Inhibition of growth of microbial mutants by trans-octadecenoates.

A series of positional isomers of trans-octadecenoic acid were tested for their ability to support growth of microbial mutants that could not synthesize unsaturated fatty acids. The bacterial strain used (a variant of Escherichia coli 30E) grew with supplements of the trans isomers only at high temperatures (38 degrees) and with acids containing the trans-ethylenic bond between carbon atoms 8 through 13. The yeast mutant (Saccharomyces cerevisiae KD46) grew only with the 9-trans-octadecenoate giving cell yields about one-fifth those obtained with oleate. Although the trans isomers had little effect on the growth of the bacteria in the presence of oleate, they inhibited the growth of yeast with oleate. Inhibition was strongest for the 4, 6, 7, 11, and 12 isomers, almost negligible for the 8 isomer and of differing intermediate degrees for the others. The inhibitory effects had no correlation with the melting points of the acids and appeared to reflect selective action(s) on the metabolism of the cell. When the net yield of the yeast cultures with oleate was lowered by the effect of added trans acids, there was a marked accumulation of triglycerides and nonesterified acids in the cells. The marked increase in triglyceride content while phospholipid per cell remained relatively constant suggest that trans acids, in addition to forming inadequate membrane lipids, may also interfere with a basic control point in lipid metabolism.

The positional isomers of trans.octadecenoic acid (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) were prepared in the laboratory of F. D. Gunstone (8,9). Since some of the preparations were methyl esters, all isomers were incubated in aqueous 1 N sodium hydroxide for 1 hour at 80" after which they were neutralized with acetic acid and extracted into petroleum ether. The ether phase was washed twice with water, evaporated to dryness, a few drops of NH,OH were added, and the excess ammonia was removed by evaporation to dryness. The ammonium soaps were then dissolved in 80% ethanol, and their concentrations were adjusted to about 3 mM. The pH of these stock solutions was about 5.0. The concentration and degree of purity of each fatty acid solution was checked by gas chromatography after esterification in BFJ methanol.
The impurities were less than 1% in all fatty acid solutions except the 3. and 4 The basal liquid medium for this strain contained Medium A (12) to which 1% Casamino acids were added. Detergent was excluded from this medium also. In the agar medium used to preserve the strain, Casamino acids were replaced by succinate and Tween 40.
Growth Measurements-To ensure that the inocula for the yeast growth tubes were of uniform character, KD46 cultures were prepared as follows. Cells were transferred from agar plates to liquid containing 50 pM oleic acid and grown to mid-log phase. Then aliquots of the cells plus medium were transferred directly to several fresh tubes in such a way that the subsequent cultures would reach a desired cell concentration (in late log phase) at the end of a chosen time interval.
The cells were then centrifuged and placed in new media which lacked oleic acid. This cell suspension was used to inoculate the experimental growth tubes. No revertants were observed while the cells were in liquid, which was normally about 50 hours. The inocula for bacterial growth tubes were prepared by transferring cells from an agar plate to liquid media containing oleic acid. The cells were grown to mid-log phase and transferred directly to experimental growth tubes without any centrifugation step. Since revertants generally appeared in the liquid culture only after 15 hours, the growth experiments were always designed to be completed before that time. The experimental growth cultures (8 ml) were shaken at 120 rpm on a rotary shaker in tubes 18 x 150 mm set at an angle of 50" at either 26" (+0. 3") or 36" (+0.  second phase of growth between 5 and 20 hours after the initial phase was completed. Control cultures without any added fatty acid normally underwent about 1.5 to 2 doublings before stopping. This increase can be attributed partly to a small amount of oleate in the yeast extract present in the medium and to some extra nutrient introduced with the cells in the inoculum, but at least one-half of the increase appears to be an endogenous growth (see Fig. 1 and Table II) not influenced by fatty acids present in the growth medium.
Of the various trans isomers, only A8 consistently supported growth above that in the control tubes under the routine conditions used. Table II illustrates the strongly inhibitory action of three of the trans acids (A7, A7, and Al2) on the total cell yields of KD46 cultures grown with 30 FM oleate. Other trans-18:1 isomers (A4, A9, All, A13, A15) were also inhibitory to differing degrees. The total cell yield of 104 x lo6 cells/ml for cultures where 30 FM oleate alone was added decreased to a level of growth, which for cultures containing 15 or 30 pM A6 alone, represented about one-half of the control growth. The doublings/hour of this early basal growth, which is represented approximately by the growth rate at 3 to 4 hours, was also unaffected by fatty acids added to the culture medium. Beyond 4 hours, the growth rate was influenced by the acids added to the medium. Fig. 1 illustrates the inhibition noted in both the rate and extent of growth of KD46 after the first 4 hours when the trans-18:1 acids (A7 and Al2 in this case) exerted their inhibitory effect on the culture. A 26", none of the trans acids, at any of the concentrations tested, supported growth of the Escherichia coli mutant above that of the controls (results not shown). At 36", a small and inconsistent net increase in cells was observed with the All and Al2 isomers. However, we confirmed that the mutant did grow on the A9-trans isomer at 38.8 i 0.5" when we tested conditions similar to those of Wilson and Fox (13). We tested other truns acids at this temperature in the concentration range of 6 to 60 FM and found that isomers A8 through Al2 also supported growth above the controls. The net cell yields were variable and small compared to those with oleate at the same concentration, but nonetheless indicated some consistent growth above the controls. The average efficiencies in cells/ fmol' were as follows: 12 + 2 for A8, 10 i 2 for A9, 8 + 1 for AlO, 9 f 2 for All, and 6 f 1 for Al2 (compared to 50 for A9-cis at 36").
The cell yields for the E. coli mutant were a linear function of the concentration of added oleate up to about 30 pM, although the oleate apparently produced a somewhat less efficient (28 cells/fmol) yield phenotype at 26" than at 36" (50 cells/fmol). Cell yields for S. cereuisiae grown on added oleate also increased linearly up to about 30 ELM nutrient acids. The linear responses at low concentrations were used to determine growth efficiencies for S. cerevisiae and E. coli grown at the two temperatures, 26" and 36". These are summarized in Table III together with the correlation coefficients of the linear regressions. Although growth on oleate for E. coli was about one-half as efficient at the lower temperature, that for S. cerevisiae was relatively unaffected. Interestingly, the A8-trans isomer, which has a melting point of 52", was somewhat more efficient for the yield of yeast cells at the lower temperature than at the higher temperature.
The growth rate (0.07 + 0.1 doubling&our) observed for S. cerevisiae grown with A8-trans alone was about 3 Small amounts of fatty acids are designated femtomole (fmol, 10-l' mol) or attomole (amol, lo-" mol). growth curves at 36". KD46 cultures were transferred from an agar plate to liquid media held at 30" with 40 FM oleate as the only fatty acid supplement. At mid-log phase the cells were inoculated into fresh medium at the same concentration and temperature. When these cells reached mid-to late log phase, they were centrifuged, resuspended in fresh medium, and inoculated into growth media held at 36" with the following fatty acid supplements: 0, oleate (30 PM Fig. 2A). Isomers with the trans bond at positions 4, 6, 7, 11, and 12 completely inhibited the growth rate (Fig. 2B) and cell yield ( Fig. 2A). The inhibition was very selective with regard to the position of the double bond; the A8-trans isomer gave little inhibition at 36" in comparison with the growth on oleic acid alone, and the other isomers showed different levels of inhibition of the growth on oleic acid. The pattern of inhibition of the rate of growth (Fig. 3B) was similar to that with the extent of growth. Additional studies (not shown) indicated stronger inhibitions at a lower temperature; the yield with oleic acid in the presence of the A6, A9, AlO, All, A12, A13, A14, and Al5 isomers at 26" was 50% less than at 36", although the yield with oleic acid alone was only about 15% lower. In further studies with a 15 fiM level of trans acid, only the 4 and 6 isomers gave complete inhibition at both temperatures, and the other isomers gave either partial or no significant inhibition (results not shown). To gain a better understanding of the inhibitory effectiveness of the trans isomers on the eukaryotic yeast cell, we examined a range of concentrations as shown in Fig. 3. The inhibition was readily observed with most acids at levels of inhibitor above 10 pM, except for the A6 isomer, which was strongly inhibitory above 2 to 5 pM. The unusually marked inhibition by the A6-tram isomer did not appear due to some nonacid contaminant, since chromatographic purification of the A6 isomer subsequently gave similar reproducible results. Growth of the E. coli mutant on oleic acid at 30 pM in the presence of 30 pM amounts of the various trans isomers is described in Fig. 4. There were no strong inhibitors of growth of this procaryotic organism among any of the trans isomers tested, and the added trans isomer seemed to have little effect on the utilization of the oleate which was present. The Al3-trans isomer consistently gave a moderate inhibition of cell yield at 26" but not at 36". The rates of growth of the bacterial cultures were not significantly different with the various fatty acid mixtures.
Incorporation of trans-Octadecenoates into Cellular Lipids-We studied the incorporation of oleic acid and the various trans-18:1 acids into cellular lipids of KD46 under  (3) 0.19 * 0.001 (3) 104 * 5 (13) 0.30 * 0.01 (13) 11 * 2 (7) 0.27 + 0.01 (7) 11 * 2 (4) 0.28 i-0.001 (4)  (3) 0.13 * 0.05 (4) o+o (3)  010 (3)   Fig. 2, and the measurement of net cell yields in this figure. The mean net cell yield (0)    octadecenoates. The inhibitions noted for certain isomers in ["C]methyl-P-thiogalactoside, and in force-area isotherms of our study do not clearly reflect the melting points of those acids extracted phosphatidylethanolamine on monolayers (17). It and suggest that selective metabolic effects occurred that were was suggested that the transition observed was due to a change not necessarily dependent upon a solid-liquid transition. in membrane lipids (from a condensed to an expanded state) as Apparently, the cell yield of the E. coli mutant was limited by the temperature increased. truns-Unsaturated fatty acids are an event very sensitive to temperature, whereas the extent of regarded as much less disruptive of hydrocarbon chain packing growth of the S. cereuisiae mutant was not. The truns isomers than cis acids (18), and would appear to cause membranes to (8 to 12) that could support growth of the bacteria were equally undergo such transitions at a higher temperature than cis effective, although they had different melting points and acids. When the E. coli mutant (civ-2 fao-6) was grown on showed dramatically different effects upon the yields of the elaidate at 37" and the temperature then lower to 27", the yeast. Thus, the growth-limiting step(s) for yeast exhibited a culture lost viability rapidly, but when oleate was added, the much more selective interaction with the positional isomers. viability loss was apparently reversed (