Cells of the obligate halophile Halobacterium cutirubrum were grown to the stationary phase in the presence of 32P-orthophosphate, 35S-sulfate, 14C-labelled acetate, malonate, mevalonate and glycerol and in the presence of 1,(3)-3H- and 1,3- 14C-glycerol mixture and 2-3H- and 1,3-14C-glycerol mixture as precursors. 32P was incorporated into the phosphatide components in proportion to their relative concentrations, thus: phosphatidyl glycerol phosphate (diether analogue) > unidentified minor phosphatide > phosphatidyl glycerol (diether analogue). 32S-sulfate was incorporated mostly into the glycolipid sulfate ester component, but small amounts of 35S also appeared in a more polar sulfolipid and in a minor unidentified sulfolipid. 14C-Mevalonate was the most efficient precursor for the phytanyl chains, followed in decreasing order by acetate and glycerol. Only traces of 14C from the malonate precursor were incorporated, and these appeared entirely in the phytanyl groups. A considerable proportion of 14C from the glycerol precursor was found in the glycerol and sugar moieties of the lipids. Little or no labelling of the water-soluble moieties of the lipids was observed with the other carbon precursors. Only traces of 14C-labelled fatty acids were detected with any of the 14C precursors; with acetate in particular, only 0.3% of the 14C in the total lipids was found in fatty acids. The synthesis of fatty acids was confirmed by the use of cell-free enzyme system and 14C-malonyl-CoA as a precursor. Although 0.5 - 0.6% of the 14C in the precursor was incorporated in fatty acids, it is believed that the fatty acid synthetase system is depressed by the high salt concentrations. It is concluded that the predominant bio-synthetic route for the lipid chains is the mevalonate pathway for synthesis of isoprenoid groups, and that the malonyl-CoA pathway for synthesis of fatty acids is operating at a very low level of activity. Studies on the metabolism of 14C and 32P-glycerol phosphate by whole cells and cell-free enzyme preparations showed that H. cutirubrum does not utilize alpha-GP as such, but it contains an active glycerol phosphatase which catalyzes the hydrolysis of alpha-GP into glycerol and Pi. The latter two components then pass into the cells where glycerol is phosphorylated by a glycerol kinase specifically to form sn-glycerol-3-phosphate. sn-Glycerol-3-phosphate was formed not only by glycerol phosphorylation, but also by reduction of dihydroxyacetone phosphate by L,alpha-glycerol phosphate dehydrogenase. Using tritiated glycerol, the incorporation of 3H into lipids of H. cutirubrum cells varied with the position of the 3H label in the precursors supplied. Total lipids and lipid moieties from cells grown in 2-3H-glycerol lost almost completely the 3H activity. In contrast, when cells were grown in 1(3)- 3H-glycerol, the phytanyl groups and the sugar residues of the lipids retained about 50% of the 3H activity while the glycerol moiety of the diphytanyl glycerol ether retained about 100% of the 3H activity. It is concluded that glycerol is metabolized via three separate pathways: (1) by the reverse of the Embden-Meyerhof cycle, to sugars; (2) by glycolysis, to acetate and then to isopentenyl pyrophosphate through the mevalonate pathway; (3) by a new pathway, in which glycerol is converted to diphytanyl glycerol ether by an as yet unknown series of reactions, probably, involving dihydroxyacetone. Direct studies on the biosynthesis of phosphatides of H. cutirubrum by short-term labelling of total lipids with 32P i was undertaken. During the first minute of labelling, an unidentified phospholipid was initiated which bears a precursor-product relationship between it and PGP-diether analogue. It is concluded that the unidentified phospholipid may be the immediate phosphorus-containing precursor of the major phosphatide PGP-diether analogue.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/10734 |
| Date | January 1969 |
| Creators | Wassef, Momtaz W. K. |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 210 p. |
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