Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1974 Jun;118(3):999–1009. doi: 10.1128/jb.118.3.999-1009.1974

Pyruvate Carboxylase Deficiency in Pleiotropic Carbohydrate-Negative Mutant Strains of Pseudomonas aeruginosa

P V Phibbs Jr a, T W Feary b, W T Blevins c,1
PMCID: PMC246850  PMID: 4208415

Abstract

Cell extracts of Pseudomonas aeruginosa strain PAO were found to contain pyruvate carboxylase activity. Specific activities were minimal when cells were grown on Casamino Acids, acetate, or succinate, but were three- to fourfold higher when cells were grown in glucose, gluconate, glycerol, lactate, or pyruvate minimal media. The reaction in crude cell extracts and in partially purified preparations was dependent on pyruvate, adenosine 5′-triphosphate, and Mg2+, but was not affected by either the presence or absence of acetyl coenzyme A. Activity was nearly totally inhibited by avidin and this inhibition was substantially blocked by free biotin in incubation mixtures. Cell extracts were shown to fix 14CO2 in a reaction that had these same characteristics. Eight pleiotropic, carbohydrate-negative mutant strains of the organism were isolated after nitrosoguanidine mutagenesis. Each mutant strain grew normally in acetate, succinate, and citrate minimal media but failed to utilize glucose, gluconate, 2-ketogluconate, mannitol, glycerol, lactate, and pyruvate as sole sources of carbon and energy. These strains were found by quantitative transductional analysis with phage F116 to form a single linkage group. Cell extracts of each mutant strain were either lacking or severely deficient in pyruvate carboxylase activity. Spontaneous revertants of five of the eight strains were isolated and found to recover simultaneously both pyruvate carboxylase activity and the ability to utilize each of the C6 and C3 compounds. A second linkage group of similar mutant strains that grew on the C3 compounds was found to contain normal levels of pyruvate carboxylase activity, but each strain was deficient in an enzyme of the Entner-Doudoroff pathway.

Full text

PDF
999

Selected References

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

  1. ASHWORTH J. M., KORNBERG H. L. FINE CONTROL OF THE GLYOXYLATE CYCLE BY ALLOSTERIC INHIBITION OF ISOCITRATE LYASE. Biochim Biophys Acta. 1963 Jul 9;73:519–522. doi: 10.1016/0006-3002(63)90457-8. [DOI] [PubMed] [Google Scholar]
  2. Ashworth J. M., Kornberg H. L. The anaplerotic fixation of carbon dioxide by Escherichia coli. Proc R Soc Lond B Biol Sci. 1966 Aug 16;165(999):179–188. doi: 10.1098/rspb.1966.0063. [DOI] [PubMed] [Google Scholar]
  3. BLOOM S. J., JOHNSON M. J. The pyruvate carboxylase of Aspergillus niger. J Biol Chem. 1962 Sep;237:2718–2720. [PubMed] [Google Scholar]
  4. Calhoun D. H., Feary T. W. Transductional analysis of Pseudomonas aeruginosa methionineless auxotrophs. J Bacteriol. 1969 Jan;97(1):210–216. doi: 10.1128/jb.97.1.210-216.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cazzulo J. J., Sundaram T. K., Dilks S. N., Kornberg H. L. Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Purification and properties of the apoenzyme and the holoenzyme synthetase. Biochem J. 1971 May;122(5):653–661. doi: 10.1042/bj1220653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cazzulo J. J., Sundaram T. K., Kornberg H. L. Mechanism of pyruvate carboxylase formation from the apo-enzyme and biotin in a thermophilic bacillus. Nature. 1970 Sep 12;227(5263):1103–1105. doi: 10.1038/2271103a0. [DOI] [PubMed] [Google Scholar]
  7. Cazzulo J. J., Sundaram T. K., Kornberg H. L. Properties and regulation of pyruvate carboxylase from Bacillus stearothermophilus. Proc R Soc Lond B Biol Sci. 1970 Oct 13;176(1042):1–19. doi: 10.1098/rspb.1970.0030. [DOI] [PubMed] [Google Scholar]
  8. Diesterhaft M. D., Freese E. Role of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme during growth and sporulation of Bacillus subtilis. J Biol Chem. 1973 Sep 10;248(17):6062–6070. [PubMed] [Google Scholar]
  9. Eyzaguirre J., Cornwell E., Borie G., Ramírez B. Two malic enzymes in Pseudomonas aeruginosa. J Bacteriol. 1973 Oct;116(1):215–221. doi: 10.1128/jb.116.1.215-221.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. FULLER R. C., SMILLIE R. M., SISLER E. C., KORNBERG H. L. Carbon metabolism in Chromatium. J Biol Chem. 1961 Jul;236:2140–2149. [PubMed] [Google Scholar]
  11. Feary T. W., Williams B., Calhoun D. H., Walker T. A. An analysis of arginine requiring mutants in Pseudomonas aeruginosa. Genetics. 1969 Jul;62(3):673–686. [PMC free article] [PubMed] [Google Scholar]
  12. GAILIUSIS J., RINNE R. W., BENEDICT C. R. PYUVATE-OXALOACETATE EXCHANGE REACTION IN BAKER'S YEAST. Biochim Biophys Acta. 1964 Dec 23;92:595–601. doi: 10.1016/0926-6569(64)90019-7. [DOI] [PubMed] [Google Scholar]
  13. HOLLOWAY B. W., EGAN J. B., MONK M. Lysogeny in Pseudomonas aeruginosa. Aust J Exp Biol Med Sci. 1960 Aug;38:321–329. doi: 10.1038/icb.1960.34. [DOI] [PubMed] [Google Scholar]
  14. Holloway B. W. Genetics of Pseudomonas. Bacteriol Rev. 1969 Sep;33(3):419–443. doi: 10.1128/br.33.3.419-443.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hylemon P. B., Krieg N. R., Phibbs P. V., Jr Transport and catabolism of D-fructose by Spirillum itersomii. J Bacteriol. 1974 Jan;117(1):144–150. doi: 10.1128/jb.117.1.144-150.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hylemon P. B., Phibbs P. V., Jr Independent regulation of hexose catabolizing enzymes and glucose transport activity in Pseudomonas aeruginosa. Biochem Biophys Res Commun. 1972 Sep 5;48(5):1041–1048. doi: 10.1016/0006-291x(72)90813-3. [DOI] [PubMed] [Google Scholar]
  17. Jacobson L. A., Bartholomaus R. C., Gunsalus I. C. Repression of malic enzyme by acetate in Pseudomonas. Biochem Biophys Res Commun. 1966 Sep 22;24(6):955–960. doi: 10.1016/0006-291x(66)90343-3. [DOI] [PubMed] [Google Scholar]
  18. LOSADA M., CANOVAS J. L., RUIZ AMIL M. OXALOACETATE, CITRAMALATE AND GLUTAMATE FORMATION FROM PYRUVATE IN BAKER'S YEAST. Biochem Z. 1964 Jul 8;340:60–74. [PubMed] [Google Scholar]
  19. 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]
  20. Large P. J., Peel D., Quayle J. R. Microbial growth on C(1) compounds. 4. Carboxylation of phosphoenolpyruvate in methanol-grown Pseudomonas AM1. Biochem J. 1962 Oct;85(1):243–250. doi: 10.1042/bj0850243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maeba P., Sanwal B. D. Feedback inhibition of phosphoenolpyruvate carboxylase of Salmonella. Biochem Biophys Res Commun. 1965 Dec 9;21(5):503–508. doi: 10.1016/0006-291x(65)90412-2. [DOI] [PubMed] [Google Scholar]
  22. McFadden B. A., Schuster E. 3-phosphoglycerate kinase from Hydrogenomonas facilis. J Bacteriol. 1972 Feb;109(2):751–756. doi: 10.1128/jb.109.2.751-756.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Phibbs P. V., Jr, Eagon R. G. Transport and phosphorylation of glucose, fructose, and mannitol by Pseudomonas aeruginosa. Arch Biochem Biophys. 1970 Jun;138(2):470–482. doi: 10.1016/0003-9861(70)90371-1. [DOI] [PubMed] [Google Scholar]
  24. Renner E. D., Bernlohr R. W. Characterization and regulation of pyruvate carboxylase of Bacillus licheniformis. J Bacteriol. 1972 Feb;109(2):764–772. doi: 10.1128/jb.109.2.764-772.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. SEUBERT W., REMBERGER U. [Purification and mechanism of action of pyruvate carboxylase from Pseudomonas citronellolis]. Biochem Z. 1961;334:401–414. [PubMed] [Google Scholar]
  26. Sundaram T. K., Cazzulo J. J., Kornberg H. L. Anaplerotic CO2 fixation in mesophilic and thermophilic bacilli. Biochim Biophys Acta. 1969 Nov 18;192(2):355–357. doi: 10.1016/0304-4165(69)90377-8. [DOI] [PubMed] [Google Scholar]
  27. Sundaram T. K. Physiological role of pyruvate carboxylase in a thermophilic bacillus. J Bacteriol. 1973 Feb;113(2):549–557. doi: 10.1128/jb.113.2.549-557.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. THEODORE T. S., ENGLESBERG E. MUTANT OF SALMONELLA TYPHIMURIUM DEFICIENT IN THE CARBON DIOXIDE-FIXING ENZYME PHOSPHOENOLPYRUVIC CARBOXYLASE. J Bacteriol. 1964 Oct;88:946–955. doi: 10.1128/jb.88.4.946-955.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tiwari N. P., Campbell J. J. Enzymatic control of the metabolic activity of Pseudomonas aeruginosa grown in glucose or succinate media. Biochim Biophys Acta. 1969 Dec 30;192(3):395–401. doi: 10.1016/0304-4165(69)90388-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES