ishinaga M., Kato M., Kito M. Temperature-sensitive formation of the phospholipid molecular species in Escherichia coli membranes. Biochim Biophys Acta. 1976 Apr 22;431(1):54–61. [PubMed] [Google Scholar]
  17. Okuyama H., Yamada K., Kameyama Y., Ikezawa H., Akamatsu Y., Nojima S. Regulation of membrane lipid synthesis in Escherichia coli after shifts in temperature. Biochemistry. 1977 Jun 14;16(12):2668–2673. doi: 10.1021/bi00631a013. [DOI] [PubMed] [Google Scholar]
  18. Prescott D. J., Vagelos P. R. Acyl carrier protein. Adv Enzymol Relat Areas Mol Biol. 1972;36:269–311. doi: 10.1002/9780470122815.ch8. [DOI] [PubMed] [Google Scholar]
  19. Rock C. O., Cronan J. E., Jr Re-evaluation of the solution structure of acyl carrier protein. J Biol Chem. 1979 Oct 10;254(19):9778–9785. [PubMed] [Google Scholar]
  20. Rock C. O., Jackowski S. Regulation of phospholipid synthesis in Escherichia coli. Composition of the acyl-acyl carrier protein pool in vivo. J Biol Chem. 1982 Sep 25;257(18):10759–10765. [PubMed] [Google Scholar]
  21. Rock C. O. Mixed disulfides of acyl carrier protein and coenzyme A with specific soluble proteins in Escherichia coli. J Bacteriol. 1982 Dec;152(3):1298–1300. doi: 10.1128/jb.152.3.1298-1300.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Semple K. S., Silbert D. F. Mapping of the fabD locus for fatty acid biosynthesis in Escherichia coli. J Bacteriol. 1975 Mar;121(3):1036–1046. doi: 10.1128/jb.121.3.1036-1046.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. YANOFSKY C., LENNOX E. S. Transduction and recombination study of linkage relationships among the genes controlling tryptophan synthesis in Escherichia coli. Virology. 1959 Aug;8:425–447. doi: 10.1016/0042-6822(59)90046-7. [DOI] [PubMed] [Google Scholar]
  24. de Mendoza D., Clark D., Cronan J. E., Jr One-step gene amplification by Mu-mediated transposition of E. coli genes to a multicopy plasmid. Gene. 1981 Oct;15(1):27–32. doi: 10.1016/0378-1119(81)90101-3. [DOI] [PubMed] [Google Scholar]
  25. de Mendoza D., Garwin J. L., Cronan J. E., Jr Overproduction of cis-vaccenic acid and altered temperature control of fatty acid synthesis in a mutant of Escherichia coli. J Bacteriol. 1982 Sep;151(3):1608–1611. doi: 10.1128/jb.151.3.1608-1611.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. de Mendoza D., Klages Ulrich A., Cronan J. E., Jr Thermal regulation of membrane fluidity in Escherichia coli. Effects of overproduction of beta-ketoacyl-acyl carrier protein synthase I. J Biol Chem. 1983 Feb 25;258(4):2098–2101. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES