Effects of Different Culture Media and Oxygen upon Lipids of Escherichia coli K-12 Wl L L I A M F. NACCARATO, Department of Biochemistry, Faculty of Arts and Sciences, and JOHN R. G I LBERTSON and ROSE A. GE LMAN, Department of Pharmacology and Physiology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

fatty acids as a more direct precursor to the 1-alkanols. The first part of this study involved characterizing the free fatty alcohols and free fatty acids (FFA) from E. coil K-12 grown aerobically and anaerobically on a chemically defined media. The second part involved isolating and characterizing the hydrocarbons from these cells. This was undertaken to evaluate the possibility of alcohol reduction to hydrocarbons, a logical step in hydrocarbon biosynthesis.

ABSTRACT

The effects of altering the chemical composition of the culture media and the oxygen content of the environment upon the lipid metabolism of Escherichia coli K-12 were investigated. When E. coli cells were grown on the same culture medium but under aerobic and anaerobic conditions, an increase in the free fatty acids of anaerobically grown cells was observed with a disproportionate increase in the unsaturated fatty acids. When glucose was the sole carbon source, both fatty alcohols and hydrocarbons were detected as component lipids of these cells, whether growth occurred under aerobic or anaerobic conditions. Based upon this observation, acetate is considered the initial precursor for fatty alcohol and hydrocarbon biosynthesis. A possible metabolic pathway involving fatty alcohols in hydrocarbon synthesis has been pestulated.

EXPERIMENTAL PROCEDURES Bacteria

The E. coli were grown as described (1), only a chemically defined medium was substituted for TSB. This medium was 0.06 M in potassium phosphate buffer (pH = 7.1), 7.5 mM (NH4)2SO4, 0.4 mM Mg SO 4, and 0.027 M glucose. Cells grown on this chemically defined media will be referred to as glucose-grown and those grown on TSB as TSB-grown. TLC

INTRODUCTION

Fatty alcohols of Escherichia coli K-12 have been characterized as l- and 2- alkanols. A preliminary investigation into the metabolism of these alcohols implicated oxygen as a regulatory factor. When E. coli were grown anaerobically, there was a quantitative decrease in the total alcohol content. Gas chromatographic analysis showed this decrease resulted mainly from a selective loss of 2-alkanols (1). Taking into consideration the effect of oxygen upon the 2-alkanols, it was hypothesized that they were synthesized by hydroCarbon oxidation (1), a process known to require molecular oxygen (2). The hydrocarbon substrates were thought to be derived from the media (analysis of the trypticase soy broth [TSB] media showed compounds that had properties of hydrocarbons on thin layer chromatography [TLC] and gas liquid chromatography [GLC]. No further characterization was carried out). If this were true, 2-alkanots should disappear when E. coIi are grown anaerobically in media with glucose as the only carbon source. Alternatively, alcohol formation would suggest acetate as the ultimate precursor and

Preparation of the chromatoplates and the techniques used for isolation and recovery of lipid have been described (1). The plates used for isolation of hydrocarbons were prewashed with ethyl ether, air-dried, and activated (1). The hydrocarbons were purified using hexane as the developing solvent. Isolation of Lipids

Lipids were extracted (3), concentrated under vacuum, and diluted to a known volume with nitrogen-equilibrated n-heptane. Ca. 400 mg lipid were applied to 18 g n-heptane-equilibrated silicic acid columns (4) and the column developed (1). Hydrocarbons eluted in the 1% ethyl ether/n-heptane fraction were purified by TLC. The free fatty alcohols, FFA, and phospholipids were isolated as before (1). Esterified fatty acids were liberated from the phospholipids by transesterification with anhydrous 2% HC1/methanol at 70 C for 1 hr, and the methyl esters extracted and isolated by TLC (1). In each instance, a blank containing the same volume of solvents as the samples was concentrated to dryness and carried through the entire lipid isolation procedure. The residue in the

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323

fractions corresponding to the individual lipid types was anlyzed to prevent artifactual assignments.

oo

GLC Conditons for GLC of free fatty alcohosl and acids are published (1). Hydrocarbons were analyzed qualitatively and quantitatively under the same conditions, only the column temperature was programed from 100 C-250 C at 2 C/min. Peak areas were related to wt using a calibration curve derived from hexadecanol.

% 0

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R ESU LTS Quantitative Changes under Growth Conditions

Table I shows the quantitative changes that occurred in cell yield and lipid when E. coli K-12 were cultured aerobically and anaerobically on TSB and on the chemically defined medium. Significant changes in the cell yield, FFA, and free fatty alcohols but no change in percent lipids or hydrocarbons were observed. Note that there was less fatty alcohol and more F F A in cells grown anaerobically than in those cultured aerobically on the same media and that cell yield always decreased under anaerobiosis. Also observe that a smaller cell yield and lower fatty alcohol content occurred in glucose-grown cells as compared to the TSB-grown cells cultured under the same oxygen environment. A decrease in F F A occurred in aerobic glucose-grown E. coli as compared to those cultured on TSB, but an increase in F F A was noted in glucsoe-grown E. coli cultured anaerobically.

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Qualitative Lipid Changes under Different Nutritional Conditions

In Table II, the chain length distribution of t h e alcohols isolated from aerobic glucoseg r o w n E. coli is compared with the distribution of alochols isolated from cells cultured on TSB. While slight differences exist in the lower percentage components, the major alcohols are 1-tetradecanol, 1-hexadecanol, and 2-pentadecanol in both cases. The F F A were isolated from aerobic glucose-grown E. coli and analyzed via GLC as described previously (1). No significant differences were noted between these values and those obtained from cells cultured on TSB (1). Methyl esters of fatty acids haying carbon numbers greater than those noted were not detected. The identification of hydrocarbons in this study was based upon three criteria. (A) A natural lipid c o m p o n e n t was eluted from a silicic acid column in a fraction known to elute

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TABLE II Qualitative Distribution Fatty Alcohols from Aerobically Grown E. Coli Cultured in Two Media Chain length a 12:0 13:0 sec 13:0 14:0 sec 14:0 15:0 sec 15:0 16:1 16:0 17:0 18:0

Glucose grown b 3.7 2.7 2.7 14.1 30.0 7.4 32.8 5.9 1.1

Wt %, Try pticase soy broth grown c (i)

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2.8 8.4 1.5 18.2 26.7 2.7 28.6 13.6

FIG. 1. Typical gas liquid chromatogram of the hydrocarbons isolated from E. coli grown on glucose as a sole carbon source in an aerobic environment. Carbon number of each component identified by gas liquid chromatography and mass spectrometry is noted in the figure. nents found in our analysis were not reported in the previous publication.

aCarbon number: number of double bonds, sec = secondary. Qualitative Lipid Changes under Anaerobiosis bAverage of two experiments. The bound fatty acids (Table IV) and CAverage of three experiments. hydrocarbons of glucose-grown E. call K-12 did hydrocarbons (4) that had the same mobility as not change qualitatively when these cells were hexadecane during TLC. (B) GLC of tiffs grown anaerobically. This is consistent with component indicated there were a number of published results (6-8). Figure 2 compares the chain length distribuspecies having relative retention times identical to known saturated aliphatic hydrocarbons. (C) tion of the F F A in aerobic and anaerobic The mass spectra of natural compounds were glucose-grown E. coli. The data show that, identical with fragmentation patterns of known under anaerobic conditions, the amount of hydrocarbons (5) and were consistent with the monoenoic fatty acids increased significantly. Hexadecenoic acid increased from 2.0% to 9.4% structure assigned to the molecule by GLC. and octadecenoic acid from 13.3% to 40.0%. A gas chromatogram of the hydrocarbon Since fatty alcohols were not detectable in fraction isolated from aerobic glucose-grown E. coli is shown in Figure 1. The components that the anaerobically cultured glucose-grown E. were identified are all members of the normal eoli, we were not able to determine the chain saturated series. Other unidentified components length distribution and no observation concernalso are present and account for 15% total peak ing the selective decrease of 2-alkanols could be area. At present, the identity of these compo- made. Analysis of the individual fractions correnents is being investigated. Identification of sponding to the F F A , fatty alcohols, and hydrocarbons in TSB-grown cells was not attempted because hydrocarbons from TSB enter hydrocarbons from t h e solvent blank by TLC the cellular pool of hydrocarbons and make it and GLC indicated that artifactual components impossible to differentiate those synthesized by were not present. This result is of particular the cell from those obtained from the medium. significance with respect to the hydrocarbon However, when the chain length distribution of fraction from glucose-grown cells, since, if hydrocarbons from aerobically, glucose-grown artifacts were not detected and glucose was the E. coli is compared with previously published sole carbon source, the hydrocarbons mast have results (6), differences in the hydrocarbon been formed biosynthetically. types are immediately obvious (Table II]). Our analysis shows that hexadecane is the major hydrocarbon of E. coli K-12, as opposed to octadecane in the previously reported data. Also, nonadecane and eicosonane were reported in significant amounts previously but our data indicate that these are not significant species in cells grown only on glucose. Tri-, tetra-, penta-, and hexacosane are the other significant hydrocarbons in the fraction we analyzed. A third difference is that the unidentifiable compoLIPIDS, VOL. 9, NO. 5

DISCUSSION

The detection of 1- and 2-alkanols in E. coli grown on a chemically defined media proves that the free fatty alcohols are not derived from oxidation of hydrocarbons in the growth media. The ultimate source of the 1- and 2-alkanols must be glucose, since it is the only carbon source in the media. It is well known that the synthesis of fatty chains from glucose proceeds through acetate condensations (9),

M E D I A , 0 2 , L I P I D O F E . COLI K - 1 2

325 T A B L E III

H y d r o c a r b o n s o f E. Colt K - 1 2 G r o w n o n Glucose under Aerobic Conditions

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wt %

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FIG. 2. Histogram depicting the chain length distribution of the free fatty acids isolated from E. coli grown on glucose as a sole carbon source in an aerobic and anaerobic environment. Ordinate-wt %; abscissa-number of carbon atoms: number of double bonds. Cyclopropane fatty acids are denoted by the number of double bonds: cyclic, n aerobic ~ anerobic The fatty chains of the alcohols probably are derived from the fatty acid, but the mechanism for the formation of the alcohol function remains unknown. Reduction of fatty acid or oxidation of endogenous hydrocarbons are possibilities. The change in alcohol content under anaerobiosis indicates that oxygen is a factor reg~alating alcohol metabolism in glucose-grown E. colt, as well as TSB-grown cells. However, it must be pointed out that the decrease in the fatty alcohols of the anaerobic glucose-.grown cells must have involved the primary, as well as the secondary, alcohols. A selective loss of only the secondary 'alcohols would have resulted in ca. 30% decrease. Our methods would have been able to measure the remaining 70%; however, no alcohol was detected chemically or by GLC. This is not the case in the TSB-grown E. colt where there is a selective decrease of secondary alcohols (1). The identification of hydrocarbons in E. colt and other microorganisms has been published (6). The qualitative differences in hydroca,'bon composition reported here and elsewhere (6) can be the result of a number of factors. Bacterial lipids vary with the culture's age, temperature, pH, and growth media (10). Since the previous report did not give the growth conditions, we cannot evaluate the difference in the two sets of data. Studies carried out with Sarcina lutea are the only extensive investigations of hydrocarbon biosynthesis in bacteria (11-13). Interestingly enough, the biosynthetic pathway in this bacteria is different from the elongation-decarboxylation pathway in plants proposed by

Chain length

Glucose growna

13:0 14:0 15:0 16:0 17:0 18:0 19:0 20:0 21:0 22:0 23:0 24:0 25:0 26:0 27:0 28:0 29:0 30:0 31:0 32:0 33:0 Uni d e n title d

0.6 1.5 3.9 21.8 '7.8 4.2 4.0 3.5 2.8 5.0 7.2 9.5 8.9 "7.4 3.8 3,4 1,6 1.4 1.1 0.6 0.7 17.8

Previous d a t a (6)

0.5 1o7 5.5 27.6 12.0 10.0 5.5 6.0 8.3 7.4 6,0 3.3 3.3 0.5 1.4

aAverage of two experiments.

Kolattukudy (14). Evidence indicates that Sarcina lutea synthesize hydrocarbons by condensation of a fatty acid and the aliphatic group of the neutral lipid 1-0-alk-l-enyl glycerols (13). The possibility that this mechanism operates in E. colt is unlikely, since this bacterium contains no alk-l-enyl glycerol ethers (1, 15). In addition, E. colt contain hydrocarbons that have chain lengths corresponding to the FFA. This suggests direct reduction of fatty acids to hydrocarbons as a biosynthetic mechanism along with a type of F F A elongation pathway for the very long chain hydrocarbons. It is conceivable that the fatty alcohols are intermediates in the reduction of fatty acids to

T A B L E IV E s t e r i f i e d F a t t y A c i d s o f G l u c o s e - G r o w n E. Colt K-12 Grown Aerobically and Anaerobically

wt % Chain length 16:1 16:0 17:cyc 18:1 18:0 19:cyc

Aerobic

Anaerobic

A e r o b i c (8)

1.4 44.6 28.3 8.2

1.9 50.0 25~ 11.1

7.0 40.0 22.0 19.0

12,2

7.7

7.4

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h y d r o c a r b o n s . An aldehyde w o u l d m o s t likely also be involved in the r e d u c t i o n , and we have isolated these c o m p o u n d s f r o m E. coli grown on TSB (1). Lipids having c h r o m a t o g r a p h i c properties o f hexadecanal on Silica Gel G were isolated f r o m the glucose-grown cells also, but the a m o u n t present m a d e any further characterization impossible. 2-Alkanols also might be intermediates in h y d r o c a r b o n biosynthesis and may arise in the following p a t h w a y : 7 (SCoA CH3--(CH2) n --CH2-CH 2 --C-(ACP + malonyl - ACF---r ? (SCoA CH3--(CH2) n - C H 2 - C H 2 - C - C H 2 - C - ( A C P

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CH3-(CH2)n-CH2-CH2-C-CH2-C-OH2

CH3-(c"2)n-C"2-C2--c.3 \2 H

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CH3--(CH2) n - C H 2 - C H 2 - C H - C H 3 "['~ H2 CH3--(CH2)n--CH2--CH = CH-CH 3 \-.-~ CH 3 - ( C H 2 ) n - - C H 2 - - C H 2 - - CH 2--CH 3 A c o m m o n m e c h a n i s m of d e c a r b o x y l a t i o n in biological systems involves a fl-keto i n t e r m e d i ate (16), and this is what is postulated here. Investigations into the m e t a b o l i s m of fatty acids in E. coli usually are c o n c e r n e d w i t h only the esterified acids (8, 17-19). However, the data in this r e p o r t indicate that changes in esterified fatty acids m a y n o t always reflect changes in F F A . The estefified fatty acid pool may remain quite constant u n d e r a given set of conditions, while the smaller pool of F F A changes quite rapidly. As a result, it must be k e p t in m i n d that measuring changes in esterifled fatty acids will n o t reflect changes in de n o v o fatty acid synthesis, It also is n o t e d f r o m the F F A analysis that c y c l o p r o p a n e fatty acids occur in the free state. Since they are biosynthesized while esterified to phospholipids (20, 21), those present as free acids must be arising f r o m chemical or enzymatic hydrolysis. Chemical hydrolysis seems rather unlikely, because the ratio of 9, 10-methylene h e x a d e c a n o i c a c i d : l 1, 12-methylene octadecanoic acid is 1.58 in the F F A and 3.5 in the bound. If chemical hydrolysis occurred during lipid e x t r a c t i o n , similar ratios w o u l d be expected, Selective e n z y m a t i c hydrolysis might be releasing this acid to initiate their degradation. A c o m p a r i s o n of the chain length of the F F A from aerobically and anaerobically grown LIPIDS, VOL. 9, NO. 5

E. coli (Fig. 2) i m m e d i a t e l y suggests a role for o x y g e n in the control of fatty acid metabolism. The m o n o e n o i c acids in E. coli are k n o w n to be synthesized by branching o f the saturated fatty acid p a t h w a y (22). a, fl D e h y d r a t i o n of the fl-hydroxyacyl derivative leads to saturated fatty acids, while 13 7 d e h y d r a t i o n of this derivative yields the monenes. Our e x p e r i m e n t s suggest that lack o f o x y g e n exerts a c o n t r o l at this point. Saturated fatty acids also were observed to increase quantitatively u n d e r anaerobiosis. The a m o u n t of h e x a d e c a n o i c acid measured by quantitative GLC in aerobically grown cells was 0 . 1 4 0 / ~ m o l e s / 1 0 0 mg total lipid. The c o n t e n t in anaerobically grown cells was 1.25 /2moles/100 mg total lipid, a ninefold increase. We have no evidence to indicate if the increased levels of F F A are due to increased synthesis or decreased degradation, thus a c o m m e n t concerning their m e t a b o l i s m c a n n o t be made. However, these changes do occur, and the causes and nature of these regulatory m e c h a n i s m are of interest. One other observation can be made from the data in Table I. When the F F A increase, the fatty alcohols decrease. It m a y be that the acyl C o A is the precursor to the 1-alkanols, as well as the F F A (23-25), and the p r o d u c t of one p a t h w a y may inhibit the o t h e r p a t h w a y at some point. It m a y be this t y p e o f m e c h a n i s m that is decreasing fatty alcohols in the anaerobic E. coli and n o t the effect of o x y g e n directly. E x p e r i m e n t s are n o w in progress to investigate this possibility.

ACKNOWLEDGMENT This work was supported in part by research grant AA00348 from the National Institute of Health, U.S. Public Health Service. REFERENCES 1. Naccarato, W.F., R.A. Gelman, J.C. Kawalek, and J.R. Gilbertson, Lipids 7 : 275 (1972). 2. Foster, J.W., in "Oxygenases," Edited by Osamu Hayaishi, Academic Press, New York, N.Y., 1962, p. 241. 3. Folch, J., M. Lees, and G.H. Sloane Stanley, J. Biol. Chem. 226:497 (1957). 4. Hirsch, J., and E.H. Ahrens, Jr., Ibid. 233:311 (19s8). 5. Budzikiewicz, H., C. Djerassi, and D.H. Williams, "Mass Spectrometry of Organic Compounds," Holden-Day, San Francisco, Calif., 1967 p. 49. 6. Han, J., and M. Calvin, Proc. Nat. Acad. Sci. 64:436 (1969). 7. Calvin, M., "Chemical Evolution," Oxford University Press, New York, N.Y. 1969 p. 55. 8. Bloch, K., P. Baronowsky, H. Goldfine, W.J. Lennarz, R. Light, A.T. Norris, and G. Scheuerbrandt, Proc. 20:921 (1961). 9. Lennarz, W.J., in "Advance in Lipid Research," Vol. 4, Edited by Rodolfo Paoletti and David

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10.

11. 12. 13. 14. 15. 16. 17. 18.

Kritchevsky, Academic Press, New York, N.Y., 1966 p. 175. Kates, M., in "Advances in Lipid Research," Vol. 2, Edited by Rodolfo Paoletti and David Kritchevsky, Academic Press, New York, N.Y., 1964, p. 17. Albro, P.W., and J.C. Dittmer, Biochemistry 8:394 (1969). Albro, P.W., and J.C. Dittmer, Ibid. 8:953 (1969). Albro, P.W., and J.C. Dittmer, Ibid 8:1913 (1969). Kolattukudy, P.E., Science 159:498 (1968). Kim, K.C., J. Gen. Appl. Microbiol. 16:291 (1970). Ingraham, L.L., "Biochemical Mechanisms," John Wiley and Son, New York, N.Y., 1962 p. 58. Cronan, J.E., Jr., J. Bacteriology 95:2054 (1968). Knivett, U.A., and J. Cullen, Biochem. J. 103:299

(1967). 19. Silbert, D.F., M. Cohen, and M.E. Harder, J. Biol. Chem. 247:1699 (1972). 20. Zalkin, H., J.H. Law, and H. Goldfine, Ibid. 238:1242 (1963). 21. Chung, A.E., and J.H. Law, Biochemistry 3:967 (1964). 22. Erwin, J., and K. Bloch, Science 143:1006 (1964). 23. Barnes, E.M., and S.J. Wakil, J. Biol. Chem. 243:2955 (1968). 24. Barnes, E.M., Jr., A.C. Swindell, and S.J. Wakil, Ibid. 245:3122 (1970). 25. Bonner, W.M.., and K. Bloch, Ibid. 247:3123 (1972). [ R e c e i v e d D e c e m b e r 21, 197 3 ]

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