Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Jan;170(1):386–392. doi: 10.1128/jb.170.1.386-392.1988

Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map.

T Chung 1, D J Klumpp 1, D C LaPorte 1
PMCID: PMC210654  PMID: 2826399

Abstract

In Escherichia coli, a single operon encodes the metabolic and regulatory enzymes of the glyoxylate bypass. The metabolic enzymes, isocitrate lyase and malate synthase, are expressed from aceA and aceB, and the regulatory enzyme, isocitrate dehydrogenase kinase/phosphatase, is expressed from aceK. We cloned this operon and determined its functional map by deletion analysis. The order of the genes in this operon is aceB-aceA-aceK, with aceB proximal to the promoter, consistent with the results of previous experiments using genetic techniques. The promoter was identified by S1 nuclease mapping, and its nucleotide sequence was determined. Isocitrate lyase and malate synthase were readily identified by autoradiography after the products of the operon clone were labeled by the maxicell procedure and then resolved by electrophoresis. In contrast, isocitrate dehydrogenase kinase/phosphatase, expressed from the same plasmid, was undetectable. This observation is consistent with a striking downshift in expression between aceA and aceK.

Full text

PDF
386

Images in this article

389Image
on p.389
390Image
on p.390

Selected References

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

  1. Belasco J. G., Beatty J. T., Adams C. W., von Gabain A., Cohen S. N. Differential expression of photosynthesis genes in R. capsulata results from segmental differences in stability within the polycistronic rxcA transcript. Cell. 1985 Jan;40(1):171–181. doi: 10.1016/0092-8674(85)90320-4. [DOI] [PubMed] [Google Scholar]
  2. Bennett P. M., Holms W. H. Reversible inactivation of the isocitrate dehydrogenase of Escherichia coli ML308 during growth on acetate. J Gen Microbiol. 1975 Mar;87(1):37–51. doi: 10.1099/00221287-87-1-37. [DOI] [PubMed] [Google Scholar]
  3. Borthwick A. C., Holms W. H., Nimmo H. G. The phosphorylation of Escherichia coli isocitrate dehydrogenase in intact cells. Biochem J. 1984 Sep 15;222(3):797–804. doi: 10.1042/bj2220797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brice C. B., Kornberg H. L. Genetic control of isocitrate lyase activity in Escherichia coli. J Bacteriol. 1968 Dec;96(6):2185–2186. doi: 10.1128/jb.96.6.2185-2186.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burton Z. F., Gross C. A., Watanabe K. K., Burgess R. R. The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein S21 and DNA primase in E. coli K12. Cell. 1983 Feb;32(2):335–349. doi: 10.1016/0092-8674(83)90453-1. [DOI] [PubMed] [Google Scholar]
  6. Garnak M., Reeves H. C. Phosphorylation of Isocitrate dehydrogenase of Escherichia coli. Science. 1979 Mar 16;203(4385):1111–1112. doi: 10.1126/science.34215. [DOI] [PubMed] [Google Scholar]
  7. Garnak M., Reeves H. C. Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli. J Biol Chem. 1979 Aug 25;254(16):7915–7920. [PubMed] [Google Scholar]
  8. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Holms W. H., Bennett P. M. Regulation of isocitrate dehydrogenase activity in Escherichia coli on adaptation to acetate. J Gen Microbiol. 1971 Jan;65(1):57–68. doi: 10.1099/00221287-65-1-57. [DOI] [PubMed] [Google Scholar]
  10. KORNBERG H. L., MADSEN N. B. Synthesis of C4-dicarboxylic acids from acetate by a glyoxylate bypass of the tricarboxylic acid cycle. Biochim Biophys Acta. 1957 Jun;24(3):651–653. doi: 10.1016/0006-3002(57)90268-8. [DOI] [PubMed] [Google Scholar]
  11. Kornberg H. L. The role and control of the glyoxylate cycle in Escherichia coli. Biochem J. 1966 Apr;99(1):1–11. doi: 10.1042/bj0990001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. LaPorte D. C., Chung T. A single gene codes for the kinase and phosphatase which regulate isocitrate dehydrogenase. J Biol Chem. 1985 Dec 5;260(28):15291–15297. [PubMed] [Google Scholar]
  14. LaPorte D. C., Koshland D. E., Jr A protein with kinase and phosphatase activities involved in regulation of tricarboxylic acid cycle. Nature. 1982 Dec 2;300(5891):458–460. doi: 10.1038/300458a0. [DOI] [PubMed] [Google Scholar]
  15. LaPorte D. C., Koshland D. E., Jr Phosphorylation of isocitrate dehydrogenase as a demonstration of enhanced sensitivity in covalent regulation. Nature. 1983 Sep 22;305(5932):286–290. doi: 10.1038/305286a0. [DOI] [PubMed] [Google Scholar]
  16. LaPorte D. C., Thorsness P. E., Koshland D. E., Jr Compensatory phosphorylation of isocitrate dehydrogenase. A mechanism for adaptation to the intracellular environment. J Biol Chem. 1985 Sep 5;260(19):10563–10568. [PubMed] [Google Scholar]
  17. LaPorte D. C., Walsh K., Koshland D. E., Jr The branch point effect. Ultrasensitivity and subsensitivity to metabolic control. J Biol Chem. 1984 Nov 25;259(22):14068–14075. [PubMed] [Google Scholar]
  18. 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]
  19. Maloy S. R., Bohlander M., Nunn W. D. Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation. J Bacteriol. 1980 Aug;143(2):720–725. doi: 10.1128/jb.143.2.720-725.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Maloy S. R., Nunn W. D. Genetic regulation of the glyoxylate shunt in Escherichia coli K-12. J Bacteriol. 1982 Jan;149(1):173–180. doi: 10.1128/jb.149.1.173-180.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  22. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nimmo G. A., Nimmo H. G. The regulatory properties of isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase from Escherichia coli ML308 and the roles of these activities in the control of isocitrate dehydrogenase. Eur J Biochem. 1984 Jun 1;141(2):409–414. doi: 10.1111/j.1432-1033.1984.tb08206.x. [DOI] [PubMed] [Google Scholar]
  24. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Summers W. C. A simple method for extraction of RNA from E. coli utilizing diethyl pyrocarbonate. Anal Biochem. 1970 Feb;33(2):459–463. doi: 10.1016/0003-2697(70)90316-7. [DOI] [PubMed] [Google Scholar]
  26. Vinopal R. T., Fraenkel D. G. Phenotypic suppression of phosphofructokinase mutations in Escherichia coli by constitutive expression of the glyoxylate shunt. J Bacteriol. 1974 Jun;118(3):1090–1100. doi: 10.1128/jb.118.3.1090-1100.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wang J. Y., Koshland D. E., Jr The reversible phosphorylation of isocitrate dehydrogenase of Salmonella typhimurium. Arch Biochem Biophys. 1982 Oct 1;218(1):59–67. doi: 10.1016/0003-9861(82)90321-6. [DOI] [PubMed] [Google Scholar]
  28. el-Mansi E. M., MacKintosh C., Duncan K., Holms W. H., Nimmo H. G. Molecular cloning and over-expression of the glyoxylate bypass operon from Escherichia coli ML308. Biochem J. 1987 Mar 15;242(3):661–665. doi: 10.1042/bj2420661. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES