Antonie van Leeuwenhoek

, Volume 63, Issue 1, pp 1–16 | Cite as

The relation of proton motive force, adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

  • Ben A. Bulthuis
  • Gregory M. Koningstein
  • Adriaan H. Stouthamer
  • Henk W. van Verseveld


The magnitude of the proton motive force (Δp) and its constituents, the electrical (Δψ) and chemical potential (-ZΔpH), were established for chemostat cultures of a protease-producing, relaxed (rel) variant and a not protease-producing, stringent (rel+) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of Δp with the specific growth rate μ was found. The calculated intracellular pH (pHin) was not constant but inversely related to μ. This change in pHin might be related to regulatory functions of metabolism but a regulatory role for pHin itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with μ for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low μ and an increased value at higher μ. For both limitations, the ATP/ADP ratio was directly related to μ.

The phosphorylation potential (ΔG'p) was invariant with μ. From the values for ΔG'p and Δp, a variable →H+/ATP-stoichiometry was inferred: →H+/ATP=1.83+0.52µ, so that at a given →H+/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for μ=0 to μmax (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of ΔG'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing μ are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible μ with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.

Key words

adenylate energy charge Bacillus chemostat energy conservation extracellular protease membrane potential phosphorylation potential proton motive force 





dry weight of biomass


Faraday's constant, 96.6 J/(mV × mol)


chemostat outflow-rate (ml/h)




phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and Pi


standard Gibbs energy change at specified conditions


number of protons translocated through


synthase in synthesis of one ATP


protons translocated during transfer of 2 electrons from substrate to oxygen


specific growth rate (1/h)


transmembrane electrochemical proton potential, J/mol


‘molar weight’ (147.6 g/mol) of bacteria with general cell formula C6.0H10.8O3.0N1.2


extracellular, intracellular pH


(intracellular) inorganic phosphate


proton motive force, mV


transmembrane pH-difference


transmembrane electrical potential, mV


number of ADP phosphorylated to ATP upon reduction of one ‘O2−’ to H2O by two electrons transferred through the electron transfer chain


(→H+/O) × (→H+/ATP)−1


P/O with the two electrons donated by resp. (NADH + H+) and FADH


specific rate of consumption or production (mol/g DW × h)


stringent, relaxed genotype


universal gas constant, 8.36 J/(mol × degree)


absolute temperature


triphenylmethylphosphonium ion


tetraphenyl phosphonium ion


growth yield, g DW/mol


conversion constant=61.8 mV for 310 K (37 °C)


transmembrane proton potential or chemical potential, mV


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  1. Atkinson DE (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7: 430–434Google Scholar
  2. Azzone GF, Pietrobon D & Zoratti M (1984) Determination of the proton gradient across biological membranes. In: Lee CP (Ed) Curr Topics Bioenergetics 13 (pp 1–77). Academic Press, Inc., Orlando, USAGoogle Scholar
  3. Bakker EP (1990) The role of alkali-cation transport in energy coupling of neutrophilic and acidophilic bacteria: an assessment of methods and concepts. FEMS. Microbiol. Rev. 75: 319–334Google Scholar
  4. Bakker EP & Randall LL (1984) The requirement for energy during export of β-lactamase inEscherichia coli is fulfilled by the total proton motive force. EMBO J. 3: 895–900Google Scholar
  5. Barrette Jr. WC, Hannum DM, Wheeler WD & Hurst JK (1988) Viability and metabolic capacity are maintained byEscherichia coli, Pseudomonas aeruginosa, andStreptococcus lactis at very low adenylate energy charge. J. Bacteriol. 170: 3655–3659Google Scholar
  6. Bierbaum G, Giesecke UE & Wandrey C (1991) Analysis of nucleotide pools during protease production withBacillus licheniformis. Appl. Microbiol. Biotechnol. 35: 725–730Google Scholar
  7. Booth IR (1985) Regulation of cytoplasmic pH in bacteria. Microbiol. Rev. 49: 359–378Google Scholar
  8. Bulthuis BA, Frankena J, Koningstein GM, van Verseveld HW & Stouthamer AH (1988) Instability of protease production in arel +/rel -pair ofBacillus licheniformis and associated morphological and physiological characteristics. Antonie van Leeuwenhoek 54: 95–111Google Scholar
  9. Bulthuis BA, Koningstein GM, Stouthamer AH & van Verseveld HW (1989) A comparison between aerobic growth ofBacillus licheniformis in continuous culture and partial recycling fermentor, with contributions to the discussion on maintenance energy demand. Arch. Microbiol. 152: 499–507Google Scholar
  10. Bulthuis BA, Rommens C, Koningstein GM, Stouthamer AH & van Verseveld HW (1991) Formation of fermentation products and extracellular protease during anaerobic growth ofBacillus licheniformis in chemostat and batch-culture. Antonie van Leeuwenhoek 60: 355–371Google Scholar
  11. Chapman AS & Atkinson DE (1977) Adenine nucleotide concentrations and turnover rates. Adv. Microbial. Physiol. 15: 253–306Google Scholar
  12. Chapman AS, Fall L & Atkinson DE (1971) Adenylate energy charge inEscherichia coli during growth and starvation. J. Bacteriol. 108: 1072–1086Google Scholar
  13. Chen PS, Toribara TY & Warner H (1956) Microdetermination of phosphorus. Anal. Chem. 28: 1756–1758Google Scholar
  14. Chen S-C, Brown PR & Rosie DM (1977) Extraction procedures for use prior to HPLC nucleotide analysis using microparticle chemically bonded packings. J. Chromat. Sci. 15: 218–221Google Scholar
  15. de Vries W & Stouthamer AH (1968) Fermentation of glucose, lactose, galactose, mannitol and xylose byBifidobacteria. J. Bacteriol. 96: 472–478Google Scholar
  16. Frankena J, van Verseveld HW & Stouthamer AH (1985) A continuous culture study of the bioenergetic aspects of growth and production of exocellular protease inBacillus licheniformis. Appl. Microbiol. Biotechnol. 22: 169–176Google Scholar
  17. Frankena J, Koningstein GM, van Verseveld HW & Stouthamer AH (1986) Effect of different limitations in chemostat cultures on growth and production of exocellular protease byBacillus licheniformis. Appl. Microbiol. Biotechnol. 24: 106–112Google Scholar
  18. Frankena J, van Verseveld HW & Stouthamer AH (1988) Substrate and energy costs of the production of exocellular enzymes byBacillus licheniformis. Biotechnol. Bioeng. 32: 803–812Google Scholar
  19. Fynn GH & Davison JA (1976) Adenine nucleotide pool and energy charge during growth of a tyrothricin-producing strain ofBacillus brevis. J. Gen. Microbiol. 94: 68–74Google Scholar
  20. Gober JW & Kashket ER (1984) H+/ATP stoichiometry of cowpeaRhizobium sp. strain 32H1 cells grown under nitrogen-fixing and nitrogen-nonfixing conditions. J. Bacteriol. 160: 216–221Google Scholar
  21. Guffanti AA & Hicks DB (1991) Molar growth yields and bioenergetic parameters of extremely alkaliphilicBacillus species in batch cultures, and growth in a chemostat at pH 10.5. J. Gen. Microbiol. 137: 2375–2379Google Scholar
  22. Hamaide F, Kushner DJ & Sprott GD (1983) Proton motive force and Na/H+ antiport in a moderate halophile. J. Bacteriol. 156: 537–544Google Scholar
  23. Hellingwerf KJ, Lolkema JS, Otto R, Neijssel OM, Stouthamer AH, Harder W, van Dam K & Westerhoff HV (1982) Energetics of microbial growth: an analysis of the relationship between growth and its mechanistic basis by mosaic non-equilibrium thermodynamics. FEMS Microbiol. Lett. 15: 7–17Google Scholar
  24. Hutchison KW & Hanson RS (1974) Adenine nucleotide changes associated with the initiation of sporulation inBacillus subtilis. J. Bacteriol. 119: 70–75Google Scholar
  25. Jones CW, Brice JM, Downs AJ & Drozd JW (1975) Bacterial respiration-linked proton translocation and its relationship to respiratory-chain composition. Eur. J. Biochem. 52: 265–271Google Scholar
  26. Kashket ER (1981a) Proton motive force in growingStreptococcus lactis andStaphylococcus aureus cells under aerobic and anaerobic conditions. J. Bacteriol. 146: 369–376Google Scholar
  27. Kashket ER (1981b) Effects of aerobiosis and nitrogen source on the proton motive force in growingEscherichia coli andKlebsiella pneumoniae cells. J. Bacteriol. 146: 377–384Google Scholar
  28. Kashket ER (1985) The proton motive force in bacteria: a critical assessment of methods. Ann. Rev. Micro. 39: 219–242Google Scholar
  29. Kashket ER, Blanchard AG & Metzger WC (1980) Proton motive force during growth ofStreptococcus lactis cells. J. Bacteriol. 143: 128–134Google Scholar
  30. Kell DB (1988) Protonmotive energy-transducing systems: some physical principles and experimental approaches. In: Anthony C (Ed) Bacterial Energy Transduction (pp 429–490). Academic Press Ltd, London, UKGoogle Scholar
  31. Khym JX (1975) An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers. Clin. Chem. 21: 1245–1252Google Scholar
  32. Knowles CJ (1977) Microbial metabolic regulation by adenine nucleotide pools. In: Haddock BA & Hamilton WA (Eds) Microbial Energetics (pp 241–283). Symp. Soc. Gen. Microbiol. 27, Cambridge University Press, Cambridge, UKGoogle Scholar
  33. Kobayashi H, Suzuki T, Kinoshita N & Unemoto T (1984) Amplification of theStreptococcus faecalis proton-translocating ATPase by a decrease in cytoplasmic pH. J. Bacteriol. 158: 1157–1160Google Scholar
  34. Konings WN & Veldkamp H (1983) Energy transduction and solute transport mechanisms in relation to environments occupied by microorganisms. In: Slater JH, Whittenbury R & Wimpenny JWT (Eds) Microbes in Their Natural Environments (pp 153–186). Symp. Soc. Gen. Microbiol. 34, Cambridge University Press, Cambridge, UKGoogle Scholar
  35. Krab K & van Wezel J (1992) Improved derivation of phosphate potentials at different temperatures. Biochim. Biophys. Acta 1098: 172–176Google Scholar
  36. Little R & Bremer H (1982) Quantitation of guanosine 5′,3′-bidiphosphate in extracts from bacterial cells by ion-pair reverse-phase high-performance liquid chromatography. Anal. Biochem. 126: 381–388Google Scholar
  37. Lolkema JS, Hellingwerf KJ & Konings WN (1982) The effect of ‘probe binding’ on the quantitative determination of the proton-motive force in bacteria. Biochim. Biophys. Acta 681: 85–94Google Scholar
  38. Maloney PC (1983) Relationship between phosphorylation potential and electrochemical H+ gradient during glycolysis inStreptococcus lactis. J. Bacteriol. 153: 1461–1470Google Scholar
  39. Marriott ID, Dawes EA & Rowley BI (1981) Effect of growth rate and nutrient limitation on the adenine nucleotide content, energy charge and enzymes of adenylate metabolism inAzotobacter beijerinckii. J. Gen. Microbiol. 125: 375–382Google Scholar
  40. Michels M & Bakker EP (1985) Generation of a large, protonophore-sensitive proton motive force and pH difference in the acidophilic bacteriaThermoplasma acidophilum andBacillus acidocaldarius. J. Bacteriol. 161: 231–237Google Scholar
  41. Mizushima S & Tokuda H (1990)In vitro translocation of bacterial secretory proteins and energy requirements. J. Bioenerg. Biomem. 22: 389–399Google Scholar
  42. Murén EM & Randall LL (1985) Export of α-amylase byBacillus amyloliquefaciens requires proton motive force. J. Bacteriol. 164: 712–716Google Scholar
  43. Neijssel OM & Tempest DW (1979) The physiology of metabolite overproduction. Symp. Soc. Gen. Microbiol. 29: 53–82Google Scholar
  44. Otto R, ten Brink B, Veldkamp H & Konings WN (1983) The relation between growth rate and electrochemical proton gradient ofStreptococcus cremoris. FEMS Microbiol. Lett. 16: 69–74Google Scholar
  45. Otto R, Klont B, ten Brink B & Konings WN (1984) The phosphate potential, adenylate energy charge and proton motive force in growing cells ofStreptococcus cremoris. Arch. Microbiol. 139: 338–343Google Scholar
  46. Otto R, Klont B & Konings WN (1985) The relation between phosphate potential and growth rate ofStreptococcus cremoris. Arch. Microbiol. 142: 97–100Google Scholar
  47. Padan E, Zilberstein D & Schuldiner S (1981) pH homeostasis in bacteria. Biochim. Biophys. Acta 650: 151–166Google Scholar
  48. Rosing J & Slater EC (1972) The value of ΔGo for the hydrolsis of ATP. Biochim. Biophys. Acta 267: 275–290Google Scholar
  49. de la Rubia T, Gonzalez-Lopez J, Moreno J, Martinez-Toledo MV & Ramos-Cormenzana A (1986) Adenine nucleotide content and energy charge ofBacillus megaterium during batch growth in low-phosphate medium. FEMS Microbiol. Lett. 35: 5–9Google Scholar
  50. Salmond CV, Kroll RG & Booth IR (1984) The effect of food preservatives on pH homeostasis inEscherichia coli. J. Gen. Microbiol. 130: 2845–2850Google Scholar
  51. Setlow B & Setlow P (1980) Measurements of the pH within dormant and germinated bacterial spores. Proc. Natl. Acad. Sci. USA 77: 2474–2476Google Scholar
  52. Shioi J-I, Matsuura S & Imae Y (1980) Quantitative measurements of proton motive force and motility inBacillus subtilis. J. Bacteriol. 144: 891–897Google Scholar
  53. Shu J & Shuler ML (1988) A mathematical model for the growth of a single cell ofE. coli in a glucose/glutamine/ammonium medium. Biotechnol. Bioeng. 33: 1117–1126Google Scholar
  54. Slonczewski JL (1992) pH-regulated genes in enteric bacteria. ASM News 58: 140–144Google Scholar
  55. Slonczewski JL, Gonzalez TN, Bartholomew FM & Holt NJ (1987) Mu d-directedlacZ fusions regulated by low pH inEscherichia coli. J. Bacteriol. 169: 3001–3006Google Scholar
  56. Sonenshein (1989) Metabolic regulation of sporulation and other stationary-phase phenomena. In: Smith I, Slepecky RA & Setlow P (Eds) Regulation of Procaryotic Development. Structural and Functional Analysis of Bacterial Sporulation and Germination (pp 109–130). American Soc. Microbiol., Washington, DC, USAGoogle Scholar
  57. Stouthamer AH (1988) Bioenergetics and yields with electron acceptors other than oxygen. In: Erickson LE & Yee-Chak Fung D (Eds) Handbook on Anaerobic Fermentations (pp 345–437). Marcel Dekker, Inc., New York, USAGoogle Scholar
  58. Stouthamer AH & van Verseveld HW (1985) Stoichiometry of microbial growth. In: Cooney CL & Humphrey AE (Eds) Comprehensive Biotechnology; the Principles, Applications and Regulations of Biotechnology in Industry, Agriculture and Medicine (pp 215–238). Pergamon Press, Oxford, UKGoogle Scholar
  59. Stouthamer AH & van Verseveld HW (1987) Microbial energetics should be considered in manipulating metabolism for biotechnological purposes. Tr. Biotechnol. 5: 149–155Google Scholar
  60. Thauer RK & Morris JG (1984) Metabolism of chemotrophic anaerobes: old views and new aspects. In: Kelly DP & Carr NG (Eds) The Microbe 1984, Part II: Prokaryotes and Eukaryotes (pp 123–168). Symp. Soc. Gen. Microbiol. 36, Cambridge University Press, Cambridge, UKGoogle Scholar
  61. Trivedi B & Danforth WH (1966) Effect of pH on the kinetics of frog muscle phosphofructokinase. J. Biol. Chem. 241: 4110–4112Google Scholar
  62. van Boven A & Konings WN (1987) A phosphate-bond driven dipeptide transport system inStreptococcus cremoris is regulated by the internal pH. Appl. Env. Microbiol. 53: 2897–2902Google Scholar
  63. van Verseveld HW, Chesbro WR, Braster M & Stouthamer AH (1984) Eubacteria have three growth modes keyed to nutrient flow; consequences for the concept of maintenance energy and maximal growth yield. Arch. Microbiol. 137: 176–184Google Scholar
  64. van Verseveld HW, de Hollander JA, Frankena J, Braster M, Leeuwerik FJ & Stouthamer AH (1986a) Modelling of microbial substrate conversion, growth and product formation in a recycling fermentor. Antonie van Leeuwenhoek 52: 325–342Google Scholar
  65. van Verseveld HW, Braster M, Kashket ER & Stouthamer AH (1986b) Proton-motive force during growth ofEscherichia coli in the recycling fermentor. FEBS Lett. 202: 319–322Google Scholar
  66. Westerhoff HV, Lolkema JS, Otto R & Hellingwerf KJ (1982) Thermodynamics of growth, non-equilibrium thermodynamics of bacterial growth, the phenomenological and the mosaic approach. Biochim. Biophys. Acta 683: 181–220Google Scholar
  67. Westerhoff HV, Hellingwerf KJ & van Dam K (1983) Thermodynamic efficiency of microbial growth is low but optimal for maximal growth rate. Proc. Natl. Acad. Sci. USA 80: 305–309Google Scholar
  68. Whatmore AM, Chudek JA & Reed RH (1990) The effects of osmotic upshock on the intracellular solute pools ofBacillus subtilis. J. Gen. Microbiol. 136: 2527–2535Google Scholar
  69. Whooley MA & McLoughlin AJ (1983) The proton motive force inPseudomonas aeruginosa and its relationship to exoprotease production. J. Gen. Microbiol. 129: 989–996Google Scholar
  70. Zaritsky A, Kihara M & Macnab RM (1981) Measurement of membrane potential inBacillus subtilis: a comparison of lipophilic cations, rubidium ion, and a cyanine dye as probes. J. Membrane Biol. 63: 215–231Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Ben A. Bulthuis
    • 1
  • Gregory M. Koningstein
    • 1
  • Adriaan H. Stouthamer
    • 1
  • Henk W. van Verseveld
    • 1
  1. 1.Department of Microbiology, Biological LaboratoryVrije UniversiteitAmsterdamThe Netherlands

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