Bacillus thuringiensis produces a 130-140 kDa insecticidal protein in the form of a bipyramidal crystal. The protein in the crystals from the subspecies kurstaki HD-1 and entomocidus was found to contain 16-18 cysteine residues per molecule, present primarily in the disulphide form as cystine. Evidence that all the cysteine residues form symmetrical interchain disulphide linkages in the protein crystal was obtained from the following results: (i) the disulphide diagonal procedure [Brown & Hartley (1966) Biochem. J. 101, 214-228] gave only unpaired cysteic acid peptides in diagonal maps; (ii) the disulphide bridges were shown to be labile in dilute alkali and the crystal protein could be released quantitatively with 1 mM-2-mercaptoethanol; (iii) the thiol groups of the released crystal protein were shown by competitive labelling [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] to have the same chemical properties as exposed groups on the surface of the protein; (iv) the thiol groups in the released crystal protein reacted quantitatively with iodoacetate or iodoacetamide. The finding that all the disulphide linkages in the protein crystal are interchain and symmetrical accounts for its alkali-lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies.

A procedure for the determination of the proportions of diacyl, alkenylacyl and alkylacyl subclasses of glycerophospholipids was developed. The procedure involves: (1) acid methanolysis of the phospholipid followed by Bligh/Dyer extraction of fatty acid methyl esters (FAME) derived from acyl chain types, dimethylacetals (DMA) derived from alkenyl ether chain types, and lysoalkyl phosphatidic acids (lysoalkyl-PA) derived from alkyl ether chain types; and (2) subsequent acetolysis to convert the lysoalkyl-PA to monoalkyl glycerol diacetates (MAGD). GLC analysis and quantitation (using internal standard, 21:0 FAME) of FAME, DMA and MAGD allowed calculation of the proportions of the three molecular subclasses. The methanolysis/acetolysis procedure gave an overall mean phospholipid recovery of 95 +/- 3%. Analysis of the major phospholipids in four separate preparations of fresh resting human platelets by this procedure showed the following range of molecular subclasses: phosphatidylcholine (PC), 86-92 mol % diacyl, 6-10 mol % alkylacyl and 2-3 mol % alkenylacyl; and phosphatidylethanoline (PE), 39-60 mol % diacyl, 5-8 mol % alkylacyl and 34-55 mol % alkenylacyl. The results of these subclass analyses were in general agreement with those reported in the literature.

Staphylococcus aureus MF31 was grown to stationary phase in a complex medium at 30, 37, and 43 degrees C in the absence of salt and at 37 and 46 degrees C in the same medium supplemented with 1 M NaCl. The principal phospholipids were cardiolipin, phosphatidylglycerol, aminoacylphosphatidyl glycerol, mono- and di-glycosyldiglyceride, and traces of phosphoglycolipid. The proportion of cardiolipin decreased with increasing growth temperature, but only slightly in the presence of 1 M NaCl, while that of aminoacylphosphatidyl glycerol was unaffected by growth temperature in absence of salt, but was about halved in the presence of 1 M NaCl. The net negative charge per mole phospholipid was greatly increased in the presence of 1 M NaCl. In the absence of salt, temperature had no effect on the total lipid content, but cells from the 46 degrees C culture in 1 M NaCl contained 25% less total lipid. The proportion of phospholipid in the total lipids, both in the absence and presence of salt, declined with increasing growth temperature. The proportion of glycolipids, however, increased with temperature both in the absence and presence of salt. It is suggested that the increase in glycolipid content and in negative charge/mole phospholipid is a part of the adaptation of S. aureus to the combination of high temperature and 1 M NaCl giving its membrane increased stability and possibly helping to exclude Cl- anion from the cell interior.

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