JOURNAL OF BACTERIOLOGY, Mar., 1967, p. 1079-1088 Vol. 93, No. 3 Copyright @ 1967 American Society for Microbiology Printed In U.S.A. Indentification and Localization of the Fatty Acids in Haemophilus parainfluenzae DAVID C. WHITE AND RICHARD H. COX Department ofBiochemistry and Department of Chemistry, University ofKentucky Medical Center, Lexington, Kentucky Received for publication 7 December 1966 Haemophilus parainfluenzae was capable of synthesizing 22 fatty acids. These fatty acids were equivalent to 4% of the bacterial dry weight. These fatty acids were localized in the membrane-wall complex, which contained the respiratory pigments, the quinone, and the phospholipids. The fatty acids which could be extracted with organic solvents comprised 86% of the total fatty acids of the cell. These fatty acids were distributed as 98% in the phospholipids and 1.9% in the neutral lipids, of which 0.5% were free fatty acids. Palmitic, palmitoleic, oleic, and vaccenic acids com- prised 72% of the total fatty acids and were found almost exclusively in the phos- pholipids. The phospholipids also contained the cyclopropane fatty acids. The neu- tral lipids contained significant proportions of the odd-numbered branched and straight-chain fatty acids. The principal free fatty acids were n-dodecanoic and penta- decenoic acids. The nonextractable wall complex contained 14% of the total fatty acids. These wall fatty acids were rendered soluble only after saponification. The wall fraction contained all of the f3-hydroxymyristic acid and most of the myristoleic and pentadecenoic acids. The significance of the distribution of fatty acids between non- esterified, neutral lipid, phospholipid, and nonextractible wall remains to be deter- mined. Haemophilus parainfluenzae has an obligatory dence will be presented to show that the fatty requirement of a membrane-bound electron acid pattern of Haemophilus is complex and that transport system for growth (22). The bacterium the localization of the fatty acids is apparently has the capacity to extensively modify the not random. composition of the membrane in response to changes in the environment (17). The changes MATERIALS AND METHODS in the electron transport system have been Growth ofbacteria. The strain of H. parainfluenzae, shown to result in a new composition that is more harvesting procedures, conditions for growth and effective in the new environment (18). These preservation of the bacteria, and medium have been modifications can be used to delineate some of the described (17). control mechanisms involved in the formation of Dry weight. The dry weight was determined after the membrane complex (20). The function of this drying in vacuo to constant weight as described electron transport system is dependent on the previously (22). structural integrity of the membranes. Any Isolation of the fatty acids. About 25 g (dry weight) of frozen bacterial pellets was suspended in 400 ml disturbance of the lipids by solvents results in the of 3 N KOH containing 50% (v/v) ethyl alcohol and loss of respiratory activity (21). However, under was refluxed for 12 hr. The cooled saponification carefully controlled conditions, the quinone can mixture was then extracted with 0.2 volume of be extracted and respiratory activity can be petroleum ether, and the organic phase was discarded. partially restored on addition of the quinone (21). The aqueous phase was then acidified to pH 2.0 with The identity of these essential lipid components HCI and extracted with 0.2 volume of petroleum ether. and the relation of changes in the composition of This was repeated with fresh petroleum ether four the lipids to changes in the proportions of the times. The combined petroleum ether extracts were respiratory pigments need to be known before dried over anhydrous Na2SO4 + NaHCO3 (4:1, w/w) and were evaporated to a small volume in vacuo. study of the role of lipid in the formation of the For studies of the localization of the fatty acids, membrane can be made. The fatty acids of 340 mg (dry weight) of bacteria harvested in the Haemophilus have been characterized, and their stationary phase of growth was saponified for 2 hr cellular localization has been determined. Evi- at 100 C in tubes sealed with Teflon-lined screw caps. 1079 1080 WHITE AND COX J. BACTERIOL. Extractions with petroleum ether were mixed vigor- wool. The pellet was re-extracted twice and the ously for 5 min on the Vortex mixer. The combined chloroform-methanol solution was partitioned against petroleum ether extracts from the acidified aqueous 0.7 M NaCI (4). The removal of nonlipid contaminants phase were taken to dryness carefully in a stream of by chromatography on Sephadex was done as de- nitrogen at 20 C. The fatty acids were extracted from scribed by Wells and Dittmer (16). The other method the lipid extracts with two portions of 0.47 M Na2CO8. of lipid isolation involved a modification of the The acids were then recovered from the aqueous phase method described by Bligh and Dyer (2). The bac- by extraction with three portions of petroleum ether teria were suspended in 30 ml of water, and 75 ml of after acidification. methanol and 37.5 ml of chloroform were added. The Methylation of the fatty acids. The method of Met- suspension was mixed vigorously and allowed to calfe and Schmitz (9), which involves borontrifluoride, stand for several hours. Then 37.5 ml of chloroform was compared with a modification of the method of and 37.5 ml of water were added, and the suspension Lorette and Brown (8). The methylation with boron was shaken and allowed to separate. The bottom trifluoride leads to the development of a brown color layer was filtered through Whatman no. 12 filter which did not appear with the dimethoxypropane- paper before lipid analysis. induced transmethylation. Consequently, the method Separation of neutral and phospholipids. Lipid involving dimethoxypropane was used routinely, al- preparations were dehydrated by evaporation to dry- though no difference could be detected between the ness and were resuspended in benzene-absolute preparations with the gas chromatograph. The modi- alcohol (4:1, v/v) several times and then resuspended fication of the method involving dimethyoxypropane in chloroform. The lipids were then applied to a was suggested by T. 0. Henderson. Fatty acids were column (1 by 6 cm) containing 2 g of silicic acid (100 suspended in 0.1 ml of benzene and, to this, 2 ml of to 200 mesh Unisil). Theneutral lipids were eluted with 1.39 M HCI in dry methanol and 0.2 ml of 2,2- 40 ml of chloroform and the phospholipids with 20 dimethoxypropane were added. After 12 hr at room ml of methanol. This method was developed by J. C. temperature, 2 volumes of water was added, and the Dittmer. mixture was extracted with three portions of petro- Phosphate analysis. Lipids were digested in 23.3% leum ether. The combined extracts were dried over (v/v) perchloric acid for 1 hr at 200 C and were solid Na2SO4 plus NaHCO, (4:1) for at least 1 hr and analyzed for phosphate by the method of Bartlett (1) the volume was reduced in a gentle stream of nitrogen. as adapted for the Technicon AutoAnalyzer by R. L. No extraneous peaks from polymerization of the Lester. dimethoxypropane could be detected when mixtures Fatty acid determination. Fatty acids were deter- of known fatty acids were methylated. mined colorimetrically (11) as modified by W. J. Hydrogenation of the fatty acid methyl esters. The Lennarz. Fatty acids were isolated from saponifica- hydrogenation was performed essentially as described tion mixtures and resuspended in chloroform-heptane by Kaneshiro and Marr (7) to differentiate between (1:1). Palmitic acid was used as the standard. olefinic and cyclopropane fatty acid esters. The olefin Fatty acids are designated as follows. The subscript esters were completely hydrogenated in 1 hr at room of C indicates the number of carbon atoms in the temperature, in methanol, at 1 atm of hydrogen with chain, the superscript B (CB) indicates a fatty acid a 5% platinum catalyst on charcoal. The cyclopropane containing two methyl groups, the superscript cyclo fatty acids were hydrogenated in glacial acetic acid at indicates a cyclopropane fatty acid, the superscript 2 atm of hydrogen, with a PtO2 catalyst at room tem- = ,9 indicates the fatty acid is monoenoic with the perature for 12 hr in a Parr hydrogenation apparatus. ,9 indicating that the olefinic double bond is between Dehydration of hydroxy fatty acids. Dehydration of carbons 9 and 10 counting from the carboxyl end, B3-hydroxymyristic acid was accomplished by heating and the superscript OH indicates the fatty acid is for 10 min at 100 C in H2SO4 as described by Slepecky hydroxylated. and Law (14). Ice was then added, and the fatty acid Gas chromatography. A dual-column gas chro- was extracted with three portions of petroleum ether. matograph (model 402, F & M Scientific Co., Avon- The fatty acid was then methylated. dale, Pa.) was used in this study. The hydrogen flame- Localization of the double bonds in the fatty acids. detectors gave a response that was within :L 1% of The method ofvon Rudloff (13) as modified by Chang linearity between detector currents of 2 X 10-11 and and Sweeley (3) was used to localize the double bonds 2 X 10- amp with serial dilutions of methyl pal- in the fatty acid methyl/ esters. The dicarboxylic acid mitate. Analytical columns were of U-shaped glass fragments were methylated and identified by their tubes that had an internal diameter of 3 mm and retention times on columns of silicone oil SE-30. The were 1.8 meters long. The glass preparative columns acid fragments from the omega end of the enoic fatty were 6 mm in internal diameter and 1.8 meters long.