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. 1997 Mar;179(5):1460–1468. doi: 10.1128/jb.179.5.1460-1468.1997

Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus.

A F Yakunin 1, P C Hallenbeck 1
PMCID: PMC178853  PMID: 9045800

Abstract

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.

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Selected References

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  1. Attwood P. V. The structure and the mechanism of action of pyruvate carboxylase. Int J Biochem Cell Biol. 1995 Mar;27(3):231–249. doi: 10.1016/1357-2725(94)00087-r. [DOI] [PubMed] [Google Scholar]
  2. Beatty J. T., Gest H. Biosynthetic and bioenergetic functions of citric acid cycle reactions in Rhodopseudomonas capsulata. J Bacteriol. 1981 Nov;148(2):584–593. doi: 10.1128/jb.148.2.584-593.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black P. N., DiRusso C. C. Molecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli. Biochim Biophys Acta. 1994 Jan 3;1210(2):123–145. doi: 10.1016/0005-2760(94)90113-9. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Cazzulo J. J., Sundaram T. K., Kornberg H. L. Regulation of pyruvate carboxylase formation from the apo-enzyme and biotin in a thermophilic bacillus. Nature. 1969 Sep 13;223(5211):1137–1138. doi: 10.1038/2231137a0. [DOI] [PubMed] [Google Scholar]
  6. Cohen N. D., Duc J. A., Beegen H., Utter M. F. Quaternary structure of pyruvate carboxylase from Pseudomonas citronellolis. J Biol Chem. 1979 Sep 25;254(18):9262–9269. [PubMed] [Google Scholar]
  7. Gibson J. Uptake of C4 dicarboxylates and pyruvate by Rhodopseudomonas spheroides. J Bacteriol. 1975 Aug;123(2):471–480. doi: 10.1128/jb.123.2.471-480.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hamblin M. J., Shaw J. G., Curson J. P., Kelly D. J. Mutagenesis, cloning and complementation analysis of C4-dicarboxylate transport genes from Rhodobacter capsulatus. Mol Microbiol. 1990 Sep;4(9):1567–1574. [PubMed] [Google Scholar]
  9. Iuchi S., Aristarkhov A., Dong J. M., Taylor J. S., Lin E. C. Effects of nitrate respiration on expression of the Arc-controlled operons encoding succinate dehydrogenase and flavin-linked L-lactate dehydrogenase. J Bacteriol. 1994 Mar;176(6):1695–1701. doi: 10.1128/jb.176.6.1695-1701.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. KNIGHT M. The photometabolism of propionate by Rhodospirillum rubrum. Biochem J. 1962 Jul;84:170–185. doi: 10.1042/bj0840170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. KORNBERG H. L., LASCELLES J. The formation of isocitratase by the Athiorhodaceae. J Gen Microbiol. 1960 Dec;23:511–517. doi: 10.1099/00221287-23-3-511. [DOI] [PubMed] [Google Scholar]
  12. LASCELLES J., RODGERS K. The intracellular concentration of succinic acid in photosynthetic bacteria. Biochem J. 1961 Aug;80:244–245. doi: 10.1042/bj0800244. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  14. Lehn D. A., Moran S. M., MacDonald M. J. The sequence of the rat pyruvate carboxylase-encoding cDNA. Gene. 1995 Nov 20;165(2):331–332. doi: 10.1016/0378-1119(95)00557-m. [DOI] [PubMed] [Google Scholar]
  15. Li S. J., Cronan J. E., Jr Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis. J Bacteriol. 1993 Jan;175(2):332–340. doi: 10.1128/jb.175.2.332-340.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lim F., Morris C. P., Occhiodoro F., Wallace J. C. Sequence and domain structure of yeast pyruvate carboxylase. J Biol Chem. 1988 Aug 15;263(23):11493–11497. [PubMed] [Google Scholar]
  17. Lowry O. H., Carter J., Ward J. B., Glaser L. The effect of carbon and nitrogen sources on the level of metabolic intermediates in Escherichia coli. J Biol Chem. 1971 Nov;246(21):6511–6521. [PubMed] [Google Scholar]
  18. MacKay N., Rigat B., Douglas C., Chen H. S., Robinson B. H. cDNA cloning of human kidney pyruvate carboxylase. Biochem Biophys Res Commun. 1994 Jul 29;202(2):1009–1014. doi: 10.1006/bbrc.1994.2029. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Modak H. V., Kelly D. J. Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography. Microbiology. 1995 Oct;141(Pt 10):2619–2628. doi: 10.1099/13500872-141-10-2619. [DOI] [PubMed] [Google Scholar]
  21. Mosley C. S., Suzuki J. Y., Bauer C. E. Identification and molecular genetic characterization of a sensor kinase responsible for coordinately regulating light harvesting and reaction center gene expression in response to anaerobiosis. J Bacteriol. 1994 Dec;176(24):7566–7573. doi: 10.1128/jb.176.24.7566-7573.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Park S. J., Cotter P. A., Gunsalus R. P. Regulation of malate dehydrogenase (mdh) gene expression in Escherichia coli in response to oxygen, carbon, and heme availability. J Bacteriol. 1995 Nov;177(22):6652–6656. doi: 10.1128/jb.177.22.6652-6656.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Park S. J., McCabe J., Turna J., Gunsalus R. P. Regulation of the citrate synthase (gltA) gene of Escherichia coli in response to anaerobiosis and carbon supply: role of the arcA gene product. J Bacteriol. 1994 Aug;176(16):5086–5092. doi: 10.1128/jb.176.16.5086-5092.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Park S. J., Tseng C. P., Gunsalus R. P. Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr. Mol Microbiol. 1995 Feb;15(3):473–482. doi: 10.1111/j.1365-2958.1995.tb02261.x. [DOI] [PubMed] [Google Scholar]
  25. Payne J., Morris J. G. Pyruvate carboxylase in Rhodopseudomonas spheroides. J Gen Microbiol. 1969 Nov;59(1):97–101. doi: 10.1099/00221287-59-1-97. [DOI] [PubMed] [Google Scholar]
  26. Qian Y., Tabita F. R. A global signal transduction system regulates aerobic and anaerobic CO2 fixation in Rhodobacter sphaeroides. J Bacteriol. 1996 Jan;178(1):12–18. doi: 10.1128/jb.178.1.12-18.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ramseier T. M., Bledig S., Michotey V., Feghali R., Saier M. H., Jr The global regulatory protein FruR modulates the direction of carbon flow in Escherichia coli. Mol Microbiol. 1995 Jun;16(6):1157–1169. doi: 10.1111/j.1365-2958.1995.tb02339.x. [DOI] [PubMed] [Google Scholar]
  28. Scrutton M. C., Taylor B. L. Isolation and characterization of pyruvate carboxylase from Azotobacter vinelandii OP. Arch Biochem Biophys. 1974 Oct;164(2):641–654. doi: 10.1016/0003-9861(74)90076-9. [DOI] [PubMed] [Google Scholar]
  29. Shaw J. G., Hamblin M. J., Kelly D. J. Purification, characterization and nucleotide sequence of the periplasmic C4-dicarboxylate-binding protein (DctP) from Rhodobacter capsulatus. Mol Microbiol. 1991 Dec;5(12):3055–3062. doi: 10.1111/j.1365-2958.1991.tb01865.x. [DOI] [PubMed] [Google Scholar]
  30. Stucka R., Dequin S., Salmon J. M., Gancedo C. DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains. Mol Gen Genet. 1991 Oct;229(2):307–315. doi: 10.1007/BF00272171. [DOI] [PubMed] [Google Scholar]
  31. Taylor B. L., Routman S., Utter M. F. The control of the synthesis of pyruvate carboxylase in Pseudomonas citronellolis. Evience from double labeling studies. J Biol Chem. 1975 Mar 25;250(6):2376–2382. [PubMed] [Google Scholar]
  32. Weaver P. F., Wall J. D., Gest H. Characterization of Rhodopseudomonas capsulata. Arch Microbiol. 1975 Nov 7;105(3):207–216. doi: 10.1007/BF00447139. [DOI] [PubMed] [Google Scholar]
  33. Wexler I. D., Du Y., Lisgaris M. V., Mandal S. K., Freytag S. O., Yang B. S., Liu T. C., Kwon M., Patel M. S., Kerr D. S. Primary amino acid sequence and structure of human pyruvate carboxylase. Biochim Biophys Acta. 1994 Oct 21;1227(1-2):46–52. doi: 10.1016/0925-4439(94)90105-8. [DOI] [PubMed] [Google Scholar]
  34. Willison J. C. Biochemical genetics revisited: the use of mutants to study carbon and nitrogen metabolism in the photosynthetic bacteria. FEMS Microbiol Rev. 1993 Jan;10(1-2):1–38. doi: 10.1111/j.1574-6968.1993.tb05862.x. [DOI] [PubMed] [Google Scholar]
  35. Wood H. G., Barden R. E. Biotin enzymes. Annu Rev Biochem. 1977;46:385–413. doi: 10.1146/annurev.bi.46.070177.002125. [DOI] [PubMed] [Google Scholar]
  36. Wurtele E. S., Nikolau B. J. Plants contain multiple biotin enzymes: discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase in the plant kingdom. Arch Biochem Biophys. 1990 Apr;278(1):179–186. doi: 10.1016/0003-9861(90)90246-u. [DOI] [PubMed] [Google Scholar]
  37. Yakunin A. F., Gennaro G., Hallenbeck P. C. Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus. J Bacteriol. 1993 Nov;175(21):6775–6780. doi: 10.1128/jb.175.21.6775-6780.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang J., Xia W. L., Brew K., Ahmad F. Adipose pyruvate carboxylase: amino acid sequence and domain structure deduced from cDNA sequencing. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1766–1770. doi: 10.1073/pnas.90.5.1766. [DOI] [PMC free article] [PubMed] [Google Scholar]

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