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Title: Active transport and energy conservation in Escherichia coli
Author: Hunter, Iain S.
ISNI:       0000 0001 3584 6944
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1977
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1. The main aim of this work was to compare the efficiencies of growth of various phenotypes of Escherichia coli. Several strains were grown in arithmetic (Wallace, 1975), chemostat and batch cultures. Values for maintenance energies were derived in order to calculate maximum growth yields in terms of carbon and oxygen. Efficiencies of ATP synthesis (expressed as P/O ratios) were calculated from yields using the assumption that the energy required for cellular biosynthesis under anaerobic conditions applied to the aerobic cultures. P/O ratios were then compared with H+/O ratios of the harvested bacteria, 2. The maintenance requirement was lower for arithmetic cultures than for chemostat cultures, suggesting that dropwise addition of nutrient in chemostat culture might uncouple cellular growth from energy supply. 3. E.coli ML30 (which is lac-inducible) had the same maintenance requirement as its lac-constitutive derivative, ML3O8, . when it was grown on a non-inducing carbon source. The gratuitous synthesis and turnover of the lac enzymes therefore make negligible contributions to maintenance. 4. There were at least two classes of reaction responsible for maintenance one of which was independent of temperature while the other was strongly temperature-dependent. The latter had an activation energy similar to that of protein denaturation, suggesting that part of the maintenance requirement may be for macromolecular organisation. 5. Bacteria grown on glucose, glycerol, maltose, galactose or mannitol had P/O ratios of 2, while those grown on lactose, gluconate or glucose 6-phosphate had P/O ratios of 3. The phenotypes with the higher efficiency of energy conservation all transported their carbon sources- by a proton-symport mechanism. 6. In pulse-oxidant determinations of H+/O ratios, cells with a P/O ratio of 3 extruded more protons than those with P/O ratio of 2, This is an independent confirmation of the conclusion derived from growth yields. 7. Growth on lactose in batch culture or in the chemostat also gave a P/O ratio of 3. High P/O ratios are therefore not peculiar to arithmetic culture. 8. Bacteria which had high P/O ratios had high maintenance coefficients. It is speculated that the additional maintenance requirement may be spent for organisation of a more efficient electron transport chain. 9. A period of logarithmic growth on the proton-symported substrate was required for the extra coupling site to be expressed, since cells which had previously been grown on glycerol with P/O ratio of 2, grew on lactose with a P/O ratio of 2 under carbon limitation in arithmetic culture. 10. E.coli ML30 grew on gluconate with a P/O ratio of 3. Therefore, the lac enzymes themselves were not required for the higher growth efficiency. 11. When bacteria which had been grown on lactose were starved for 2 h, they lost the additional energy-coupling site as judged by H+/O ratios. The lac permease also decayed, indicating a possible relationship between the two processes. 12. Cultures of E.coli ML3O8 grown on either lactose or glycerol had active lac permease, but only the lactose phenotype had its H+/O ratio enhanced by adding non-metabolisable galactosides. Transport in cells growing on lactose can therefore affect energy conservation. 13. Addition of salt (NaCl, Na2SO4) to growth media reduced the P/O ratios of cultures but did not significantly change their H+/O ratios. Salt must therefore either decrease the efficiency of the ATPase machinery or have an un-coupling effect on the membrane. 14. Cultures grew on lactose/glucose mixtures with a P/O ratio of 3. Therefore when the additional coupling site was present, all carbon sources were metabolised through the same system with high efficiency. 15. The additional coupling site found in lactose/grown bacteria was not due to energy-conserving transhydrogenase activity nor to an alternative energy-conserving terminal oxidase. In the absence of other experimental data on the composition of the electron transport chain of this phenotype, models are presented to explain the results in terms of novel electron carriers or rearrangement within the membrane of preexisting electron carriers. 16. It is concluded that an additional site of energy conservation is synthesised by E.coli in response to transport of carbon sources which enter the cell via proton symport, This conclusion implies a relationship between active transport and electron transport which have hitherto been assumed to work independently of each other.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available