Photosynthesis Research

, Volume 41, Issue 1, pp 75–88

An enzyme and13C-NMR study of carbon metabolism in heliobacteria

  • Mark W. Pickett
  • Michael P. Williamson
  • David J. Kelly
Group 3: New Organisms, Ecology and Biochemistry Regular Papers


Heliobacteria are a group of anoxygenic phototrophs that can grow photoheterotrophically in defined minimal media on only a limited range of organic substrates as carbon sources. In this study the mechanisms which operate to assimilate carbon and the routes employed for the biosynthesis of cellular intermediates were investigated in a newHeliobacterium strain, HY-3. This was achieved using two approaches (1) by measuring the activities of key enzymes in cell-free extracts and (2) by the use of13C nuclear magnetic resonance (NMR) spectroscopy to analyze in detail the labelling pattern of amino-acids of cells grown on [13C] pyruvate and [13C] acetate.Heliobacterium strain HY-3 was unable to grow autotrophically on CO2/H2 and neither (ATP)-citrate lyase nor ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPcase) were detectable in cell-free extracts. The enzyme profile of pyruvate grown cells indicated the presence of a pyruvate:acceptor oxidoreductase at high specific activity which could convert pyruvate to acetyl-Coenzyme A. No pyridine nucleotide dependent pyruvate dehydrogenase complex activity was detected. Of the citric-acid cycle enzymes, malate dehydrogenase, fumarase, fumarate reductase and an NADP-specific isocitrate dehydrogenase were readily detectable but no aconitase or citrate synthase activity was found. However, the labelling pattern of glutamate in long-term 2-[13C] acetate incorporation experiments indicated that a mechanism exists for the conversion of carbon from acetyl-CoA into 2-oxoglutarate. A 2-oxoglutarate:acceptor oxidoreductase activity was present which was also assayable by isotope exchange, but no 2-oxoglutarate dehydrogenase complex activity could be detected. Heliobacteria appear to use a type of incomplete reductive carboxylic acid pathway for the conversion of pyruvate to 2-oxoglutarate but are unable to grow autotrophically using this metabolic route due to the absence of ATP-citrate lyase.

Key words

photosynthesis carbon assimilation photosynthetic bacteria citric-acid cycle pyruvate synthase fermentation heliobacteria 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Allison MJ, Robinson IM and Baetz AL (1979) Synthesis of α-ketoglutarate by reductive carboxylation of succinate inVeillonella, Selenomonas andBacterioides species. J Bacteriol 140: 980–986Google Scholar
  2. Beatty JT and Gest H (1981) Generation of succinyl-CoA in photosynthetic bacteria. Arch Micobiol 129: 335–340Google Scholar
  3. Beer-Romero P and Gest H (1987)Heliobacillus mobilis, a peritrichously flagellated anoxyphototroph containing bacteriochlorophyll g. FEMS Microbiol Lett 41: 109–114Google Scholar
  4. Beer-Romero P, Favinger JL and Gest H (1988) Distinctive properties of bacilliform photosynthetic heliobacteria. FEMS Microbiol Letts 49: 451–454Google Scholar
  5. Brandis-Heep A, Gebhardt NA, Thauer RK, Widdel F and Pfennig N (1983) Anaerobic acetate oxidation to CO2 byDesulfobacter postgatei. 1. Demonstration of all enzymes required for the operation of the citric-acid cycle. Arch Microbiol 136: 222–229Google Scholar
  6. Brockman H and Lipinski A (1983) Bacteriochlorophyll g. A new bacteriochlorophyll fromHeliobacterium chlorum. Arch Microbiol 136: 17–19Google Scholar
  7. Cooper RA and Kornberg HL (1974) Phosphoenolpyruvate synthetase and pyruvate phosphate dikinase. In: Boyer PD (ed) The Enzymes, Vol X, pp 631–649, 3rd edn. Academic Press, New YorkGoogle Scholar
  8. Dixon GH and Kornberg HL (1959) Assay methods for key enzymes of the glyoxylate cycle. Biochem J 72: 3PGoogle Scholar
  9. Ekiel I, Smith ICP and Sprott GD (1983) Biosynthetic pathways inMethanospirillum hungatei as determined by13C nuclear magnetic resonance. J Bacteriol 156: 316–326Google Scholar
  10. Englander SW, Calhoun DB and Englander JJ (1987) Biochemistry without oxygen Anal Biochem 161: 300–306Google Scholar
  11. Evans MCW, Buchannan BB and Arnon DI (1966) A new ferredoxin dependent carbon reduction cycle in a photosynthetic bacterium. Proc Natl Acad Sci USA 55: 928–934Google Scholar
  12. Eyzaguirre J, Jansen K and Fuchs G (1982) Phosphoenolpyruvate synthetase inMethanobacterium thermoautotrophicum. Arch Microbiol 132: 67–74Google Scholar
  13. Gest H and Favinger JL (1983)Heliobacterium chlorum, an anoxygenic brownish-green photosynthetic bacterium containing a ‘new’ form of bacteriochlorophyll. Arch Microbiol 136: 11–16Google Scholar
  14. Gottschalk G (1968) The stereospecificity of the citrate synthase in sulfate reducing and photosynthetic bacteria. Eur J Biochem 5: 346–351Google Scholar
  15. Gottschalk G (1986) Bacterial Metabolism, 2nd edn. Springer-Verlag, New YorkGoogle Scholar
  16. Guest JR and Greaghan IT (1973) Gene-protein relationships of the α-keto acid dehydrogenase complexes ofEscherichia coli K12: Isolation and characterisation of lipoamide dehydrogenase mutants J Gen Microbiol 75: 197–210Google Scholar
  17. Hacking AJ and Quayle JR (1990) Malyl-CoA lyase fromMethylobacterium extorquens AM1. Meths Enzymol 188: 379–386Google Scholar
  18. Harris MA and Reddy CA (1977) Hydrogenase activity and the H2-fumarate electron transport system inBacteroides fragilis. J Bacteriol 131: 922–928Google Scholar
  19. Holo H and Sirevåg R (1986) Autotrophic growth and CO2 fixation ofChloroflexus aurantiacus. Arch Microbiol 145: 173–180Google Scholar
  20. Ivanofsky RN, Sintsov NV and Kondratieva EN (1980) ATP-linked citrate lyase activity in the green sulphur bacteriumChlorobium limicola formathiosulphatophilum. Arch Microbiol 128: 239–241Google Scholar
  21. Jones RW and Garland PB (1977) Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes ofEscherichia coli: Effects of permeability barriers imposed by the cytoplasmic membrane. Biochem J 164: 199–211Google Scholar
  22. Kimble L and Madigan M (1992) Nitrogen fixation and nitrogen metabolism in heliobacteria. Arch Microbiol 158: 155–161Google Scholar
  23. Kisumi M, Komatsubara S and Chibata I (1977). Pathway for isoleucine formation from pyruvate by leucine biosynthetic enzymes in leucine accumulating isoleucine revertants ofSerratia marcescens. J Biochem 82: 95–103Google Scholar
  24. Madigan MT (1992) The family Heliobacteriaceae. In: Balows A, Truper HG, Dworkin M, Harder W, Schliefer KH (eds) The Prokaryotes, pp 1981–1992, 2nd edn. Springer-Verlag, BerlinGoogle Scholar
  25. Meinecke B, Bertram J and Gottschalk G (1989) Purification and characterisation of the pyruvate-ferredoxin oxidoreductase fromClostridium acetobutylicum. Arch Microbiol 152: 244–250Google Scholar
  26. Möller D, Schauder R, Fuchs G and Thauer RK (1987) Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation inDesulfobacter postgatei growing on acetate and sulphate. Arch Microbiol 148: 202–207Google Scholar
  27. Nesbakken T, Kolsaker P and Ormerod J (1988) Mechanism of biosynthesis of 2-oxo-3-methylvalerate inChlorobium vibrioforme. J Bacteriol 170: 3287–3290Google Scholar
  28. Oberlies G, Fuchs G and Thauer RK (1980) Acetate thiokinase and the assimilation of acetate inMethanobacterium thermoautotrophicum. Arch Microbiol 128: 248–252Google Scholar
  29. Ormerod J, Nesbakken T and Torgersen Y (1990) Phototrophic bacteria that form heat resistant endospores. In: Baltscheffsky M (ed) Current Research in Photosynthesis, Vol 4, pp 935–938. Kluwer Academic Publishers, DordrechtGoogle Scholar
  30. Reeves HC, Rabin R, Wegener WS and Ajl SJ (1971). Assays of enzymes of the tricarboxylic acid and glyoxylate cycles. Methods Microbiol 6A: 437–439Google Scholar
  31. Rosenthal SN and Fendler JH (1976)13C NMR spectroscopy in macromolecular systems of biological interest. Adv Phys Org Chem 13: 279–423Google Scholar
  32. Schauder R, Widdel F and Fuchs G (1987) Carbon assimilation pathways in sulphate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophicDesulfobacter hydrogenophilus. Arch Microbiol 148: 218–225Google Scholar
  33. Sirevåg R and Ormerod JG (1970) Carbon dioxide fixation in green sulphur bacteria. Biochem J 120: 399–408Google Scholar
  34. Taylor DP, Cohen SN, Clark WG and Marrs BL (1983) Alignment of the genetic and restriction maps of the photosynthesis regions of theRhodopseudomonas capsulata chromosome by a conjugation-mediated marker rescue technique. J Bacteriol 154: 580–590Google Scholar
  35. Umbarger HE (1978) Amino-acid biosynthesis and its regulation. Ann Rev Biochem 47: 533Google Scholar
  36. Vollbrecht D (1978) Three pathways of isoleucine biosynthesis in mutant strains ofSaccharomyces cerevisiae. Biochim Biophys Acta 362: 382–389Google Scholar
  37. Weaver PF, Wall JD and Gest H (1975) Characterisation ofRhodopseudomonas capsulata. Arch Microbiol 105: 207–216Google Scholar
  38. Williamson JR and Corkey BE (1969) Assays of the intermediates of the citric-acid cycle and related compounds by fluorometric enzyme methods. Meth Enzymol 13: 434–513Google Scholar
  39. Woese CR, Debrunner-Vossbrinck BA, Oyaizu H, Stackebrandt E and Ludwig W (1985) Gram-positive bacteria: Possible photosynthetic ancestry. Science 229: 762–765Google Scholar
  40. Wood HG, Davis JJ and Willard JM (1969) PEP carboxytransphosphorylase fromPropionibacterium shermanii. In: Lowenstein JM (ed) Methods in Enzymology, Vol XIII, pp 297–309. Academic Press, New YorkGoogle Scholar
  41. Zeikus JG, Fuchs G, Kenealy W and Thauer RK (1977) Oxidoreductases involved in cell carbon synthesis ofMethanobacterium thermoautotrophicum. J Bacteriol 132: 604–613Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Mark W. Pickett
    • 1
    • 2
  • Michael P. Williamson
    • 1
  • David J. Kelly
    • 1
    • 2
  1. 1.Krebs Institute, Department of Molecular Biology and BiotechnologyUniversity of SheffieldSheffieldUK
  2. 2.Robert Hill Institute, Department of Molecular Biology and BiotechnologyUniversity of SheffieldSheffieldUK

Personalised recommendations