Aspects of methanol metabolism in methylotrophs.
thesisposted on 19.11.2015, 09:07 by Shirley Margaret. Burton
The environmental regulation of methanol and formaldehyde metabolism by Methylophilus methylotrophus was examined by varying the growth conditions imposed upon cells in continuous culture and measuring the activities of key metabolic enzymes. Methanol dehydrogenase was repressed by the presence of high standing concentrations of methanol, whereas hexulose 6-phosphate synthase was constitutive and the glucose 6-phosphate and 6-phosphogluconate dehydrogenases were regulated only by the growth rate. It is concluded that the observed regulation of these enzymes occurs in order to achieve the required in situ rates of energy conservation and carbon flux demanded by the imposed growth rate. The control of flux through the methanol oxidase system of M. methylotrophus was examined in oxygen-limited cultures (D = 0.04 h-1). It was determined that the terminal oxidase (cytochrome oxidase co) does not exert significant control over flux in these cells. In methanol-limited cultures (D = 0.18 h-1) major control of flux through the methanol oxidase system was exerted by the cytochrome c pool. Hexulose 6-phosphate synthase of M. methylotrophus was found to be loosely associated with the cytoplasmic side of the membrane, mirroring the location of methanol dehydrogenase on the periplasmic side of the membrane. It is suggested that juxtapositioning of the formaldehyde-producing and -fixing enzymes on opposite sides of the membrane may help to reduce the amount of this potentially toxic metabolite which is released into the cytoplasm. Methanol dehydrogenase, hexulose 6-phosphate synthase and cytochrome Cl were purified from M. methylotrophus and antibodies raised against them. A wide variety of methylotrophs (including obligate or restricted-facultative and facultative bacteria and methylotrophic yeasts) were screened, by SDS-PAGE and Western blotting, for proteins which cross-react with the antisera. Only organisms which were closely related to M. methylotrophus (i.e. Gram-negative bacteria possessing the ribulose monophosphate pathway) gave any cross-reaction with the antisera. Two serologically distinct types methanol dehydrogenase and hexulose 6-phosphate synthase were therfore distinguished.