EFFECT OF TEMPERATURE ON THE COMPOSITION OF FATTY ACIDSIN ESCHERICHIA COLI

ALLEN G. MARR AND JOHN L. INGRAHAM

Department of Bacteriology, University of California, Davis, California

Received for publication July 19, 1962

ABSTRACT

MARR, ALLEN G. (University of California,Davis) AND JOHN L. INGRAHAM. Effect of temper-ature on composition of fatty acids in Escherichiacoli. J. Bacteriol. 84:1260-1267. 1962.-Variationsin the temperature of growth and in the com-position of the medium alter the proportions ofindividual fatty acids in the lipids of Escherichiacoli. As the temperature of growth is lowered,the proportion of unsaturated fatty acids (hex-adecenoic and octadecenoic acids) increases. Theincrease in content of unsaturated acids with adecrease in temperature of growth occurs in bothminimal and complex media. Cells harvested inthe stationary phase contained large amounts ofcyclopropane fatty acids (methylenehexadec-anoic and methylene octadecanoic acids) incomparison with cells harvested during ex-ponential growth. Cells grown in a chemostat,limited by the concentration of ammonium salts,show a much higher content of saturated fattyacids (principally palmitic acid) than do cellsharvested from an exponentially-growing batchculture in the same medium. Cells grown in achemostat, limited by the concentration ofglucose, show a slightly higher content of un-saturated fatty acids than cells from the cor-responding batch culture. The results do notindicate a direct relation between fatty acidcomposition and minimal growth temperature.

It is a common observation that the lipids ofpoikilothermic organisms vary with the temper-ature of growth. Plants (Howell and Collins,1957), insects (Fraenkel and Hopf, 1940), andmicroorganisms (Terroine, Hatterer, and Roehrig,1930; Gaughran, 1947) all appear to containincreased proportions of unsaturated fatty acidsor more highly unsaturated fatty acids if they aregrown at low temperatures. Homiothermicorganisms also contain a greater proportion ofunsaturated fatty acids in the surface lipids if theenvironmental temperature is low (Dean andHilditch, 1933).

Stemming from these observations, the concepthas been developed that composition of the lipidmay set the limits of temperature for growth ofmicroorganisms. Gaughran (1947), who investi-gated the lipid composition of steno- and euri-thermophilic bacteria, suggested that cells cannot grow at temperatures below the solidificationpoint of their lipids; i.e., the temperature atwhich the lipids solidify is the minimal temper-ature for growth. Heilbrunn (1924) and Beleh-rddek (1931) proposed the antithetical argumentthat melting of lipids at high temperature de-stroys essential structures of the cell; i.e., thetemperature at which the lipid melts is themaximal temperature for growth.The variation in the composition of lipids of

microorganisms with temperature of growth hasbeen established by measurement of iodinenumber, as a means of estimating the proportionof unsaturated fatty acids. The development ofthe technique of gas-liquid chromatographypermits the quantitative measurement of indi-vidual fatty acids. In this investigation wedetermined individual fatty acids in Escherichiacoli by gas-liquid chromtography. The resultsshow a progressive increase in saturated fattyacids and a corresponding decrease in unsaturatedfatty acids as the temperature of growth isincreased. However, the results suggest that fattyacid composition is not directly related to thelimits of temperature of growth.

MATERIALS AND METHODS

Media. The basal medium was medium 56 ofMonod, Cohen-Bazire, and Cohn (1951). Glucoseor succinate (0.2%) was added as a carbon sourcefor minimal media. Complex media were preparedby supplementing the glucose-minimal mediumwith 0.2% Casamino Acids (Difco) or with 0.2%yeast extract (Difco). The Casamino Acids andyeast extract were extracted with diethyl etherto remove lipids.

Organism. E. coli ML30, obtained from JacquesMonod, was used in all these experiments.

1260

on January 28, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

TEMPERATURE AND FATTY ACIDS OF E. COLI

Growth conditions. Batch cultures were grownin 5-liter bottles containing 3 liters of medium.The cultures were immersed in water held at thestated temperature i 0.05 C and sparged througha sintered-glass thimble with air saturated withwater vapor at the temperature of the culture.

Continuous cultures were grown in a chemostatoperated aerobically at 30 C at a specific dilutionrate of 0.20 hr-' (Marr and Marcus, 1962). Forcarbon limitation, the concentrationi of glucosein the minimal medium was decreased to 0.05%;and for nitrogen limitation, the concentration of(NH4)2SO4 was decreased to 0.0185%. Theoverflow from the culture was collected in areceiver immersed in an ice bath.

Analysis of fatty acids. Cells were harvested bycentrifugation and washed twice with water.Approximately 1 g (dry wt) of cells was hydro-lyzed for 2 hr at 120 C in a sealed tube containing15 ml of 2 N HCl. The hydrolysate was extractedthree times with 2 vol of diethyl ether. Thecombined ether extract was dried with anhydrousNa2SO4, and the ether was evaporated by astream of N2. The residue was esterified in 5 mnl of0.2 N HCl in anhydrous methanol under refluxfor 2 hr. After evaporating the solution to 1 mlunder a stream of N2, 2 ml of water were added;and the methyl esters were extracted with threesuccessive 5-ml portions of petroleum ether. Theextract was dried with anhydrous Na2SO4, andthe petroleum ether was evaporated under astream of N2. The residue was dissolved in drybenzene.The methyl esters were determined by gas-

liquid chromatography on a column (0.25 in. by9 ft) of 25% diethylene glycol-succinate polyesteron fire brick at 189 C (Kaneshiro and Marr,1961).The amount of each component was computed

from the area of the recorded peak, which wasestimated from the area of the inscribed triangle.Over the range of concentrations encountered inthe analysis of samples, the relationship betweenarea and amount of a given methyl ester wasfound to be linear; however, the area obtainedwith different esters was not related linearlyeither to mole fraction or to weight fraction.Under the conditions used in the analysis, therelative areas corresponding to equal weights ofmethyl esters were: myristate, 1.1; palmitateand hexadecenoate, 1.0; octadecenoate (oleate),0.825; methylene hexadecanoate, 0.90; ,3-hydroxy-myristate, 0.55; methylene octadecanoate,

0.775. The relative areas for methylene hexa-decanoate and methylene octadecanoate werenot determined experimentally; the areas wereestimated by interpolation from their retentionvolumes.

RESULTS

Identification of the fatty acid esters. Theprincipal fatty acids of E. coli were identified bygas-liquid chromatography of the methyl esters

O 2 3 4 5 6 7 8

LITERS OF HELIUM

FIG. 1. Tracings of gas-liquid chromatograms ofthe methyl esters of fatty acids from Escherichia coliharvested during exponential growth in glucose-mini-mal medium at 35, 20, and 10 C. A, laurate; B, myris-tate; C, palmitate; D, hexadecenoate; E, methylenehexadecanoate; F, octadecenoate; G, ,3-hydroxy-myristate.

35C

D

E ,

20C

(

VOL. 84, 1962 1261

on January 28, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

MARR AND INGRAHAM

(Fig. 1) as lauric, myristic, palmitic, hexa-decenoic, octadecenoic, methylene hexadecanoic,and f-hydroxymyristic acids. The retentionvolumes of the esters of the fatty acids from E.coli agreed with the retention volume of authenticmethyl esters to i 2%.The identity of the esters of the unsaturated

acids, methyl hexadecenoate and methyl octa-decenoate, was confirmed by hydrogenation tomethyl palmitate and methyl stearate, re-spectively. The position of the double bond wasnot determined; however, the octadecenoic acidof E. coli has been reported to be a mixtuire ofcis-vaccenic and oleic acids (Kaneshiro and Marr,1961).Methyl ,B-hydroxymyristate was recovered

from the mixed methyl esters of E. coli bytrapping component G from the effluent of thegas-liquid chromatogram (Fig. 1). This methylester was purified by chromatography on acolumn of silicic acid; the ester eluted in thefraction characteristic of methyl esters of hydroxyacids. Methyl f-hydroxymyristate was identifiedby dehydration and catalytic hydrogenationthe product of which was identical with methylmyristate in gas-liquid chromatography. Theinfrared spectrum of the recovered methyl,3-hydroxymyristate was identical with thespectrum of synthetic methyl /3-hydroxymyris-tate, and the retention volumes in gas-liquidchromatography of the recovered ester and syn-thetic methyl 3-hydroxymyristate were iden-tical. Although Ikawa et al. (1953) found /-hydroxymyristic acid as a major component of apolar lipid of E. coli, this acid was not detectedin the alcohol-soluble lipid of E. coli (Kaneshiroand Marr, 1961). Presumably, f-hydroxymyris-tic acid is released from the bound lipid by acidhydrolysis of the cells.

In many of the samples of the methyl estersfrom E. coli, methyl laurate was only partiallyresolved from an unidentified component bygas-liquid chromatography, making the quanti-tative determination of the small amount ofmethyl laurate difficult (Fig. 1). Because of thisdifficulty in precise estimation of the amount ofmethyl laurate, and because lauric acid is aminor component of the lipids of E. coli, lauricacid was omitted from all calculations of thecomposition of the fatty acids. Several minorcomponents are also evident in the chromato-

grams between the peaks of methyl myristateand methyl palmitate (Fig. 1, 3, 4). These com-ponents were not identified, and their areas werenot considered in computing the percentages offatty acids.

Effect of growth temperature on fatty acidcomposition of cells grown in glucose-minimalmedium. Cultures of E. coli ML30 were grown inglucose-minimal medium at 10, 15, 20, 25, 30, 35,40, and 43 C, and were harvested by centrif-ugation of an exponentially growing culture.Three tracings of representative analyses areshown in Fig. 1. From such analyses, the amountof each fatty acid was computed from the areaunder each peak (Table 1, Fig. 2). As growthtemperature is decreased, the proportion ofunsaturated acid increases. The percentage of themost abundant unsaturated fatty acid, octa-decenoic acid, decreases continuously withincreasing growth temperature over the entire

FIG. 2. Effect of growth temperature on the fattyacid composition of Escherichia coli MLSO harvestedduring exponential growth in glucose-minimal me-dium. Per cent by weight of the total fatty acids iscalculated as methyl esters, neglecting lauric acid.A, palmitic acid; B, sum of hexadecenoic acid andmethylene hexadecanoic acid; C, octadecenoic acid.

1262 J. BACTERIOL.

on January 28, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

TEMPERATURE AND FATTY ACIDS OF E. COLI

TABLE 1. Effect of growth temperature onfatty acid composition of Escherichia coli ML30 grown in glucose-minimal medium and harvested during exponential growth

Temp (C)Fatty acid

10 15 20 25 30 35 40 43

%t % % % % % % %*Myristic........................... 3.9 3.8 4.1 3.8 4.1 4.7 6.1 7.7Palmitic ........................... 18.2 21.9 25.4 27.6 28.9 31.7 37.1 48.0Hexadecenoic .................... 26.0 25.3 24.4 23.2 23.3 23.3 28.0 9.2Methylene hexadecanoic........... 1.3 1.1 1.5 3.1 3.4 4.8 3.2 11.6Octadecenoic ...................... 37.9 35.4 34.2 35.5 30.3 24.6 20.8 12.2Methylene octadecanoic.. 0 0 0 0 0 0 0 3.7,3-Hydroxymyristic................ 12.6 12.5 10.4 6.9 10.1 11.0 4.8 7.5

Hexadecenoic-palmitict ............ 1.43 1.55 0.96 0.84 0.81 0.74 0.75 0.19Octadecenoic-palmitict............ 2.08 1.62 1.35 1.29 1.05 0.78 0.56 0.25

* Calculated as the weight per cent of the methyl ester. Lauric acid is not included in total (see text).t Ratio of weight per cent.

growth range. The most abundant saturatedfatty acid, palmitic acid, increases continuouslywith increasing growth temperature. The highestrate of change of these fatty acids occurs at hightemperature. The percentage of the cyclopropanefatty acid, methylene hexadecanoic acid, alsodecreases with decreasing growth temperature,and ,3-hydroxymyristic acid is almost constant.The percentage of hexadecenoic acid increasesonly slightly at low temperature, but decreasesdramatically above 40 C. Ratios of unsaturatedto saturated fatty acids (Table 1) serve as usefulindices of the degree of shift in fatty acid com-position of the cells; e.g., the hexadecenoic-palmitic and octadecenoic-palmitic acid ratiosboth decrease approximately eightfold over thetemperature range of 10 to 43 C.

Effect of growth rate on fatty acid composition.The changes in proportions of fatty acids mighthave resulted from differences in growth rateimposed by temperature. This possibility wastested by varying the growth rate at constanttemperature by altering the composition of themedium. Cultures were grown at 25 C in thefollowing media: succinate-minimal, glucose-minimal, glucose-minimal supplemented withCasamino Acids, and glucose-minimal supple-mented with yeast extract.The cells were harvested during exponential

growth and were analyzed to determine thecomposition of fatty acids (Table 2). The pro-portion of various fatty acids is somewhat de-pendent on the medium, but does not correlate

with the growth rate (Table 2). The specificgrowth rate in glucose-minimal medium sup-plemented with yeast extract at 25 C is almostidentical to the growth rate in glucose-minimalmedium at 35 C, but the composition of fattyacids of cells grown at 25 C with a supplement ofyeast extract (Table 2) corresponds more closelyto cells grown in minimal medium (Table 1)at about 10 C as judged by the hexadecenoic-palmitic acid ratio or at about 20 C as judged bythe octadecenoic-palmitic acid ratio.The percentage of palmitic acid is significantly

higher in cells grown in glucose-minimal than inthe other three media.

Effect of growth temperature on fatty acidcomposition of cells grown in complex medium.Since cells grown at 25 C in glucose-minimalmedium supplemented with yeast extract have afatty acid composition similar to that of cellsgrown near the minimal growth temperature inglucose-minimal medium, it was of interest todetermine whether growth at low temperature incomplex medium would result in an even greaterproportion of unsaturated fatty acids. Cultureswere grown in glucose-minimal medium sup-plemented with yeast extract at 10, 20, 25, and35 C. The cells were harvested during exponentialgrowth and were analyzed for fatty acids (Table3). The results are comparable to minimalmedium except that at any given temperaturethe percentage of unsaturated fatty acids ishigher in complex than in minimal medium. Asgrowth temperature is lowered, the percentage

1263VOL. 84, 1962

on January 28, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

MARR AND INGRAHAM

TABLE 2. Effect of growth rate on the fatty acidcomposition of Escherichia coli harvested dur-

ing exponential growth at 25 C

Glucose GlucoseSuc- Glucose minimal minimal

Medium................... cinate + +minimal milnimal Casamino yeast

Acids extract

k (hr-1) * ................. 0.35 0.48 0.64 0.88

Equivalent tempt......... 22 C 25 C 32 C 36 C

% % % %Fatty acidMyristic .......... 4. 2t 4.4 5.7 4.4Palmitic .......... 21.0 25.6 24.8 22.2Hexadecenoic... 28.7 27.3 29.6 31.7Methylene hexa-

decanoic ........ 1.3 2.1 O.7 0.8Octadecenoic ..... 34.6 31.5 30.6 31.3f-Hydroxymyris-

tic ............. 10.2 9.2 8.5 9.6

Ratios of weight percent

Hexadecenoic-palmitic ........ 1.34 1.07 1.19 1.43

Octadecenoic-palmitic ........ 1.65 1.23 1.23 1.41

* Specific growth rate, calculated from the2.303(log X2 -log xl).

equation, k = in which xit2-t1and X2 are optical densities at times t1 and t2.

t Temperature at which the same growth rateis attained in glucose minimal medium.

t Calculated as the weight per cent of themethyl ester. Lauric acid is not included in thetotal (see text).

of palmitic and methylene hexadecanoic acidsdecrease and the percentage of octadecenoic acidincreases. The change in hexadecenoic acid isslight, and the percentage of ,B-hydroxymyristicacid remains essentially constant.

Fatty acids of cells harvested frcm cultures in thestationary phase. The preceding measurementsdiffer from earlier reports of the fatty acidcomposition of E. coli. We found only smallamounts of methylene hexadecanoic acid and nomethylene octadecanoic (lactobacillic) acidexcept in cells grown near the maximal temper-ature. Previously, Dauchy and Assilineau (1960)and Kaneshiro and Marr (1961) reported thecyclopropane acids as major components of thelipid of E. coli. This apparent discrepancy results

TABLE 3. Effect of growth temperature on the fattyacid composition* of Escherichia coli MLSOgrown in glucose-minimal medium supple-

mented with yeast extract

Temp of growth (C)Fatty acid

10 20 25 35

%o % % %

Myristic ........ 3.9 4.8 5.0 5.6Palmitic ........ 16.4 22.4 21.8 33.1Hexadecenoic... 30.4 32.9 30.0 28.4Methylenehexadecanoic. 0.7 1.3 1.1 1.8

Octadecenoic ... 38.8 30.1 31.3 20.2j-Hydroxymyr-

istic .......... 10.0 8.6 10.9 10.9

Hexadecenoic-palmitict ..... 1.85 1.47 1.38 0.86

Octadecenoic-palmitict ..... 2.37 1.34 1.44 0.61

* Calculated as the weight per cent of methylester. Lauric acid is not included in the total (seetext).

t Ratio of weight per cent.

from an increase in the cyclopropane acids at theexpense of the corresponding unsaturated acidsafter the end of exponential growth.

Figure 3 shows the analyses of cells harvestedfrom succinate-minimal medium during expon-ential growth and of cells harvested several hoursafter the growth of the culture ceased fromexhaustion of the carbon source. The percentageof methylene hexadecanoic acid increases ap-proximately 12-fold in the cells from thestationary phase, with a corresponding decreasein hexadecenoic acid. Methylene octadecanoicacid is present in cells from the stationary phasebut is not detectable in cells from the culturegrowing exponentially. A third significant differ-ence is the presence in the cells from thestationary-phase culture of an unresolvedcomponent between palmitate and hexadeceno-ate. This component is clearly revealed inanalyses of hydrogenated samples. The retentionvolume of this component is identical with thatof the methyl ester of a branched chain C17 acid.

Effect of carbon or nitrogen limitation. Theprevious experiments suggested a significantinfluence of nutrition on the proportions of fattyacids produced by E. coli. The influence ofnutrition and of reduced growth rate was tested

J. BACTERIOL.1264

on January 28, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

TEMPERATURE AND FATTY ACIDS OF E. COLI

EXPONENTIAL

F

B

G

I\-l-j~~~~~

STATIONARY

D E F

G' G

IIIZLJ1 2 3 4 5 6 7

LITERS OF HELIUM

FIG. 3. Tracing of gas-liquid chromatograms of themethyl esters of fatty acids from Escherichia coligrown in succinate-minimal medium at 25 C andharvested during the exponential phase and duringthe stationary phase. A, laurate; B, myristate; C,palmitate; D, hexadecenoate; E, methylene hexa-decanoate; F, octadecenoate; G', methylene octa-decanoate; G, f-hydroxymyristate.

further by cultivation of E. coli at 30 C in achemostat, limited by the concentration eitherof the carbon source or of the nitrogen source.The flow rate was adjusted to provide a specificgrowth rate of 0.2 hr-'. The analyses of the cellsfor fatty acids are shown in Fig. 4. Cells harvestedfrom the glucose-limited culture differ onlyslightly in fatty acid composition from cells

LITERS OF HELIUM

FIG. 4. Tracings of gas-liquid chromatograms ofthe methyl esters of fatty acids from Escherichia coligrown in a chemostat at 30 C under conditions of car-bon and of nitrogen limitation. A, laurate; B,myristate; C, palmitate; D, hexadecenoate; E,methylene hexadecanoate; F, octadecenoate; G,f3-hydroxymyristate.

grown in batch culture. Thus, a reduction inspecific growth rate from 0.65 to 0.2 hr-1 does notsignificantly alter the proportions of fatty acids.The fatty acid composition of the cells from thenitrogen-limited chemostat is strikingly different.The amount of palmitic and methylene hexa-decanoic acids is increased, and the amount ofunsaturated fatty acids is decreased. The effectof nitrogen limitation mimics the effect of growthat high temperature.

Changes in fatty acid composition of cells after a

shift in temperaturefrom 40 to 10 C. Ng, Ingraham,

A

D

C- LIMITED

AB

F

N- LIMITED

2 3 4 5 61

\J B.iL

I

l

I

VOL. 84, 1962 1265

on January 28, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

MARR AND) INGRAHAM

and Marr (1962) reported that, when a culture ofE. coli growing exponentially at 40 C is cooledto 10 C, growth ceases for approximately 4 hr,after which the culture grows exponentially for 10hr at approximately twice the normal growthrate at 10 C. To determine whether the fatty acidcomposition changes during the lag, cells wereharvested just after the period of rapid exponen-tial growth began and were analyzed for fattyacids. The fatty acid composition of these cellsdiffered from the typical composition of cellsgrown at either 40 or 10 C. The composition wassimilar to that of cells grown exponentially in thesame medium at 25 C, indicating that the fattyacid composition had changed significantly duringthe period of lag.

DISCUSSION

The proportion of unsaturated fatty acids of E.coli decreases continuously as growth tempera-ture is increased in both minimal and complexmedia. The fatty acids in bacteria are mainly con-tained in phospholipids, which are an essentialpart of the structure of the cell membrane ratherthan mere storage products. Thus, it is temptingto speculate that changes in fatty acid composi-tion with growth temperature are adaptations toenvironments of different temperature. However,growth at a particular temperature does notresult in a unique fatty acid composition, sincealtering the nutrition independently of tempera-ture also results in major changes in fatty acidcomposition.

This fact argues against the hypothesis thatsolidification of lipids sets the minimal growthtemperature (Gaughran, 1947). The minimaltemperature for growth, which is slightly lessthan 10 C for E. coli ML30 (Ng et al., 1962), isknown to be independent of medium. However,the fatty acid composition of cells grown inglucose-minimal medium at 10 C differs markedlyfrom that of cells grown at the same temperaturein this medium supplemented with yeast extract.If the fatty acid composition of cells grown inminimal medium did indeed set the minimaltemperature for growth, the minimal temperaturein complex medium would have been between 15and 20 C.The striking difference in fatty acid composi-

tion between cells grown at 20 and at 35 C (Table1) is not demonstrably adaptive. Ng et al.(1962) have shown that the specific growth rate

of cultures of E. coli ML30 responds immediatelyto a change in temperature in the range of 20 to37 C. Cells which have a fatty acid compositioncharacteristic of the steady state of growth at 20C grow at 37 C at the same rate as cells with afatty acid composition characteristic of the steadystate of growth at 37 C. Thus, rather largechanges in the fatty acid composition have nomeasurable effect on the specific growth rate. Thephysiological effects of these differences in fattyacid composition appear to be trivial.The mechanism by which a change in tempera-

ture regulates the synthesis of fatty acids hasnot been established. The effect might be (i) adirect effect of temperature on the relative ratesof two or more enzymes which synthesize satu-rated and unsaturated acids, respectively, (ii) anindirect effect resulting from a change in growthrate, or (iii) an indirect effect resulting from achange in the concentration of intermediates.The possibility that temperature alters the

fatty acid composition through changing thegrowth rate is not, a priori, an attractive hy-pothesis, and it can be readily eliminated by theresults of the experiments. Increasing the growthrate by supplementation of minimal medium withCasamino Acids or yeast extract results in ahigher proportion of unsaturated fatty acids, butincreasing the growth rate by increasing tempera-ture results in a lower proportion of unsaturatedfatty acids. The most direct demonstration thatgrowth rate, per se, does not control the fatty acidcomposition comes from a comparison of cellsgrown in glucose-minimal medium at 30 C inbatch culture (k = 0.64 hr-i) and in glucose-limited chemostat (k = 0.20 hr-'). Despite thelarge difference in growth rates, the fatty acidcomposition was quite similar.The effect of nitrogen limitation may offer a

clue to the means by which temperature controlsthe proportions of fatty acids. Limitation of thenitrogen source imposed by cultivation in achemostat at 30 C with ammonium as the limitingnutrient (Fig. 4) resulted in a dramatic increasein the proportion of palmitic acid at the expenseof both hexadecenoic and octadecenoic acids. Thefatty acid composition under this condition ofnitrogen limitation is equivalent to the composi-tion in a batch culture grown at about 40 C. Theincrease in proportions of unsaturated fatty acidswhich results from supplementation of minimalmedium with amino acids or yeast extract can

1266 J. BACTERIOL.

on January 28, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

TEMPERATURE AND FATTY ACIDS OF E. COLI

also be explained as the relief of a partial defi-ciency in the intermediates in nitrogenmetabolism.An interesting but unexpected result of this

study is the finding that the cyclopropane fattyacids, methylene hexadecanoic and methyleneoctadecanoic acids, are formed in substantialamounts only after the cessation of exponentialgrowth. Both appear to be formed at the expense

of the unsaturated homologue in accord with thehypothesis that the cyclopropane ring is formedby addition of a methylene carbon to an olefin.

ACKNOWLEDGMENTS

This work was aided by a grant from the Whirl-pool Corporation, by grant-in-aid RG-6807 fromthe National Institutes of Health, U.S. PublicHealth Service, and by grant-in-aid 9860 fromthe National Science Foundation.We are indebted to Raymond Combs for

technical assistance.

LITERATURE CITED

BMLEHRkDEK, J. 1931. Le mechanisme physico-chemique de l'adaptation thermique. Proto-plasma 12:406-434.

DAUCHY, S., AND J. ASSELINEAU. 1960. Sur lesacides gras des lipid de Escherichia coli.Compt. Rend. 250:2635-2637.

DEAN, H. K., AND T. P. HILDITCH. 1933. The bodyfats of the pig. III. The influence of bodytemperature on the composition of depot fats.Biochem. J. 27:1950-1956.

FRAENKEL, G., AND H. S. HOPF. 1940. Tempera-

ture adaptation and degree of saturation ofthe phosphatides. Biochem. J. 34:1085-1092.

GAUGHRAN, E. R. L. 1947. The saturation of bac-terial lipids as a function of temperature. J.Bacteriol. 53:506.

HEILBRUNN, L. V. 1924. The heat coagulation ofprotoplasm. Am. J. Physiol. 69:190-199.

HOWELL, R., AND F. I. COLLINS. 1957. Factorsaffecting linolenic and linoleic acid content ofsoybean oil. Agron. J. 49:593-597.

IKAWA, M., J. B. KOEPFLI, S. G. MUDD, AND C.NIEMANN. 1953. An agent from E. coli causinghemorrhage and regression of an experimentalmouse tumor. III. The component fatty acidsof the phospholipide moiety. J. Am. Chem.Soc. 75:1035-1038.

KANESHIRO, T., AND A. G. MARR. 1961. Cis-9,10-methylene hexadecanoic acid from the phos-pholipids of Escherichia co 'i. J. Biol. Chem.236:2615-2619.

MARR, A. G., AND L. MARCUS. 1962. Kinetics ofinduction of mannitol dehydrogenase in Azoto-bacter agilis. Biochim. Biophys. Acta 64:65-82.

MONOD, J., G. COHEN-BAZIRE, AND M. COHN. 1951.Sur la biosynthese de la ,3-galactosidase (lac-tase) chez Escherichia coli. La spdcificit6 del'induction. Biochim. Biophys. Acta 7:585-599.

NG, H., J. L. INGRAHAM, AND A. G. MARR. 1962.Damage and derepression in Escherichia coliresulting from growth at low temperatures.J. Bacteriol. 84:331-339.

TERROINE, E. F., C. HATTERER, AND P. ROEHRIG.1930. Les acides gras des phosphatides chezanimaux poikilothermes, les v6getaux sup6-rieurs et les microorganismes. Bull. Soc.Chim. Biol. 12:682-702.

1267VOL. 84, 1962

on January 28, 2020 by guesthttp://jb.asm

.org/D

ownloaded from