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Title: Regulation of C₁ metabolism in Methylococcus capsulatus (Bath)
Author: Hay, Steven C.
ISNI:       0000 0001 3547 8512
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1990
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The aim of this project was to examine aspects of the regulation of C1 metabolism in M.capsulatus (Bath). To achieve this, steady-state cultures were set up under defined conditions using a gas-limited chemostat, coupled to an on-line mass spectrometer. During periods of steady-state growth, the conditions of culture were perturbed via the addition of various C1 metabolites and the response of the cells monitored with respect to their capacity to attain a new steady-state. An initial series of continuous cultivation experiments were carried out to determine the physiological state of the cells prior to making such perturbations. Gas exchange rates, carbon distribution and levels of in vitro enzyme activity monitored within the culture during these periods provided a base-line with which to compare the effect of the addition of C1 metabolites. Examination of the response of cells to the addition of formate showed that under carbon-limiting conditions, added formate was oxidised to C02. Under oxygen-limiting conditions cells were capable of assimilating low levels of formate carbon, although under such circumstances the cultures were also susceptible to formate-induced uncoupling of oxidative phosphorylation. Additional formate oxidation also resulted in an observed increase in cell yield on methane, especially when the cells expressed soluble MMO. It was concluded that this was a consequence of extra NAD(P)H being generated by the NAD(P)+ - linked oxidation of formate, which in turn relieved the apparent NAD(P)H-limitation of cells growing on methane. The ability of cells to metabolize exogenously supplied formaldehyde was linked to the type of MMO expressed by the cells. Soluble MMO- containing cells showed an increased sensitivity to formaldehyde accumulation compared with particulate MMO- containing cells. At one stage it was possible to maintain particulate MMO- containing cells on formaldehyde as their sole source of carbon, albeit for a limited period of time. This period of time appeared to be linked to the cell's ability to maintain an active MMO. Results showed that the synthesis of soluble MMO was repressed in the presence of additional formaldehyde metabolism, the loss of enzyme coinciding with formaldehyde accumulation and ultimately cell death. Subsequent analysis of the intracellular levels of NAD+and NADH in the culture implied that the MMO played an active role in the regulation of the NAD+ : NADH ratio within the cell. Loss of MMO activity in the presence of additional formaldehyde metabolism effectively compromised the cell's ability to regulate it's intracellular NAD+ : NADH ratio. It was shown during this study that cultures of M.capsulatus (Bath) could be transferred directly from methane-limited growth to growth on methanol as a sole source of carbon, without any prior period of physiological adaptation. During this switch in carbon sources, the MMO appeared to be actively involved in the in vivo metabolism of methanol. Studies concerning the environmental regulation of methanol metabolism showed that the level of methanol dehydrogenase activity in the culture was inversely related to the standing concentration of methanol in the culture. Similarly, higher levels of methanol dehydrogenase activity were recorded at lower dilution rates, during methanol-limited conditions. It was concluded that the methanol dehydrogenase enzyme is subject to regulation via catabolic repression and at low dilution rates the synthesis of the enzyme is effectively derepressed.
Supervisor: Not available Sponsor: Science and Engineering Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology