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. 1984 Dec;160(3):1088–1092. doi: 10.1128/jb.160.3.1088-1092.1984

Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme.

C Grabau, J E Cronan Jr
PMCID: PMC215823  PMID: 6209262

Abstract

The pyruvate oxidase structural gene (poxB) of Escherichia coli was cloned into derivatives of plasmid pBR322. The gene was first cloned into a cosmid vector by selection for the tetracycline resistance determinant of a closely linked Tn10 insertion (no direct selection for the gene was available). Subsequent subcloning resulted in localization of the gene to a 3.1-kilobase-pair DNA segment. Two of the smaller poxB plasmids were shown to cause the overproduction of oxidase activity (by six- to eightfold), and one of these plasmids was shown to encode a protein having the size and antigenic determinants of pyruvate oxidase. Introduction of poxB plasmids into strains (aceEF) lacking pyruvate dehydrogenase activity relieved the aerobic growth requirement of the strains for exogenous acetate.

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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., Low K. B., Taylor A. L. Recalibrated linkage map of Escherichia coli K-12. Bacteriol Rev. 1976 Mar;40(1):116–167. doi: 10.1128/br.40.1.116-167.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bochner B. R., Savageau M. A. Generalized indicator plate for genetic, metabolic, and taxonomic studies with microorganisms. Appl Environ Microbiol. 1977 Feb;33(2):434–444. doi: 10.1128/aem.33.2.434-444.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang Y. Y., Cronan J. E. An Escherichia coli mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the enzyme. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4348–4352. doi: 10.1073/pnas.81.14.4348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang Y. Y., Cronan J. E., Jr Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase. J Bacteriol. 1983 May;154(2):756–762. doi: 10.1128/jb.154.2.756-762.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cobbett C. S., Pittard J. Formation of a lambda (Tn10) tyrR+ specialized transducing bacteriophage from Escherichia coli K-12. J Bacteriol. 1980 Dec;144(3):877–883. doi: 10.1128/jb.144.3.877-883.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cunningham C. C., Hager L. P. Crystalline pyruvate oxidase from Escherichia coli. 3. Phospholipid as an allosteric effector for the enzyme. J Biol Chem. 1971 Mar 25;246(6):1583–1589. [PubMed] [Google Scholar]
  7. Cunningham C. C., Hager L. P. Crystalline pyruvate oxidase from Escherichia coli. II. Activation by phospholipids. J Biol Chem. 1971 Mar 25;246(6):1575–1582. [PubMed] [Google Scholar]
  8. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  9. HENNING U. EIN REGULATIONSMECHANISMUS BEIM ABBAU DER BRENZTRAUBENSAEURE DURCH ESCHERICHIA COLI. Biochem Z. 1963 Jul 26;337:490–504. [PubMed] [Google Scholar]
  10. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Livingston D. M. Immunoaffinity chromatography of proteins. Methods Enzymol. 1974;34:723–731. doi: 10.1016/s0076-6879(74)34094-3. [DOI] [PubMed] [Google Scholar]
  13. MUNKRES K. D., RICHARDS F. M. THE PURIFICATION AND PROPERTIES OF NEUROSPORA MALATE DEHYDROGENASE. Arch Biochem Biophys. 1965 Mar;109:466–479. doi: 10.1016/0003-9861(65)90391-7. [DOI] [PubMed] [Google Scholar]
  14. Maquat L. E., Reznikoff W. S. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters. J Mol Biol. 1978 Nov 15;125(4):467–490. doi: 10.1016/0022-2836(78)90311-x. [DOI] [PubMed] [Google Scholar]
  15. Moyed H. S., Nguyen T. T., Bertrand K. P. Multicopy Tn10 tet plasmids confer sensitivity to induction of tet gene expression. J Bacteriol. 1983 Aug;155(2):549–556. doi: 10.1128/jb.155.2.549-556.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Russell P., Hager L. P., Gennis R. B. Characterization of the proteolytic activation of pyruvate oxidase. Control by specific ligands and by the flavin oxidation-reduction state. J Biol Chem. 1977 Nov 10;252(21):7877–7882. [PubMed] [Google Scholar]
  17. Russell P., Schrock H. L., Gennis R. B. Lipid activation and protease activation of pyruvate oxidase. Evidence suggesting a common site of interaction on the protein. J Biol Chem. 1977 Nov 10;252(21):7883–7887. [PubMed] [Google Scholar]
  18. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  19. Sancar A., Kacinski B. M., Mott D. L., Rupp W. D. Identification of the uvrC gene product. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5450–5454. doi: 10.1073/pnas.78.9.5450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schrock H. L., Gennis R. B. High affinity lipid binding sites on the peripheral membrane enzyme pyruvate oxidase. Specific ligand effects on detergent binding. J Biol Chem. 1977 Sep 10;252(17):5990–5995. [PubMed] [Google Scholar]
  21. 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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