Fatty Acid Composition of Lipid Extracts of

Transcription

1 JOURNAL OF BACrERIOLOGY, Jan. 1970, p Copyright a 1970 American Society for Microbiology Vol. 101, No. 1 Printed in U.S.A. Fatty Acid Composition of Lipid Extracts of a Thermophilic Bacillus Species HARLOW H. DARON Department of Animal Science, Agricultural Experiment Station, Auburn University, Auburn, Alabama Received for publication 6 October 1969 Fatty acids having 16 or 17 carbon atoms accounted for over 80% of the fatty acids produced by a thermophilic Bacillus species. Under most conditions, branched-chain fatty acids were more abundant than normal fatty acids. The proportion of unsaturated fatty acids varied inversely with the growth temperature and was never greater than 14%. When acetate was used as a carbon source, the percentage of fatty acids having 15 or 17 carbon atoms was about twice that found when glucose was used as a carbon source. Increasing the growth temperature from 40 to 60 C resulted in a threeto fourfold increase in the ratio of the normal to branched-chain hexadecanoic acids. Two normal hexadecenoic acids were found and their relative abundance was influenced by the growth temperature. The extensive work of Kaneda (10, 15-17) has established that branched-chain fatty acids are the principal fatty acids in a number of mesophilic species of the genus Bacillus. Since the preponderance of branched-chain fatty acids appears to be characteristic of this genus, it was of interest to see if it extends to thermophilic species as well. However, fatty acid distribution patterns are often influenced by such factors as physiological age of the culture (13), temperature (19, 20, 24), and composition of the growth medium (5, 13, 18, 25). Therefore, it seemed desirable to examine the fatty acid composition under several environmental conditions to determine the extent of allowable phenotypic variation of the branched-chain fatty acid content. This paper describes the fatty acids in lipid extracts of the vegetative cells of a thermophilic Bacillus species. This organism has been previously characterized (4) and is similar to B. stearothermophilus, but differs primarily in its inability to hydrolyze starch. It utilizes either glucose or acetate as a carbon source; the effect of these two compounds, as well as the growth temperature, on the fatty acid composition is reported here. MATERIALS AND METHODS Growth of microorganisms. Stock cultures were maintained on slants of acetate medium agar supplemented with 0.1% tryptone (4). Cultures for lipid analysis were grown in Fernbach flasks containing 1,500 ml of medium that had either acetate (1%) or glucose (1%) as a carbon source (4). The flasks were incubated in an incubator-shaker (Lab-Line Instruments, Inc.) at a temperature of 40, 50, or 60 C (A1 C) with vigorous shaking (about 120 cycles/min) to ensure adequate aeration. Cells were harvested by centrifugation in mid-exponential phase (except as noted), examined microscopically to ensure that only vegetative cells with no refractile bodies were present, freeze-dried, and stored at 1 C until further use. Samples of the cells were analyzed for residual moisture content by drying at 105 C and for ash content by heating at 700 C to constant weight. Preparation of fatty acid methyl esters. Lipids were extracted from the dry cells with acetone and chloroform-methanol mixtures at room temperature (9), and nonlipid material was removed by the procedure of Folch et al. (6). The amount of lipid was determined by weighing the residue after solvent evaporation. The fatty acid methyl esters were prepared by heating the lipid with 2% H2S04 in methanol in culture tubes sealed with Teflon-lined caps at 100 C for 12 to 15 hr. The methyl esters were extracted with hexane after an equal volume of water was added, and the extract was dried over anhydrous Na2SO4. After treatment of the esters with mercuric acetate (8), the saturated fatty acid methyl esters were separated from the mercuric acetate derivatives of the unsaturated fatty acid methyl esters on small columns of silicic acid (3). Identification of fatty acids. The fatty acid methyl esters were analyzed with a gas chromatograph (Micro Tek, model DSS-162) equipped with a flame ionization detector. Helium was used as the carrier gas at a flow rate of 70 ml per min. The injector and detector temperatures were approximately 215 and 225 C, respectively. Chromatography was conducted on both polar columns consisting of stainless-steel tubing [6 ft, 5-inch diameter (1.83 m, 35-cm diameter)] packed with 15% EGSS-X on Chromosorb W (AW), 80 to 100 mesh (Applied Sciences), and on nonpolar columns consisting of stainless-steel tubing 145

2 146 DARON J. BACTERIOL. [4 ft, 5-inch diameter (1.22 m, 35-cm diameter)] packed with 5% SE-30 on Chromport, 80 to 100 mesh (Applied Sciences). The nonpolar columns did not resolve the isomeric branched-chain fatty acids and were used only to confirm identifications made on the polar columns. Most analyses were conducted isothermally at an oven temperature of 150 C, but several analyses were also made at 175 and 200 C to aid in distinguishing between unsaturated and branchedchain fatty acids (21). Fatty acid esters were identified by compaiing their retention times with those of standard fatty acid methyl esters (Applied Sciences) and by the linear relationship within an homologous series between the logarithm of the retention time and the number of carbon atoms (23). The chromatographic standards used were the methyl esters of caprylic (n-8:0), capric (n-10:0), lauric (n-12:0), myristic (n-14:0), pentadecanoic (n-15:0), palmitic (n-16:0), stearic (n-18:0), arachidic (n-20:0), behenic (n-22 :0), lignoceric (n-24:0), 12-methyl-tridecanoic (i-14:0), 12-methyl-tetradecanoic (a-15:0), 14- methyl-pentadecanoic (i-16:0), 14-methyl-hexadecanoic (a-17:0), palmitoleic (n-16: 1), and oleic (n-18:1) acids. The designations for the unsaturated fatty acids are ambiguous in that they do not specify the position or configuration of the double bond; they are used in their general sense throughout this report. Most of the unsaturated fatty acids were identified as their saturated counterparts after hydrogenation in hexane with 5% palladium on charcoal as a catalyst. Quantitative determination of fatty acids. The weight per cent of the fatty acids present in the lipid extract was calculated from data obtained by multiplying the height of the recorded peak by its width at half height. A linear relationship was found when the width at half height was compared with the retention time (measured in millimeters). The regression line determined by least squares analysis of the data from 187 well-resolved chromatographic peaks had a slope of and an intercept of with a standard error of estimate of This information was used as a guide in estimating the width at half height of such overlapping peaks as the isomeric branchedchain fatty acids. The unsaturated fatty acids represented a small fraction of the total fatty acids, and most of them were completely or partially obscured by the larger amounts of the saturated fatty acids. To obtain the total fatty acid composition, the relative amounts of the saturated and unsaturated fatty acids were determined from separate chromatograms and then related by the amount of n-18:1 (also by n-16: la in the case of extracts from cells grown at 40 C with glucose as a carbon source) relative to saturated fatty acids (usually n-18 :0, i-18: 0, and n-i4:0) in chromatograms of the total fatty acid methyl esters. RESULTS Extractable lipid represented approximately 3 to 8% of the organic matter of the bacterial cells (corrected for moisture and ash content). Increasing the temperature at which the cells were grown resulted in an increase in the lipid content of cells utilizing glucose as a carbon source (Table 1). The fatty acids which have been identified in the bacterial lipid extracts are listed in Table 2. The saturated fatty acids produced by this organism include all fatty acids with an even number of carbon atoms from 12 to 18 inclusive having either the iso or the normal configuration, and all fatty acids with an odd number of carbon atoms from 15 to 19 inclusive having either the iso, anteiso, or normal configuration. In addition to those listed, small amounts of substances having the same retention times as n-9:0, n-10:0, n-11:0, i-13:0, a-13:0, n-13:0, i-20:0, and n-20:0 have been observed in some of the extracts. After separation from the saturated fatty acids, the unsaturated fatty acid fractions were chromatographed and found to contain a number of compounds. Two of these had retention times identical with those of authentic samples of methyl palmitoleate and methyl oleate. The rest were identified from their saturated counterparts after hydrogenation. The fatty acid composition of the unsaturated fatty acid methyl ester fraction of several extracts both before and after hydrogenation is presented in Table 3. An extract from cells grown on acetate medium at 60 C and harvested in the late-exponential phase contained a component with the same retention time as methyl palmitoleate, and comparison of the relative amount of this component showed that hydrogenation converted it to methyl palmitate. This component is presumably palmitoleic acid and has been designated n-16:1. Extracts from cells harvested in the mid-exponential phase (Table 3) contained a major component with a retention time different from methyl palmitoleate. After hydrogenation, this fraction contained methyl palmitate as the major component, so apparently another 16-carbon, normal, mono-unsaturated carboxylic acid is produced by this organism. This fatty acid has been designated as TABLE 1. Lipid content of vegetative cells of a thermophilic Bacillus species Cells' Per cent lipidb (average 4 SD) A40(2) 3.6 i 0.4 A50(3) 3.9 -i A60(2) 4.7 i 0.4 G40(3) 2.9 : G60(2) 7.7 ± a Cells or extracts from cells are designated by an "A" or "G", indicating whether acetate or glucose was the carbon source, followed by the growth temperature. Thus, A40 indicates cells grown on acetate medium at 40 C. The number in parentheses is the number of cell cultures on which analyses were made. b Corrected for moisture and ash content; SD, standard deviation.

3 VOL. 101, 1970 Fatty acid FATrY ACIDS OF A BACILLUS SPECIES TABLE 2. Fatty acid composition of a thermophilic Bacillus species Weight per cent of fatty acids (average 4 SD) A40(2)- AS0(3)a A60(2)a G40(3)a G60(2)a 147 i-12: i n-12: = i-14: A t n-14: i-15: : : t 7 a-15: it n-15:0 0 : : L L i-16: L n-16: i I i-17: it : a-17: ± b ± n-17:0 1 = _ i-18: n-18: it i-19: d 0.01 a-19: i : i n-19: i 0.02 <0.01 Saturated : d n-14: i ± <0.01 n-15: Olb i b <0.01 i-16: Oob i 0.00b n-16:1a : 8 2 b n-16:1 9 i i-17:1 8 i : i i i a-17: i = n-17: i O.Olb 0.02 i 0.00 i-18: i 0.01 <0.01 n-18:1a n-18:1 5 i i ± : Unsaturated i I a The lipid extracts are designated according to carbon source and growth temperature (see Table 1, note a). The number in parentheses is the number of cell cultures on which analyses were made. b These may contain more than one isomeric form (see text). n-16: la and probably differs from palmitoleic acid in the position or configuration, or both, of the double bond. An extract from cells grown on acetate medium at 50 C and harvested in the late-exponential phase contained both components, and the data obtained from quantitative hydrogenation are consistent with their assignment as isomers of hexadecenoic acid. Chromatograms of this fraction before and after hydrogenation are presented in Fig. 1 to show the relative retention times of the two isomeric hexadecanoic acids and the branched-chain unsaturated fatty acids. The data in Table 3 suggest that there are also two isomeric forms of 18-carbon, monounsaturated carboxylic acids. The compound designated n-18:1 had the same retention time as methyl oleate. The compound designated n-18:1a probably differs from oleic acid in the same manner that n-16: la differs from palmitoleic acid, since its chromatographic relationship to oleic acid is similar to that of n-16: la to palmitoleic acid. Semilogarithmic plots of retention time versus carbon number gave a straight line for n-14:1, n-15:1, n-16:1, n-17:1, and n-18:1, so presumably the position and configuration of the double bond in these fatty acids is the same as in palmitoleic acid and oleic acid. Similar treatment of the data for i-16:1, i-17:1, and i-18:1 gave a straight line which was parallel to that for the normal components, so the position and configuration of the double bond in these fatty acids is presumably the same, although not necessarily the same as the normal fatty acids. In a few rare instances, small chromatographic peaks were observed with retention times which suggested that they might be homologues of n-16:1a and n-18:1a. These are noted in Table 2. Other chromatographic peaks have been observed which suggest that branched-chain and normal unsaturated fatty acids with 19 to 22

4 148 DARON J. BACTERIOL. TABLE 3. Hydrogenation of unsaturated fatty acids Fatty acid Before hydrogenation n-14:1 n-is: I i-16:1 n-16: la n-16:1 i-17:1 a-17:1 n-17:1 i-18:1 n-18: la n-l8:1 After hydrogenation n-14:0 n-15:0 i-16:0 n-16:0 i-17:0 a-17:0 n-17:0 i-18:0 n-18:0 Weight per cent of fatty acidsa A40(2)b G40(3)b A50(3)b A5Ob. C A60b, c < <0.1 < a Calculated on the basis of the isolated ununsaturated fatty saturated or hydrogenated acids only. An average value is presented where more than one cell culture was analyzed. bthe lipid extracts are designated according to carbon source and growth temperature (see Table 1, note a). When analyses were made on extracts from more than one cell culture, the number is given in parentheses. c These cell cultures were harvested in the late exponential phase and microscopic examination showed that many cells contained refractile bodies. No mature spores were observed. carbon atoms may also be produced by this organism. These are not included in Table 2 because their identification is open to question, they were always minor constituents of the unsaturated fatty acid fraction, and they were not consistently observed. These components represented less than 5% of the material in the unsaturated fatty acid fraction of extracts from cells grown at 40 or 50 C, but accounted for as much as 25% of the unsaturated fatty acid fraction of extracts from cells grown at 60 C. Chromatographic peaks which corresponded to fatty acids with 21 and 22 carbon atoms were observed only when the growth temperature was 60 C. The fatty acid composition of lipid extracts r- 0 0 I af ter hydrogenotion TIME (MINUTES) FIG. 1. Gas chromatograms of the unsaturated fatty acidfraction, before and after hydrogenation, from extracts of late-exponential phase cells grown at 50 C on a medium containing acetate as the carbon source. from cells grown at various temperatures with either acetate or glucose as a carbon source is shown in Table 2. The representative chromatograms in Fig. 2 and 3 show, somewhat more graphically, the influence of growth conditions on the fatty acid composition. In all extracts examined i-16:0, n-16:0, and a-17:0 accounted for over 70% of the total fatty acids. The relative amount of a-17:0 in cells grown in acetate medium was a little over twice that in cells grown in glucose medium at the same temperature. This was generally the case with nearly all branchedchain fatty acids having an odd number of carbon atoms, and is the most characteristic feature of the fatty acid composition which distinguishes cells utilizing acetate as a carbon source from those utilizing glucose. Although n-15 :0 and n-17 :0 were never present in large amounts, their relative abundance was greater in extracts of glucose-grown cells than in those of acetategrown cells. The generally observed inverse relationship between the proportion of unsaturated fatty acids and growth temperature was also found with this organism. The influence of growth temperature was more pronounced with cells utilizing 0 *3 a 3o

5 VOL. 101, 1970 FATTY ACIDS OF A BACILLUS SPECIES 149 glucose than with cells utilizing acetate. At the lower growth temperatures, n-16: la was the. predominant unsaturated fatty acid. At 60 C, the other isomeric hexadecenoic acid, n-16 :1 (probably palmitoleic acid), was also present. Data in Table 3 indicate that a similar change in the relative contributions of these two isomers 40 also occurred when the physiological age of the 0 culture was increased. Apparently increasing! ' either the "age" of the culture or the growth tem- 0 - perature results in a relative increase in n-16:1 **! * I and a relative decrease in n-16:1a. With the V V 7S saturated fatty acids, the most obvious effect of increasing growth temperature was the increase in the ratio of n-16:0 to i-16:0. This ratio increased three and four times for acetate-grown cells and glucose-grown cells, respectively, as the 600 growth temperature was increased from 40 to 60 C. Similar increases of approximately the same magnitude were also found for the ratios of the isomeric tetradecanoic acids and the isomeric octadecanoic acids. Because of their large con- I,, I>J 6 lb io to TIME (MINUTES) FIG. 3. Gas chromatograms of the total fatty acids from extracts of cells grown at the temperatures shown i O: 400on a medium containing glucose as the carbon source. 00 ge, 0@ tribution, this increase in the proportion of h o o 0 o normal fatty acids with an even number of carbon I \ 2 / \ z \ & O,, atoms resulted in a decrease in the proportion of. V branched-chain fatty acids and of fatty acids with an odd number of carbon atoms as the growth temperature was raised. 500 DISCUSSION In extracts from this thermophilic Bacillus species, the most abundant fatty acids were those having 16 or 17 carbon atoms. They accounted for 80 to 90% of the total fatty acids in all -extracts examined regardless of the growth conditions of the cells. In contrast, fatty acids with 15 carbon atoms predominated in all of the mesophilic Bacillus species examined by Kaneda (15- l ), and fatty acids with 17, 18, and 19 carbon atoms were the major constituents in some extremely thermophilic bacteria (2). This seems to suggest that the chain length of the most abundant fatty acids produced by an organism is related to the optimal growth temperature of that 6 co10 20 o organism. It should be noted, however, that in- TIME (MINUTES) creasing the temperature at which this organism FIG. 2. Gas chromatograms of the total fatty acids was grown did not significantly increase the averfrom extracts ofcells grown at the temperatures shown age size of the fatty acids produced. on a medium containing acetate as the carbon source. Branched-chain fatty acids represented over

6 150 DARON 50% of the total for all but one of the growth conditions investigated. The single exception was with cells grown in glucose medium at 60 C, where palmitic acid alone accounted for over 60%. This seems to support Kaneda's contention that the preponderance of branched-chain fatty acids is a characteristic feature of the genus Bacillus. The biosynthesis of the branched-chain fatty acids is related to the biosynthesis of the branched-chain amino acids (11, 12, 14, 22), although some details need further clarification. The two systems probably compete for the common intermediates a-ketoisovalerate, a-ketoisocaproate, and a-keto-f3-methyl valerate. Upon transamination, these intermediates yield valine, leucine, and isoleucine, respectively, whereas oxidative decarboxylation yields isobutyryl coenzyme A (CoA), isovaleryl CoA, and a-methyl butyryl CoA, which are the terminal precursors for iso fatty acids having an even number of carbon atoms (i-c20), iso fatty acids having an odd number of carbon atoms (i-c20+l), and anteiso fatty acids having an odd number of carbon atoms (a-c20+o). The reversibility of the transamination reaction accounts for the observation that added branched-chain amino acids act as precursors for the branched-chain fatty acids (1, 11, 12, 26). When the demands of protein synthesis are more than adequately met by the supply of branched-chain amino acids, or their immediate precursors, it might be expected that the excess is channeled into the biosynthesis of the branched-chain fatty acids. In this respect, it should be noted that the percentage of branchedchain fatty acids decreased from 79 to 57% in the case of acetate-grown cells and from 56 to 30% in the case of glucose-grown cells as the growth temperature was increased from 40 to 60 C. This may be the result of increased protein turnover at the higher temperatures. The ratio of a-c2f+1 to i-c2n+l was essentially independent of either growth temperature or carbon source for the conditions investigated. With extracts from cells grown in acetate medium at various temperatures the ratio of i-c2. to i-c2n+l or i-c2n to a-c2n+l was also essentially constant. However, the ratio of i-c2n to i-c2n+l or i-c2n to a-c2n+l was greater in glucose-grown cells than in acetate-grown cells, and, furthermore, this ratio decreased as the growth temperature was raised. It would be of interest to know the relative amounts of valine, leucine, and isoleucine in this organism under these growth conditions, since it has been suggested that the relative abundance of the fatty acids reflects the availability of the corresponding terminal precursors (15, 17). However, the observation that J. BACrERIOL. the proportion of normal fatty acids with an even number of carbon atoms is greater in cells utilizing glucose than in those utilizing acetate suggests that some modification of this postulate is required. The system(s) involved in the biosynthesis of the unsaturated fatty acids apparently favors normal fatty acids rather than branched-chain fatty acids as substrates, since the percentage of branched-chain fatty acids was constantly lower in the unsaturated fatty acid fraction than in the saturated fatty acid fraction. Similar results for B. thuringiensis and B. anthracis have been reported by Kaneda (16). Recently, Fulco (7) has reported two distinct systems which desaturate added palmitic acid in several Bacillus species. One system produced A5-hexadecenoic acid exclusively and was temperature-sensitive. The other system produced mixtures of Al-, A9-, and A'0-hexadecenoic acids and was not significantly affected by temperature. Most of the strains examined contained only one of the two systems, but one strain (B. licheniformis 9259) produced both A5- and A'0-hexadecenoic acids. This suggests several possible explanations for the present finding of two isomeric hexadecenoic acids and the effect of growth temperature on their relative concentrations. Any critical appraisal, however, must await the determination of the locations of the double bonds. ACKNOWLEDGMENT I wish to thank Bobby J. Roberson for able technical assistance. This research was supported by a Biomedical Science support grant from Auburn University. LITERATURE CITED 1. Albro, P. W., and J. C. 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