Biochemical Genetics of the -Keto Acid Dehydrogenase Complexes of 12: Isolation and Biochemical Properties of Deletion Mutants Free

Abstract

Summary: Mutants of with deletions in the region of the chromosome were obtained for use in studies on the expression of the (pyruvate dehydrogenase complex, specific components) and (lipoamide dehydrogeriase) genes. These were isolated by selecting spontaneous mutants (lacking the general aromatic amino-acid permease and thus resistant to inhibitory aromatic amino-acid analogues) and screening for auxotrophy due to deletions extending into neighbouring genes. From 2892 isolates tested, the AroP- phenotypes of 2322 were confirmed and, of these, 28 stable and independently-derived auxotrophs were designated as deletion mutants.

Six nutritionally-distinct categories were recognized: Nad- (8 strains); NadAce (7); Nad ‘Ace’ (3); Ace (8); ‘Ace’ (1); Lpd (1). The Ace phenotypes of four isolates designated ‘Ace’ were leaky and enzymological studies confirmed that they had less than 7 % of parental pyruvate dehydrogenase complex activity.

Enzymological studies showed that the 15 Ace or NadAce strains all lacked the pyruvate dehydrogenase complex and pyruvate dehydrogenase (Eip) activities and only three retained detectable dihydrolipoamide acetyltransferase (E2p). The one Lpd- strain lacked pyruvate dehydrogenase, dihydrolipoamide acetyltransferase and lipoamide dehydrogenase (E3) activities as well as the activities of the pyruvate and -ketoglutarate dehydrogenase complexes.

The results confirmed the gene order and indicated that no other essential functions are determined by genes within the region. Resistance to lactate during growth of mutants on acetate was directly related to the specific activity of the pyruvate dehydrogenase complex. None of the deletions promoted the high degree of resistance characteristically associated with constitutive expression of the dehydrogenase complex. Six mutants having Ace or ‘Ace’ phenotypes were more sensitive than the parental strains and expression of their operons appeared to be affected; most sensitive were the Ace- strains which lacked pyruvate dehydrogenase complex and phosphoenolpyruvate synthetase activities.

The lipoamide dehydrogenase activities of the deletion strains (Lpd) ranged between 30 % and 100 % of parental levels indicating that expression of the gene may be affected by the operon but can be independent.

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1977-04-01
2024-03-29
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References

  1. Alwine J. C., Russell F. M., Murray K. N. 1973; Characterization of an Escherichia coli mutant deficient in dihydrolipoyl dehydrogenase activity. Journal of Bacteriology 115:1–8
    [Google Scholar]
  2. Ames G. F., Roth J. R. 1968; Histidine and aromatic permeases of Salmonella typhimurium. Journal of Bacteriology 96:1742–1749
    [Google Scholar]
  3. Brice C. B., Kornberg H. L. 1967; Location of a gene specifying phosphopyruvate synthase activity on the genome of Escherichia coli K12. Proceedings of the Royal Society B168:281–292
    [Google Scholar]
  4. Brown K. D. 1970; Formation of aromatic amino acid pools in Escherichia coli K-12. Journal of Bacteriology 104:177–188
    [Google Scholar]
  5. Carwlo-Castaneda G., Ortega M. V. 1970; Mutants of Salmonella typhimurium lacking phosphoenol-pyruvate carboxykinase and α-ketoglutarate dehydrogenase activities. Journal of Bacteriology 102:524–530
    [Google Scholar]
  6. Creaghan I. T., Guest J. R. 1972; Amber mutants of the α-ketoglutarate dehydrogenase gene of Escherichia coli K12. Journal of General Microbiology 71:207–220
    [Google Scholar]
  7. Demerec M. 1960; Frequency of deletions among spontaneous and induced mutations in Salmonella. Proceedings of the National Academy of Sciences of the United States of America 46:1075–1079
    [Google Scholar]
  8. Flatgaard J. E., Hoehn B., Henning U. 1971; Mutants of Escherichia coli KI2 which synthesize the pyruvate dehydrogenase complex constitutively. Archives of Biochemistry and Biophysics 143:461–470
    [Google Scholar]
  9. Guest J. R. 1974; Gene-protein relationships of the a-keto acid dehydrogenase complexes of Escherichia coli K12: chromosomal location of the lipoamide dehydrogenase gene. Journal of General Microbiology 80:523–532
    [Google Scholar]
  10. Guest J. R., Creaghan I. T. 1972; Lipoamide dehydrogenase mutants of Escherichia coli KI2. Biochemical Journal 130:8P
    [Google Scholar]
  11. Guest J. R., Creaghan I. T. 1973; Gene-protein relationships of the a-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and characterization of lipoamide dehydrogenase mutants. Journal of General Microbiology 5:197–210
    [Google Scholar]
  12. Guest J. R., Creaghan I. T. 1974; Further properties of lipoamide dehydrogenase mutants of Escherichia coli KI2. Journal of General Microbiology 81:237–245
    [Google Scholar]
  13. Gunsalus I. C., Razzell W. E. 1957; Preparation and assay of lipoic acid and derivatives. Methods in Enzymology 3:941–946
    [Google Scholar]
  14. Hager L. P., Kornberg H. L. 1961; On the mechanism of a-oxoglutarate oxidation in Escherichia coli. Biochemical Journal 78:194–198
    [Google Scholar]
  15. Henning U., Herz C. 1964; Ein Strukturgen-Komplex für den Pyruvat-Dehydrogenase-Komplexvon Escherichia coli K12. Zeitschrift für Vererbungslehre 95:260–275
    [Google Scholar]
  16. Henning U., Herz C., Szolyvay K. 1964; Polarisation und Disproportionalität der Synthese von Enzymkomponenten des Pyruvat-Dehydrogenase-Komplexes als Mutationsfolge in Escherichia coli K12. Zeitschrift für Vererbungslehre 95:236–259
    [Google Scholar]
  17. Henning U., Dennert G., Hertel R., Shipp W. S. 1966; Translation of the structural gene of the E. coli pyruvate dehydrogenase complex. Cold Spring Harbor Symposium for Quantitative Biology 31:227–234
    [Google Scholar]
  18. Henning U., Dietrich J., Murray K. N., Deppe G. 1968; Regulation of pyruvate dehydrogenase synthesis: substrate induction. In Molecular Genetics pp. 223–236 Wittman H. G., Schuster H. Edited by Berlin: Springer-Verlag.;
    [Google Scholar]
  19. Henning U., Busch W., Deppe G., Marek R. 1969; Pyruvate dehydrogenase synthesis in E. coli K12. Federation of European Biochemical Societies Symposia 19:19–28
    [Google Scholar]
  20. Herbert A. A., Guest J. R. 1969; Studies with α-ketoglutarate dehydrogenase mutants of Escherichia coli. Molecular and General Genetics 105:182–190
    [Google Scholar]
  21. Kornberg H. L. 1966; The role and control of the glyoxylate cycle in Escherichia coli. Biochemical Journal 99:1–11
    [Google Scholar]
  22. Kornberg H. L., Smith J. 1967; Genetic control of the uptake of pyruvate by Escherichia coli. Biochimica et biophysica acta 148:591–592
    [Google Scholar]
  23. Langley D., Guest J. R. 1974; Biochemical and genetic characteristics of deletion and other mutant strains of Salmonella typhimurium LT2 lacking a-keto acid dehydrogenase complex activities. Journal of General Microbiology 82:319–335
    [Google Scholar]
  24. Langley D., Guest J. R. 1975; Deletion mutants of Escherichia coli K12 lacking a-keto acid dehydrogenase complex activities. Proceedings of the Society for General Microbiology 2:66–67
    [Google Scholar]
  25. Lennox E. S. 1955; Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206
    [Google Scholar]
  26. Lipmann F., Tuttle L. C. 1945; A specific micromethod for the determination of acyl phosphates. Journal of Biological Chemistry 159:21–28
    [Google Scholar]
  27. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. 1951; Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193:265–275
    [Google Scholar]
  28. Thorne G. M., Corwin L. M. 1975; Mutations affecting aromatic amino acid transport in Escherichia coli and Salmonella typhimurium. . Journal of General Microbiology 90:203–216
    [Google Scholar]
  29. Vogel H., Bonner D. M. 1956; A convenient growth medium for Escherichia coli and some other micro-organisms. Microbial Genetics Bulletin 13:43–44
    [Google Scholar]
  30. Willms C. R., Oliver R. M., Henney H. R., Mukherjee B. B., Reed L. J. 1967; α-Keto acid dehydrogenase complexes. VI. Dissociation and reconstitution of the dihydrolipoyl transacetylase of E. coli. Journal of Biological Chemistry 242:889–897
    [Google Scholar]
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