1. The major phospholipids extracted from Bacillus amylolique - faciens were cardiolipin, phosphatidylycerol and phosphatidylethanolamine. 2. The distribution of these phospholipids between the two halves of the cytoplasmic membrane bilayer was studied using phospholipase C ( B. cereus ), phospholipase A2 ( Crotalus ) and the non - penetrating chemical probe trinitrobenzenesulphonic acid ( TNBS ). After treatment of intact protoplasts of B. amylolique - faciens with either phospholipase, approximately 70 % of total membrane phospholipid was hydrolysed ; specifically approximately 90 %, 90 % and 30 % of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin respectively. Under these conditions, protoplasts remained intact and sealed. However, when protoplasts that were permeabilized by cold shock treatment were incubated with either of the phospholipases, up to 80 % of cardiolipin was hydrolysed and phosphatidylglycerol and phosphatidylethanolamine were hydrolysed virtually to completion. In intact cells, 92 % of the phosphatidylethanolamine could be labelled with TNBS under conditions in which the reagent did not penetrate the membrane to any significant extent. 3. These results suggest that 70 % of total phospholipid of this bacillus exists in the outer half of the bilayer. The distribution of phosphatidylethanolamine in this bilayer is highly asymmetric with it being located predominantly in the outer half. The results with phospholipases suggest that the distributions of cardiolipin and phosphatidylglycerol are also asymmetric but independent confirmation or this is required. 4. The fatty acid composition of cells grown at different temperatures was investigated. When cells were grown at 30 ° C, branched - chain saturated fatty acids made up over 80 % of the total fatty acids. Saturated straight - chain fatty acids made up the bulk of the remainder. Less than 1 % of the total fatty acids were unsaturated. Decrease in growth temperature was accompanied by an increase in the ratio of branched to straight - chain fatty acids and a marked increase in the level of unsaturation of branched - chain fatty acids. 5. When cells of this organism, grown at 30 ° C, were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween - 80 to cells caused the critical temperature zone for cold shocking to be significantly lowered. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock. 6. The role of lipids in the process of extracellular enzyme secretion was studied using cerulenin, an antibiotic known to inhibit fatty acid synthesis in microorganisms. Cerulenin inhibited the secretion of alpha - amylase and protease in washed cell suspensions by 80 % and 75 % respectively over 3 hours. The effect was a general one since secretion of all protein species into the medium was drastically reduced by the antibiotic. At the concentration of cerulenin used ( 100 . µ g / ml ), [ 14C ] - acetate incorporation into cellular lipid was inhibited by approximately 50 % but total cellular protein and RNA synthesis were virtually unaffected. The inhibitory effect of cerulenin on alpha - amylase and protease secretion could be partially reversed if cell suspensions were supplemented with either fatty acids prepared from the lipids extracted from B. amyloliquefaciens, or various individual pure fatty acids. These results suggest that fatty acid synthesis may be required for protein secretion by this organism. 7. Attempts were made to detect precursors to extracellular enzymes either associated with the cells or in the culture medium, employing immunological techniques. These experiments, however, were not successful. / Thesis (Ph.D.)--Department of Biochemistry, 1979.
| Identifer | oai:union.ndltd.org:ADTP/263878 |
| Date | January 1979 |
| Creators | Paton, James Cleland |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_US |
| Detected Language | English |
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