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Mechanism of ethanol-induced changes in lipid composition of Escherichia coli

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Title:
Mechanism of ethanol-induced changes in lipid composition of Escherichia coli preferential inhibition of saturated fatty acid synthesis
Creator:
Buttke, Thomas Martin, 1952-
Publication Date:
Copyright Date:
1978
Language:
English
Physical Description:
viii, 57 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Acetates ( jstor )
Chromatography ( jstor )
Enzymes ( jstor )
Ethanol ( jstor )
Fatty acids ( jstor )
In vitro fertilization ( jstor )
Lipids ( jstor )
Nonesterified fatty acids ( jstor )
Phospholipids ( jstor )
Unsaturated fatty acids ( jstor )
Alcohol -- Physiological effect ( lcsh )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Escherichia coli ( lcsh )
Fatty acid synthesis ( lcsh )
Lipids -- Metabolism ( lcsh )
Microbiology and Cell Science thesis Ph. D
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 54-56.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Thomas Martin Buttke.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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05313015 ( OCLC )
AAK0596 ( NOTIS )

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MECHANISM OF ETHANOL-INDUCED CHANGES IN LIPID
COMPOSITION OF ESCHERICHIA COLI: PREFERENTIAL
INHIBITION OF SATURATED FATTY ACID SYNTHESIS




By



THOMAS MARTIN BUTTKE


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY






UNIVERSITY OF FLORIDA

















ACKNOWLEDGEMENTS


Le chemin aet tong du project a la close.
Moliere


The author would like to thank Dr. L.O. Ingram for his help and

encouragement during the author's graduate career. Dr. Ingram's

knowledge as well as his friendship has been invaluable and his

willingness to share both is greatly appreciated.

Thanks are also due to Drs. J.F. Preston and C.M. Allen for

donating their time and suggestions during the course of this research.

The author is especially grateful to his wife, Mary, for providing

companionship during late night experiments, and for typing this

dissertation. Her encouragement and good humor have contributed

immeasurably towards the completion of this research.

Part of this work was performed while the author was the recipient

of a fellowship from the Center for Gerontological Studies, and this

support is gratefully acknowledged.


















TABLE OF CONTENTS



Page

ACKNOWLEDGMENTS .............................................. ii

LIST OF TABLES ................................................ iv

LIST OF FIGURES............................................... v

ABBREVIATIONS USED ... ........................................ vi

ABSTRACT ...................................................... vii

INTRODUCTION ................................................ 1

EXPERIMENTAL PROCEDURE....................................... 4

RESULTS................ ....................................... 13

DISCUSSION....................... ....... ..................... 47

REFERENCES................................................... 54

BIOGRAPHICAL SKETCH ......................................... 57

















LIST OF TABLES


Table Page

1 Bacterial strains.................................... 5

2 Effect of ethanol on the fatty acid composition of
newly synthesized phospholipids in strain TB4......... 14

3 Effect of incubation temperature on ethanol-
induced changes in the fatty acid composition
of newly synthesized phospholipids................... 17

4 Effect of fatty acid degradation on ethanol-
induced changes in fatty acid composition............ 20

5 Effect of ethanol on the fatty acid composition
of an unsaturated fatty acid auxotroph, strain
K1060................................................ 25

6 Effect of ethanol on the incorporation of fatty
acids into phospholipids............................. 28

7 Effect of exogenous 16:0 on ethanol-induced changes
in fatty acid composition in strain TB4.............. 30

8 Effect of ethanol on the incorporation of exogenous
16:0 [ H] and endogenous 16:0 [14C] into the
phospholipids of strain TB4.......................... 32

9 Effect of ethanol on the incorporation of 17:0 into
the phospholipids of an unsaturated fatty acid
auxotroph, strain K1060 ............................. 33

10 Effect of ethanol on the fatty acids synthesized in
vitro by a crude fatty acid synthetase ............... 35

11 Effect of purification of fatty acid synthetase on
ethanol-induced fatty acid changes in vitro.......... 37

12 Effect of ethanol on the chain lengths of the fatty
acid products synthesized in vitro ................... 44

13 Effect of incubation temperature on the fatty acid
products synthesized in vitro ........................ 46

















LIST OF FIGURES


Figure Page

1 Biosynthetic pathway for fatty acids in E. coli
showing the location of the defects in fatty
acid synthesis mutants................................. 7

2 Effect of ethanol on free fatty acid synthesis
and total lipid synthesis in strains LW1 and LW3...... 23

3 Effect of ethanol on the rates of synthesis of
saturated and unsaturated fatty acids in vitro........ 40

4 Argentation chromatography of fatty acid products
synthesized in vitro................................... 42

















ABBREVIATIONS USED


UFA

SFA

PFA

ACP

16:0

16:1

17:0

cis-A9-18:1

cis-11-18:1

A17

20:2

A19


Unsaturated fatty acid

Saturated fatty acid

Polar fatty acid

Acyl carrier protein

Palmitic acid

Palmitoleic acid

Heptadecanoic acid

Oleic acid

cis-Vaccenic acid

cis-9,10-Methylene hexadecanoic acid

Eicosadienoic acid

cis-11,12 Methylene octadecanoic acid


Morpholinopropane sulfonic acid


MOPS

















Abstract of Dissertation Presented to the Graduate Council of
the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy

MECHANISM OF ETHANOL-INDUCED CHANGES IN LIPID
COMPOSITION OF ESCHERICHIA COLI: PREFERENTIAL
INHIBITION OF SATURATED FATTY ACID SYNTHESIS

By

Thomas Martin Buttke

June 1978

Chairman: Lonnie 0. Ingram

Major Department: Microbiology and Cell Science


Experiments were performed to determine the mechanisms by which

ethanol induces fatty acid changes in Escherichia coli. Mutants

deficient in various aspects of lipid metabolism were used to determine

the mechanisms of ethanol interaction in vivo. Under conditions which

uncoupled fatty acid synthesis from phospholipid synthesis, ethanol

decreased the amount of saturated fatty acids synthesized, but had

little effect on their incorporation into phospholipids. In the

absence of fatty acid degradation and unsaturated fatty acid synthesis,

E. coli still demonstrated ethanol-induced fatty acid changes, while

the inhibition of total fatty acid synthesis eliminated this response.

Experiments with the fatty acid synthetase enzymes in vitro supported

the in vivo results. Fatty acid synthetase preparations from three

levels of purification demonstrated a preferential inhibition of

saturated fatty acid synthesis by ethanol, similar to that observed


vii











in vivo. The fatty acid changes in vitro did not result from a total

arrest of saturated fatty acid synthesis, nor were they due to a

premature termination of saturated fatty acid elongation. These

results indicate that ethanol-induced fatty acid changes in E. coli

result from a preferential inhibition of the rate of saturated fatty

acid synthesis.

















INTRODUCTION


In a previous report Ingram demonstrated that Escherichia coli

will alter its membrane fatty acid composition when grown in the

presence of straight chain alcohols (Ingram, 1976). These alcohol-

induced fatty acid changes are similar to those induced by changes

in growth temperature (Marr and Ingraham, 1962). Short chain alcohols

such as ethanol cause a decrease in the amounts of saturated fatty

acids, similar to a shift down in growth temperature. Long chain

alcohols such as hexanol induce changes analogous to a shift up in

temperature, an increase in the proportion of saturated fatty acids

(Ingram, 1976). Like temperature adaptation, these changes in fatty

acid composition are independent of major changes in phospholipid

composition (Ingram, 1977a). These results with alcohols have been

extended to show that a wide variety of other lipophylic agents

(organic solvents and food preservatives) also induce fatty acid

changes in E. coli (Ingram, 1976). The diverse structures of these

compounds as well as their lipophylic nature support the theory that

these changes in fatty acid composition result from a general inter-

action with a hydrophobic site, rather than a result of specific

catabolic processes (Ingram, 1977b). The intercalation of these

agents into the membrane would be expected to alter membrane fluidity.

The resulting changes in fatty acid composition suggest that the cell

may be able to detect changes in membrane fluidity, per se, and

compensate for the direct actions of drugs.











In view of the importance of ethanol, ethanol-tolerance and with-

drawal syndrome in society, the ability of ethanol to induce dramatic

changes in the fatty acid composition is of particular interest. A

membrane whose lipid composition has been altered in response to

ethanol could be considered as being adapted to the presence of the

alcohol (tolerance), much as membrane lipid changes allow a membrane

to adapt to growth temperature (Shaw and Ingraham, 1965). The removal

of ethanol would cause additional changes in membrane organization

which would be compensated for by a return to the original composition

(withdrawal). Thus, an understanding of the mechanisms responsible for

ethanol-induced changes in fatty acid composition in E. coli could

provide a model for the development of experiments in mammalian

systems.

The similarity of the changes in fatty acid composition induced

by ethanol and temperature adaptation suggests that these processes

may share a common mechanism. This regulation of fatty acid composition

could be carried out at any of three levels: (1) fatty acid degrada-

tion; (2) fatty acid synthesis; and (3) phospholipid synthesis (Cronan

and Vagelos, 1972). E. coli mutants blocked in one or more of these

processes have proven useful in studying the control of thermal

regulation of membrane lipid composition (Cronan and Gelmann, 1975).

These same mutants were used in this study to elucidate the mechanisms

by which ethanol induces membrane fatty acid changes in E. coli. The

results of these experiments indicate that the ethanol-induced

changes in fatty acid composition in vivo result primarily from the

inhibition of saturated fatty acid synthesis.











Experiments were also conducted with the fatty acid synthesis

enzymes in vitro to determine if the relative amounts of fatty acids

synthesized in vitro were also affected by ethanol. In a fashion

analogous to that observed in vivo, ethanol inhibited total fatty

acid synthesis in vitro but with a preferentially greater inhibition

of saturated fatty acid synthesis. Taken collectively, the results

of both the in vivo as well as the in vitro experiments suggest that

ethanol-induced fatty acid changes in E. coli result from a decreased

availability of saturated fatty acids for phospholipid synthesis.

Further, the decreased availability of saturated fatty acids reflects

a preferential inhibition of saturated fatty acid synthesis by

ethanol.
















EXPERIMENTAL PROCEDURE


Bacterial Strains. The E. coli K12 derivatives used in this

study are listed in Table 1. Figure 1 illustrates the biosynthetic

pathway for fatty acids in E. coli, showing the location of the defects

in fatty acid synthesis. Strain TB4 is derived from strain CSH2 and is

defective in fatty acid degradation (fadE ). Strain CSH2 was made

proB by mutagenesis with ultraviolet light followed by penicillin

selection (Miller, 1972). A proB fadE lysate was made from strain

L43 by the method of Marsh and Duggan (1972) using the Plvir (Cold

Spring Harbor Laboratory), and this was used to introduce the fadE

gene into strain CSH2. Potential fadE transductants were selected

by their ability to grow without proline supplementation.

Growth Conditions. Unless otherwise indicated, all bacterial

strains were grown in a gyrotory shaker at 300C. The standard medium

used was E medium (Vogel and Bonner, 1956). Supplements differed for

each strain. Strains CSH2, TB4 and L43 were grown with sodium

succinate (0.2%), thiamine (0.001%), tryptophan (0.004%), and yeast

extract (0.0005%). Strains LW1 and LW3 were supplemented with sodium

succinate (0.2%), thiamine (0.001%), casein hydrolysate (vitamin free)

(0.05%), and sn-glycerol-3-phosphate (0.01%). Glycerol-3-phosphate

deprivation was performed by filtration as described by Cronan et al.

(1975). For strains L8 and L48, the media was supplemented with

glycerol (1.0%), thiamine (0.001%), monosodium glutamate (0.1%),








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Figure 1. Biosynthetic pathway for fatty acids in
Escherichia coli showing the location of
the defects in the fatty acid synthesis
mutants.












CHCO-S-CoA CHyC -S-CoA

HCA fabE

H00-CHfCO-S-COA
o*C.A ACPJ,._ fabD
r*
CHSCO-S-ACP HOOO-CH!CO-S-ACP
L --------------;
L *14-kC02
CHICHiCHI-CO-S-ACP

NADP


NADPH2

H *0
CHSC=CCO-S-ACP CH3C-CHTCO-S-ACP
H

NADPH2

H20
H NADP
*0
CHSC-CHTCO-S-ACP
H LN


CHS(CH)BCHDCHC0-S-AC P
H
H20 H2


HH H
C HS(CH2)AC-=CHC0-S-AC P CH15(CH2)6 CHCO-S-ACP
fab B? NAOPH2- P
HH NADPHL
CHj(C H2) C'O(CH2)4C0 S -ACP
3HS (C H2)ACH2CH2C 0-S-ACP

H I H I
&H(CHA)C O(CH2)C0-S-ACP CH-3(CH2)TI S-C











yeast extract (0.0001%), Brij 58 (0.4%), potassium palmitate

(0.001%) and potassium oleate (0.001%). Cell growth was monitored

by measuring the absorbance at 550 nm. using a Beckman model 25

spectrophotometer. Cultures were routinely used in the experiments

at a cell density of 2x108 cells/ml (0.36 absorbance units at 550 nm.)

to insure that cells were in log phase.

Cells grown for in vitro fatty acid synthesis studies were

grown into late log phase (0.70 absorbance units at 550 nm.) in Medium

63 mineral salts (Miller, 1972) supplemented with glucose (0.2%),

thiamine (0.001%), tryptophan (0.02%) and casamino acids (vitamin free;

0.1%). These cells were also grown in a gyrotory shaker, except the

incubation temperature was 370C.

Preparation of Fatty Acid Synthetase Enzymes. Cells grown into

late log phase were harvested by centrifugation and washed once with

10 mM phosphate buffer pH 7.0. The cells were disrupted by passage

through a French pressure cell (12,500 psi) and centrifuged at 5,000

rpm for ten minutes to remove unbroken cells. The supernate contained

the fatty acid synthesis enzymes (Lennarz et al., 1962) and is called

the "crude" enzyme preparation. A membrane free enzyme preparation

was obtained by centrifuging the "crude" enzyme preparation at

100,000 x g for two hours to pellet the membranous material. The

supernate which contained the fatty acid synthesis enzymes after

this step was referred to as the "100 Kg" preparation. This prepara-

tion was further purified by ammonium sulfate fractionation as

described by Lennarz et al. (1962). Ammonium sulfate was added to the

"100 Kg" preparation to 55% saturation and the precipitated protein

was removed by centrifuging at 10,000 rpm for ten minutes. The











supernate was brought to 75% saturation with additional ammonium

sulfate and the precipitated fatty acid synthetase enzymes were

harvested by centrifugation. The resultant pellet was resuspended

in 10 mM phosphate buffer, pH 7.0, containing 1 mM dithiothreitol, .and

frozen at -200C in 0.2 ml aliquots.

In Vitro Fatty Acid Synthesis Assay. The conditions for assaying

fatty acid synthesis in vitro were a modification of the procedure

of Silbert (1976). Standard incubation mixtures contained MOPS

buffer, pH 7.1 at 370C (50 moless, dithiothreitol (1 pmole), NADH2

(100 nmoles), NADP+ (100 nmoles), glucose-6-phosphate (1 lmole),

glucose-6-phosphate dehydrogenase (0.65 units), acetyl CoA (15 nmoles),

acyl carrier protein (25 ug), and [2-14C] malonyl CoA (30 moles; 3.8

pCi/pmole) in a final volume of 0.48 ml. The reaction was started by

the addition of 100 pg of enzyme protein, and the reaction mixtures

were incubated at 370C in screw-cap tubes sealed with Teflon-lined

caps. The reaction was quenched by the addition of 1 ml of 15% KOH in

methanol, and the samples were heated for two hours at 700C. The

samples were acidified with 0.6 ml of 6N HC1, carrier fatty acids

were added, and the mixture was extracted two times with pentane.

Combined pentane extracts were washed twice with water, added to pre-

weighed screw-cap tubes, and the pentane was evaporated under nitrogen

at 45 C until one gram of pentane remained. Aliquots of 100 pl were

removed for measuring total lipid synthesis, and the remainder was

taken to dryness prior to transesterification. The labeled methyl

esters were separated into saturated, unsaturated and polar fatty

acids by argentation chromatography.











Analysis of Fatty Acids by Gas Chromatography. Cells were

harvested by centrifugation, inactivated by the addition of 5%

trichloroacetic acid, and extracted into chloroform-methanol as

described by Kanfer and Kennedy (1973). For the separation of

neutral lipids (includes free fatty acids) from phospholipids, washed

lipid extracts were chromatographed on unactivated silica gel G

impregnated glass-fiber paper (Gelmann ITLC-SG, Fisher Scientific Co.,

Pittsburg, Pa.) using solvent 1 as described by Freeman and West (1966).

Phospholipids were eluted with chloroform-methanol (2:1), and

transesterified using 2% H2SO4 in methanol, as described by Silbert

et al. (1973a). The methyl esters were extracted into pentane and

concentrated under N2 prior to analysis. The conditions for gas

chromatography have been described (Ingram, 1976).

Analysis of Radioactive Lipids. The incorporation of labeled

acetate into fatty acids was assayed essentially by the procedure of

Cronan and Wulff (1969). One milliliter samples of cells were added

to tubes containing 10 pCi of [ 14C] acetate (61 mCi/mmole) and incubated

at 37 C for ten minutes. The reaction was stopped by the addition of

chloroform-methanol (1:2) and extracted overnight. The resulting lipid

extract was separated into two phases by the addition of chloroform

and water; radioactive lipids were present in the lower (chloroform)

phase. An aliquot (0.2 ml) was removed from the chloroform phase and

counted to determine total lipid synthesis. The remaining radio-

active lipids were transesterified as described above, and the radio-

active methyl esters were analyzed by argentation chromatography. When
the incorporation of 14 acetate into free (non-esterified) fatty
the incorporation of [ C] acetate into free (non-esterified) fatty











acids was measured, the lipid extract was separated into phospholipids

and neutral lipids by thin layer chromatography using either the

solvent system of Klein et al. (1971) or the two step procedure

described by Cronan et al. (1975). Both the phospholipid and the

neutral lipid fractions were eluted, transesterified and analyzed by

argentation chromatography. Incorporation of exogenously supplied
14 3 3
[14C] or [ H] palmitic acid and 3 HI oleic acids were performed in

essentially the same fashion, except phospholipids were separated

from neutral lipids by thin layer chromatography using the solvent

diethyl ether-acetic acid (100:1). The phospholipid region was scraped

into vials containing toluene scintillation solution (Omnifluor;

New England Nuclear) and counted on a Beckman model 133 liquid

scintillation counter. When the counter was adjusted for dual-label

counting, the efficiency was 58.2% for [3H] and 72.5% for [14C].

Argentation Chromatography. Thin layer chromatography plates

coated with silica gel G were impregnated with 10% aqueous silver

nitrate and activated as described by Cubero and Mangold (1965). The

labeled methyl esters were applied to the argentation plate in pentane

and developed twice in toluene at -170C (Morris et al., 1967).

Radioactive regions were scraped into vials and counted.

Reversed Phase Chromatography. The chain lengths of the saturated

and unsaturated fatty acids synthesized in vitro were determined by

reversed phase chromatography (Bergelson et al., 1964). Regions of

argentation plates corresponding to saturated and unsaturated fatty

acids were scraped and the methyl esters were eluted with pentane. The

methyl esters were applied in pentane to silica gel G plates impregnated










with dodecane. Impregnation was achieved by ascending a 10% dodecane

in light petroleum ether solution up a silica gel G plate and the

petroleum ether evaporated. The solvent system used for development

consisted of acetone-acetonitrile (1:1) saturated with dodecane

(Bergelson et al., 1964). Labeled methyl esters were located by

autoradiography using Kodak X-OMAT R film.

Chemicals. Heptadecanoic acid was purchased from Applied Science

Laboratories Inc. (State College, Pa.). All other unlabeled fatty

acids were obtained from the Sigma Chemical Co. (St. Louis, Mo.).

[l-14C] Acetate, [9,10(n)- H] oleic acid, and [1-14 C palmitic acid

are products of Amersham Corp. (Arlington Heights, II.). [9,10(n)- 3H]

Palmitic acid and [2- C] malonyl CoA were purchased from New England

Nuclear (Boston, Ma.). Unlabeled malonyl CoA, acetyl CoA and dodecane

are the products of Sigma Chemical Co. Acyl carrier protein (ACP)

was purified by the procedure of Majerus et al. (1968) from one

kilogram of E. coli K12 (Grain Processing Co., Muscatine, Iowa).
















RESULTS


Effect of Ethanol on the Fatty Acid Composition of Newly

Synthesized Phospholipids. Previous experiments have shown that

growth in the presence of ethanol results in the synthesis of phos-

pholipids rich in unsaturated fatty acids (Ingram, 1976). To

determine if these changes represent a direct effect of ethanol on

lipid synthesis (fatty acid synthesis and utilization) or a secondary

effect mediated through such processes as enzyme induction or other

alternatives in metabolism, we have examined the immediate effects of

ethanol addition on newly synthesized lipids. Strain TB4 (fadE )

14
was pulse-labeled with [ 14C] acetate to measure the effects of various

concentrations of ethanol on the incorporation of newly synthesized

fatty acids into phospholipids.

The addition of ethanol caused an immediate increase in the

abundance of newly synthesized unsaturated fatty acids which were

incorporated into phospholipids (Table 2). This resulted in a dose-

related increase in the UFA/SFA ratios, and is analogous to the

changes previously observed in the bulk lipid composition (Ingram,

1976). High concentrations of ethanol (4% and 6%) strongly inhibited

the synthesis and utilization of both unsaturated and saturated fatty

acids, and this inhibition may be related to the "lipid poor" cells

described earlier (Ingram, 1977). The ethanol-induced increase in

the UFA/SFA ratio was immediately reversed by ethanol removal (washing),










Table 2. Effect of ethanol on the fatty acid composition of
newly synthesized phospholipids in strain TB4





14
S4C] Acetate Incorporation (cpm)

EtOH(%) Total Saturated Unsaturated UFA/SFA Ratioc


Oa 14,400 4,100 10,300 2.5

1 8,800 1,700 7,200 4.2

2 9,300 1,400 7,900 5.6

3 8,600 1,100 7,500 6.8

4 2,600 360 2,300 6.4

6 1,600 350 1,300 3.7

3%, 16 hrb 4,800 900 3,900 4.3

No EtOH 10,800 2,800 8,000 2.9



aCells were grown in the absence of ethanl. Samples were removed and
added to tubes containing 10 pCi of [1- C] acetate (61 mCi/mmole) with
various concentrations of ethanol. The samples were incubated for ten
min. Fatty acids were separated using argentation chromatography.

Cells were grown in the presence of 3% ethanol for 16 hr (with appro-
priate dilutions). The culture was split into two parts. One part
was rapidly washed using membrane filtration and resuspended to its
o~aginal volume. Samples were removed from both parts, pulsed with
[ C] acetate and analyzed by argentation chromatography.


CUFA, unsaturated fatty acid; SFA, saturated fatty acid











even after 16 hours of growth in the presence of 3% ethanol. The

immediacy of action of ethanol on the UFA/SFA ratio of newly

synthesized lipids coupled with its equally rapid reversibility

provide evidence for the direct action of ethanol on lipid synthesis.

Pulse-labeling experiments typically yielded UFA/SFA ratios

which were higher than those of bulk lipids. This probably resulted

from the incorporation of newly synthesized fatty acids into phospha-

tidyl glycerol. In E. coli this phospholipid is enriched in unsatu-

rated fatty acids and turns over rapidly during normal growth. Thus,

the pulse-labeling studies may reflect both the de novo synthesis as

well as the turnover of phosphatidyl glycerol which would give rise to

the increased UFA/SFA ratios observed. However, the UFA/SFA ratios

following the initial addition of ethanol (Table 2) were much higher

than expected based upon determinations of bulk lipid composition

(Ingram, 1976). During growth in the presence of ethanol, the ratios
14
observed in log phase cells pulsed with [ 14C] acetate approached that

of bulk lipid, 7.1, 6.1, 5.8, and 4.4 after 0, 1, 2, and 16 hours,

respectively. Thus the initial increase in the UFA/SFA ratio

following the addition of ethanol represented an overcompensation

in newly synthesized lipids which was subsequently decreased as the

cells adjusted to the presence of ethanol.

Effect of Incubation Temperature on Ethanol-Induced Changes in

the Fatty Acid Composition of Newly Synthesized Lipids. E. coli alters

its membrane fatty acid composition in response to changes in growth

temperature (Marr and Ingraham, 1962). A shift to a higher growth











temperature leads to an increase in the proportion of palmitic acid

while a shift to a lower growth temperature leads to an increase in the

proportion of unsaturated fatty acids. These changes in lipid composi-

tion are regulated both at the level of fatty acid synthesis (Cronan,

1975; Okuyama et al., 1977) and fatty acid utilization for phos-

pholipid synthesis (Esfahani et al., 1969; Sinensky, 1971). We have

shown that the addition of ethanol causes changes in fatty acid

composition similar to a reduction in growth temperature. To further

explore this relationship, we have investigated the simultaneous

effect of ethanol addition and a shift in incubation temperature on

the fatty acid composition of newly synthesized lipids. Samples from

an exponential culture of strain TB4 grown at 300C were incubated at

230C, 300C and 370C, and pulse-labeled with [14C] acetate in the

presence and absence of 3% ethanol. Esterified fatty acids were

separated by argentation chromatography, scraped and counted.

Strain TB4 altered its fatty acid composition (UFA/SFA ratio) in

response to temperature (Table 3) as previously reported (Marr and

Ingraham, 1962). This same trend was reflected among samples containing

ethanol indicating that ethanol did not disrupt the normal thermo-

regulatory systems. The addition of ethanol resulted in an increase

in the proportion of unsaturated fatty acids at all incubation

temperatures. Thus the effects of ethanol and a shift down in temper-

ature were roughly additive while the effects of ethanol and a shift

up in temperature were opposing.

The increases in the proportion of unsaturated fatty acids in

response to ethanol addition and a shift to lower temperature were











Table 3. Effect of incubation temperature on ethanol-
induced changes in the fatty acid composition
of newly synthesized phospholipids


Fatty Acids (cpm)
Incubation
Temperature Conditions Saturated 16:1 cis-l -18:1 UFA/SFA Ratio


23 C No EtOH 1,600 3,600 5,300 5.6

3% EtOlI 520 2,900 2,800 11.0


300C No EtOH 2,500 2,700 3,800 2.6

3% EtOH 580 1,900 1,700 6.2


370C No EtOH 580 580 480 1.8

3% EtOH 300 540 420 3.2




aSamples frm an exponential culture of strain TB4 were pulse-labeled with
10 pCi 11- C] acetate (61 mCi/mmole) at various temperatures in the
presence and absence of 3% ethanol. Fatty acids from purified
phospholipids were separated by argentation chromatography.


UFA, unsaturated fatty acid; SFA, saturated fatty acid











primarily the result of an absolute decrease in esterified saturated

fatty acids. Unlike ethanol addition, a shift to lower temperature

was also accompanied by an absolute increase in esterified unsaturated

fatty acids. A shift to higher temperature caused an inhibition of

acetate incorporation into both saturated and unsaturated fatty acids

which was further inhibited by the inclusion of ethanol.

The stimulation of lipid synthesis by a shift to lower temperature

and its inhibition by a shift to higher temperature are difficult to

explain. These changes may be related to changes in the levels of

guanosine-5'-diphosphate-3'-diphosphate (ppGpp). High levels of

ppGpp inhibit both phospholipid synthesis (Merlie and Pizer, 1973) and

fatty acid synthesis (Nunn and Cronan, 1976). Recent studies by

Gallant et al. (1977) have shown that E. coli rapidly accumulates

this nucleotide upon shifting to a higher temperature.

Effect of Fatty Acid Degradation (fadE) on Ethanol-Induced Changes ''

in Fatty Acid Composition. Since fatty acid synthesis is tightly

coupled to phospholipid synthesis, the changes observed in UFA/SFA

ratios could be due to the direct action of ethanol on the avail-

ability of fatty acids, or their utilization by the acyltransferase

system, or both. E. coli possesses a multi-enzyme complex capable

of fatty acid degradation (Binstock et al., 1977). This enzyme complex

may partially determine the fatty acid composition of membrane lipids

by altering the relative amounts of fatty acid species available

for incorporation into phospholipids. Thus, the ethanol-induced

reduction in palmitic acid previously reported in E. coli (Ingram, 1976)











could result from a preferential degradation of endogenously

synthesized palmitic acid. To determine if this was indeed the case,

the effects of ethanol on strain CSH2, proficient in fatty acid

degradation (fadE ), and strain TB4, defective in fatty acid

degradation (fadE ), were compared (Table 4). In both strains the

proportion of palmitic acid (16:0) was reduced by 10%, with an equal

increase in the proportion of cis-vaccenic acid during growth in the

presence of ethanol. Neither strain showed a significant change in

the level of palmitoleic acid (16:1). These results indicate that

preferential fatty acid degradation is not responsible for ethanol-

induced changes in fatty acid composition.

Effect of Ethanol on Free Fatty Acid Synthesis. Although fatty

acid degradation is not essential for ethanol-induced fatty acid

changes (Table 4), the availability of fatty acids for phospholipid

synthesis could also be modified by a direct effect of ethanol on fatty

acid synthesis. In E. coli, fatty acid synthesis is normally tightly

coupled with phospholipid synthesis (Cronan, 1974) and prevents an

evaluation of the effect of ethanol on either of these systems indepen-

dent of the other. In order to determine if ethanol directly affects

fatty acid synthesis, we have used two mutants in which fatty acid

synthesis can be uncoupled from phospholipid synthesis in vivo by

glycerol-3-phosphate starvation. The removal of exogenous glycerol-3-

phosphate from growing cells of strains LW1 and LW3 causes an inhibition

of phospholipid synthesis and an accumulation of free (non-esterified)

fatty acids (Cronan et al., 1975). These are easily labeled by pulsing.






















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14
the cells with [ C] acetate during starvation. Subsequent analysis

of the free fatty acids provides an in vivo method for examining the

regulation of fatty acid synthesis, per se, independent of the

acyltransferase system.

The addition of ethanol to strains LW1 and LW3 altered the

proportions of saturated and unsaturated free fatty acids which

accumulated (Figure 2A and C). Although the synthesis of both

saturated as well as unsaturated fatty acids was inhibited by ethanol
14
([ C] acetate incorporation), 3% ethanol caused only a 23% decrease

in the synthesis of unsaturated fatty acids while causing a 73%

reduction in the synthesis of saturated fatty acids. Thus the ethanol-

induced increase in the UFA/SFA ratio during the free fatty acid

synthesis results from the preferential inhibition of saturated fatty

acid synthesis.

In order to determine whether ethanol affects the incorporation

of fatty acids into newly synthesized phospholipids in strain LW1 and

LW3, we also examined the fatty acid composition of the phospholipids.

In the absence of exogenous glycerol-3-phosphate, a small amount of

residual phospholipid synthesis continues in these strains (Cronan

et al., 1975). The ratio of unsaturated to saturated fatty acids either

in these residual phospholipids or in the phospholipids of cells

supplemented with glycerol-3-phosphate can be compared with the

ratios in the accumulated free fatty acids to determine the effect of

ethanol on the selectivity of the phospholipid synthesizing enzymes.

Figure 2 (B and D) demonstrates that phospholipids extracted from

glycerol-3-phosphate supplemented cells as well as from glycerol-3-





























Figure 2. Effect of ethanol on free fatty acid synthesis and
total lipid synthesis in strains LW1 and LW3.
Exponential cultures grown with glycerol-3-phosphate
supplementation were filtered, washed and resuspended
in fresh media minus glycerol-3-phosphate. Cultures
were split and one-half of each culture was supple-
mented with glycerol-3-phosphate. Growth was continued
for 3 hours at which time one-milliliter samples were
removed from the cultures4and added to tubes
containing 10 pCi of [1- C] acetate (61 mCi/mmole)
and various concentrations of ethanol. After ten
minutes the reaction was stopped by the addition of
chloroform-methanol (1:2). Lipids were extracted and
analyzed by argentation chromatography as described in
the "Experimental Procedure" section. A, Free fatty
acid synthesis in strain LW3 starved for glycerol-3-
phosphate. B, Incorporation of fatty acids into phos-
pholipids of strain LW3 starved for glycerol-3-
phosphate ( ) and supplemented with glycerol-3-
phosphate ( 0 ). C, Free fatty acid synthesis in
strain LWl starved for glycerol-3-phosphate. D,
Incorporation of fatty acids into phospholipids of
strain LWl starved for glycerol-3-phosphate ( 0 )
and supplemented with glycerol-3-phosphate ( O ).














S55 A 75 C
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n- 45-
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0 I 2 3 0 1 2
ETHANOL (%) ETHANOL (%)


55 B 70
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45- 60
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35 50



(n 25 40- o


ETHANOL (%)


I 2
ETHANOL (%)











phosphate starved cells contained reduced levels of saturated fatty

acids as a result of ethanol addition. Further, the dose-related

reductions of saturated fatty acids incorporated into phospholipids

are very similar to the dose-dependent inhibition of de novo

saturated fatty acid biosynthesis. This suggests that the ethanol-

induced changes in the fatty acid composition of newly synthesized

phospholipids arise prior to phospholipid synthesis and that ethanol

may have little effect upon the selectivity of fatty acid utilization

for phospholipids. Our results with strains LW1 and LW3 demonstrate

that ethanol acts directly at the level of fatty acid synthesis,

preferentially inhibiting the synthesis of saturated fatty acids.

Effect of Ethanol on the Fatty Acid Composition of an Unsaturated

Fatty Acid Auxotroph. Our results thus far demonstrate that the

addition of ethanol causes a reduction in the proportion of saturated-

fatty acids available for incorporation into phospholipids. This

reduction could reflect an increased availability of unsaturated fatty

acids for phospholipid synthesis resulting from a stimulation of

unsaturated fatty acid synthesis by ethanol. To test this, we used

an unsaturated fatty acid auxotroph, strain K1060, which derives all

of its esterified unsaturated fatty acids from exogenous supplements

(Overath et al., 1970).

Strain K1060 was grown in the presence and absence of ethanol with

several unsaturated fatty acid supplements (Table 5). When

supplemented with either palmitoleic acid (16:1) or oleic acid (cis-

A 9-18:1), the addition of ethanol resulted in an increase in the









25












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proportion of esterified unsaturated fatty acids. Further, this

ethanol-induced change was not dependent upon specific unsaturated

acyl chains. E. coli does not normally synthesize di-unsaturated

fatty acids or fatty acids of chain lengths greater than 18 carbons.

However, strain K1060 supplemented with eicosadienoic acid (20:2) still

underwent significant changes in the proportion of unsaturated fatty

acids in response to ethanol (Table 5). These results demonstrate

that ethanol does not act by stimulating unsaturated fatty acid

synthesis, and that the regulation of unsaturated fatty acid synthesis

is not essential for the ethanol response.

Effect of Ethanol on the Incorporation of Exogenous Fatty Acids

into Phospholipids in the Absence of de novo Fatty Acid Synthesis. We

have shown that ethanol increases the UFA/SFA ratio produced during

de novo fatty acid synthesis by preferentially inhibiting the

synthesis of saturated fatty acids. The dose-dependent shift in this

ratio is almost identical to that observed following incorporation into

phospholipids, suggesting that the acyltransferase system may not be

affected directly by ethanol (Figure 2). This differs from temperature

adaptation of fatty acid composition, which has been shown by other

workers to be regulated by both fatty acid synthesis (Cronan, 1975;

Okuyama et al., 1977) and the selectivity of the acyltransferase

system (Esfahani et al., 1969; Sinensky, 1971). However, it is

possible that in vivo the selectivity of the acyltransferase enzymes

is only a secondary means of regulating fatty acid composition. Cells

which can regulate their fatty acid composition at the level of fatty

acid synthesis may not require or exhibit additional changes in











selectivity of incorporation of fatty acids into phospholipids

(Okuyama et al., 1977). Indeed, the similarity between synthesized

fatty acids and phospholipid composition in E. coli in response to

growth temperature (Cronan, 1975) would seem to support this theory.

Thus the effect of ethanol on fatty acid synthesis may obscure any

effect of the alcohol on the acyltransferase enzymes. To test this

possibility, the effect of ethanol on the incorporation of exogenous

fatty acids into phospholipids in vivo was examined under conditions

which minimize de novo fatty acid synthesis. Strains L8 and L48

synthesize fatty acids at one-fourth the normal rate when shifted

to 370C, and therefore require supplementation with both saturated and

unsaturated fatty acids for phospholipid synthesis (Harder et al.,

1972).

The results in Table 6 show that ethanol caused a slight inhibi-

tion of the incorporation of exogenous 16:0 and cis-A -18:1 fatty

acids into phospholipids, but did not alter the ratio of their

incorporation. These results demonstrate that ethanol has little

direct effect upon the selectivity of the acyltransferase enzymes.

Effect of Exogenous Fatty Acids on Ethanol-Induced Changes in

Fatty Acid Composition. Our results have shown that ethanol

preferentially inhibits the synthesis of saturated fatty acids. The

ethanol-induced reduction in the proportion of saturated fatty acids

in cellular phospholipids could simply result from a reduced avail-

ability. To examine this possibility, we have determined the fatty

acid composition of phospholipids from strain TB4 grown with a series

of concentrations of 16:0 in the presence and absence of ethanol.
















Table 6. Effect of ethanol on the incorporation of fatty
acids into phospholipids


Fatty Acids Incorporatedb


No EtOH 3% EtOH

Strain Total Saturated Unsaturated Total Saturated Unsaturated


L8 298 216 82 266 193 73


L48 283 204 79 225 161 64




Cells were grown wAth palmitic acid and oleic acid supplements at 370C,
to a density of 10 cells/ml. The cells were harvested by centrifugation
and resuspended in one-half volume of fresh media. Samples (1 ml) were
removed agd added to tubes containing 1 ml of media, 50 Pl of
[9,10(n)- H] oleic acid (2.2 Ci/mmole) and 1.3 pCi of [1- CJ palmitic
acid (56 mCi/mmole) in the presence and absence of ethanol. Phospho-
lipids were separated by thin layer chromatography and analyzed by
liquid scintillation counting.


bpicames f fatty acid/sampe.
picamoles of fatty acid/sample.











Supplementation with exogenous palmitic acid increased the

proportion of esterified 16:0 with decreases in both cis-All-18:1 and

16:1 fatty acids (Table 7). A similar pleiotropic effect of 16:0

was previously reported by Silbert et al. (1973a). The increase in

abundance of esterified 16:0 was nearly constant at all levels of

supplementation. Further, supplementation with palmitic acid did not

prevent the ethanol-induced decrease in esterified 16:0. This decrease

in esterified palmitic acid was compensated for primarily by an increase

in 16:1 fatty acid rather than cis-All-18:1, in contrast to the previous

experiments without supplements.

The results in Table 7 would seem to suggest that the ethanol-

induced reduction in esterified 16:0 is not due solely to a

limitation of 16:0 available to the cell. However, it is not clear to

what extent the activated form of 16:0 is available to the acyltrans-

ferase system. Thus the apparent failure of excess 16:0 to eliminate

the ethanol-induced reduction in esterified palmitic acid could result

from a limited ability to transport or activate exogenous 16:0, consis-

tent with the lack of a dose-dependent response to 16:0 supplement.

Alternatively, ethanol could directly inhibit the transport or

activation of exogenously supplied 16:0. Either of these coupled

with a high affinity of the acyltransferase enzymes for 16:0 would

lead to changes in fatty acid composition which still predominantly

reflect the biosynthetic production of 16:0. To investigate these

possibilities, we have examined the effect of ethanol on the contri-

bution of de novo biosynthesis and of exogenously supplied 16:0 to

























































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esterified lipids in both pulse-label experiments and also in bulk

lipid composition.

Double label experiments were performed with strain TB4 using
14
[14C] acetate to label esterified palmitic acid derived from biosyn-

thesis and [3H] palmitic acid to label that derived from supplements

(Table 8). In strain TB4, the incorporation of exogenously supplied

16:0 [ H] was slightly stimulated by the addition of ethanol while

the incorporation from biosynthesis [ 14C] was strongly inhibited.

These results confirm that ethanol inhibits saturated fatty acid

synthesis and demonstrate that ethanol does not decrease the avail-

ability of exogenously supplied 16:0 to the acyltransferase enzymes.

The results of the pulse-label experiments were further

confirmed in a second organism, strain K1060, using exogenously

supplied 17:0 and cis-A -18:1 fatty acids. Heptadecanoic acid (17:0)

is used by E. coliasa 16:0 equivalent (Silbert et al., 1973b; Ingram

et al., 1977). In the absence of a direct effect of ethanol on the

utilization of exogenous fatty acids for esterification, the ratio

of esterified (14:0 + 16:0)/17:0 provides a measure of saturated fatty

acid biosynthesis. These fatty acids are easily resolved by gas

chromatography. As observed in pulse-label experiments, the

utilization of saturated fatty acids derived from biosynthesis

(16:0 + 14:0) and of that derived from exogenous supplements (17:0) did

not follow the same trends in response to ethanol addition (Table 9).

The addition of ethanol resulted in a dramatic increase in the

proportion of exogenously supplied saturated fatty acid (17:0)
















Table 8. Effect of ethanol on the incorporation
of exogenous 16:0 [3H] and endogenous
16:0 [14C] into the phospholipids of
strain TB4a


Palmitic Acid Incorporated into Phospholipids (cpm)

Conditions Endogenous [14 C] Exogenous [3H] Ratio



No EtOH 2,500 18,200 7.3

3% EtOH 310 20,800 67.1



aSamples from an exponential culture were pulsed for 10 minutes with
10 pCi of [l-14C] acetate (61 mCi/mmole) and 10 uCi/ml of 19,10(n)- H]
palmitic acid (442.5 mCi/mmole). Lipids were extracted and the phos-
pholipids separated using thin layer chromatography.


Ratio of the I H] cpm to [14 C] cpm.
















Table 9. Effect of ethanol on the incorporation of 17:0
into the phospholipids of an unsaturated fatty
acid auxotroph, strain K1060a


Fatty Acid Composition (%)

Conditions 14:0 16:0 17:0 cis-9-18:l(A19) Ratio (14:0+16:0)/17:0



No Ethanol 7.0 40.7 25.4 26.9 1.9

3% Ethanol 3.4 25.8 41.2 29.6 0.7



aCells were grown with both 17:0 and 18:1 fatty acids in the presence
and absence of ethanol. Phospholipids were purified by thin layer
chromatography and analyzed by gas chromatography. Results are expressed
as a percentage of total fatty acids.











and a simultaneous decrease in the proportion of saturated fatty

acids derived from biosynthesis (16:0 + 14:0). Further, the ethanol-

induced increase in the proportion of unsaturated fatty acid (18:1)

was also reduced in strain K1060 as compared to strain TB4 grown with

a saturated fatty acid supplement (Table 7). This may be due to the

more efficient transport or utilization of exogenous fatty acids by

strain K1060. Spontaneous secondary mutations appear to be selected

for in fatty acid auxotrophs (Fox et al., 1970). Thus the exogenous

supply of saturated fatty acids to this mutant partially compensates

for the direct inhibition of saturated fatty acid synthesis by ethanol.

These results provide further evidence that the ethanol-induced

changes in fatty acid composition do not result from a direct effect

of ethanol on the selectivity of the acyltransferase system in vivo.

Effect of Ethanol on Fatty Acid Synthesis in Vitro. If ethanol

is acting at the level of fatty acid synthesis in E. coli, it should

be possible to demonstrate an effect on the fatty acids synthesized

in vitro. A crude preparation of fatty acid synthesizing enzymes was

prepared from E. coli, and the fatty acids synthesized in vitro were

separated by argentation chromatography. The effect of ethanol on

fatty acid synthesis in vitro is demonstrated in Table 10.

Ethanol preferentially inhibited saturated fatty acid synthesis

in vitro in a manner similar to the preferential inhibition observed

in vivo. The addition of divalent cations (5 mM) could not prevent

this inhibition (Table 10), suggesting that ethanol does not disrupt

an essential ion-protein interaction. Therefore, to determine the
















Table 10. Effect of ethanol on the fatty acids synthesized
in vitro by a crude fatty acid synthetasea




Fatty Acids Synthesized (cpm)

Experiment SFAb UFA PFA UFA/SFA




1 (Na+K+)
No EtOH 266,389 351,847 49,406 1.32

3% EtOH 182,426 396,106 58,682 2.17


2 (+Mg)
No EtOH 313,563 434,349 51,194 1.38

3% EtOH 181,812 424,006 57,729 2.33


3 (+Ca+ )
No EtOH 194,379 299,044 60,363 1.54

3% EtOH 85,067 254,799 61,393 2.99



Fatty acids were synthesized in vitro by a crude enzyme preparation.
Incubation was carried out at 370C for 45 minutes in the presence
and absence of ethanol. Fatty acids were transesterified and separated
by argentation chromatography.

SFA, saturated fatty acids; UFA, unsaturated fatty acids; PFA,
polar fatty acids.











mechanism by which ethanol affects fatty acid synthesis, the crude

enzymes were taken through two additional steps of purification.

Crude enzymes were centrifuged at 100,000 x g for two hours to

remove most of the membrane fragments, and the resulting enzyme is

referred to as the 100 Kg enzyme preparation. An aliquot of the 100

Kg preparation was further purified by ammonium sulfate fractionation

as described by Lennarz et al. (1962), and this enzyme represents the

(NH )2SO4 preparation. The three different preparations were then

assayed in the presence and absence of ethanol, and the products were

analyzed by argentation chromatography.

The results in Table 11 demonstrate that all three enzyme

preparations were markedly affected by the presence of ethanol. In

each case, the addition of ethanol resulted in a preferential inhibi-

tion of saturated fatty acid synthesis, resulting in an increased UFA/

SFA ratio. Thus ethanol seems to be exerting its effect directly at

the level of the fatty acid synthesizing enzymes.

It seemed possible that ethanol could be affecting saturated fatty

acid synthesis in a manner which would lead to the arrest of further

saturated fatty acid synthesis without affecting unsaturated fatty

acid synthesis. To test this, ammonium sulfate enzyme preparations

were incubated for increasing periods of time in the presence and

absence of ethanol. The synthesized fatty acids were separated by

argentation chromatography and the amounts of saturated and unsaturated

fatty acids determined.

Figure 3 demonstrates the effects of ethanol on fatty acid

synthesis in vitro. Both saturated fatty acid synthesis as well as
















Table 11. Effect of purification of fatty acid synthetase
on ethanol-induced fatty acid changes in vitro




Fatty Acids Synthesized (cpm)

Enzyme Conditions SFAb UFA PFA UFA/SFA



Crude -EtOH 42,463 106,240 17,651 2.50

3% EtOH 24,983 84,586 17,247 3.39


100 Kg -EtOH 69,341 112,312 13,477 1.62

3% EtOH 48,904 124,379 19,061 2.54


(NH4)2SO4 -EtOH 76,396 108,604 11,391 1.42

3% EtOH 51,779 116,187 12,449 2.24




aFatty Acids were synthesized in vitro by enzymes of different levels of
purification. Incubation was carried out at 370C for 45 minutes in the
presence and absence of ethanol. Fatty acids were transesterified and
separated by argentation chromatography.

SFA, saturated fatty acids; UFA, unsaturated fatty acids; PFA, polar
fatty acids.











unsaturated fatty acid synthesis were inhibited by ethanol, but the

inhibition of saturated fatty acid synthesis was greater. In the

presence of 4% ethanol, the rate of unsaturated fatty acid synthesis

was decreased by 30%, while the rate of saturated fatty acid synthesis

was inhibited by 48%. In a duplicate experiment, unsaturated fatty

acid synthesis was not inhibited, while saturated fatty acid synthesis

was inhibited by 30%. Thus saturated fatty acid synthesis is not

totally arrested, but rather it occurs at a slower rate than unsaturated

fatty acid synthesis.

In the presence of ethanol, short-chained saturated fatty acids

(decanoic and dodecanoic) may be synthesized, but their subsequent

elongation to myristic or palmitic acids could be inhibited. To

examine this possibility, fatty acids were synthesized in vitro in

the presence and absence of 4% ethanol. The products were separated

into saturated, unsaturated and polar fatty acids by argentation

chromatography (Figure 4). Regions corresponding to these fatty acids

were eluted with pentane and rechromatographed. Saturated and unsat-

urated fatty acids were separated on the basis of chain length by

reversed phase chromatography; polar fatty acids were rechromatographed

on argentation thin layer plates. The results of this experiment are

shown in Table 12.

In agreement with a previous report (Lennarz et al., 1962), the

fatty acids synthesized in vitro were found to be of greater chain

lengths than fatty acids synthesized in vivo. This is thought to

result from an absence of phospholipid synthesis, which would normally

utilize the palmitic, palmitoleic and vaccenic acids before they can

































Figure 3. Effect of ethanol on the rates of synthesis of
saturated and unsaturated fatty acids in vitro.
Fatty acid synthetase enzymes purified by ammonium
sulfate fractionation were added to the standard
reaction mixture. The assay mixtures were incubated
at 37 C for various periods of time in the presence
and absence of 4% ethanol. Labeled fatty acids were
extracted and analyzed by argentation chromatography.
Symbols: ( A ), unsaturated fatty acid synthesis
in the absence of ethanol; ( A ), unsaturated fatty
acid synthesis in the presence of ethanol; ( N ),
saturated fatty acid synthesis in the absence of
ethanol; ( O ), saturated fatty acid synthesis in
the presence of ethanol.



















O
0




0




LU












2O
C/)




z



I-





20
I-















0 10 20 30
TIME (min)



































Figure 4. Argentation chromatography of fatty acid products
synthesized in vitro. Fatty acid synthesis
enzymes, purified by ammonium sulfate fractionation,
were used to synthesize fatty acids in vitro. Assay
mixtures were incubated for 30 minutes in the presence
and absence of 4% ethanol. Labeled fatty acids were
extracted and analyzed by argentation chromatography.
Regions corresponding to the radioactive fatty acids
were located by autoradiography. Designated regions
represent: SFA, Saturated fatty acids; UFA,
Unsaturated fatty acids; PFA, Polar fatty acids.












No EtOH 4% EtOH






SFA











cis-A 5-22:1


cis- 13-20:1 UFA

cis-A118:1

cis-A -16:1













A

B
PFA











be further elongated (Cronan et al., 1975). In the absence of

ethanol, large amounts of stearic, arachidic and eicosenoic fatty

acids were synthesized due to the continued elongation of palmitic

and vaccenic acids. In the presence of 4% ethanol there is a shift to

shorter fatty acids, as demonstrated by the marked reduction observed

in arachidic and eicosenoic acids. However, the high levels of

palmitic and stearic acids synthesized as well as the absence of any

shorter chain intermediates (lauric or myristic acids) demonstrate

that ethanol is not preventing the complete elongation of saturated

fatty acids. Since ethanol also causes a reduction in the chain

lengths of the unsaturated fatty acids observed in vitro (Table 12),

the observed reduction in chain lengths may result from an interaction

of ethanol with the condensing enzymes responsible for elongating

the fatty acid intermediates.

Despite the separation of the polar fatty acids (8-hydroxy inter-

mediates) into three distinct regions by argentation chromatography,

we were unable to identify these different polar acids. Although

there was an increase in fraction C in the presence of ethanol (Table

12), we were not able to demonstrate an accumulation of any specific

intermediate. Thus the inhibition by ethanol of a specific enzyme

cannot be demonstrated by the accumulation of a specific enzyme's

precursor. These results suggest that the ethanol-induced reduction

in saturated fatty acid synthesis is due to a greater inhibition of

the rate of saturated fatty acid synthesis as compared to the rate

of unsaturated fatty acid synthesis.




















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The fatty acid changes induced by alcohols are similar to the

fatty acid changes induced by temperature shifts (Marr and Ingraham,

1962). Recent evidence by Cronan (1975) suggests that in vivo,

fatty acid synthesis is one site of thermal control of phospholipid

fatty acid composition. Therefore, it seemed appropriate to investi-

gate the effects of incubation temperature on fatty acid synthesis

in vitro. Enzymes purified by ammonium sulfate fractionation were

used to synthesize fatty acids at three incubation temperatures, and

the results of this experiment are given in Table 13.

Increasing incubation temperature resulted in a corresponding

increase in the relative amounts of saturated fatty acids synthesized

in vitro. These results are in agreement with those reported by

Okuyama et al. (1977), and provide evidence that temperature

adaptation of fatty acid composition does not depend solely upon

interactions with the cell membrane per se. The effect of temperature

is also exerted directly upon the fatty acid synthetase enzymes. These

results with temperature suggest that the fatty acid changes induced

by temperature shifts and the fatty acid changes induced by ethanol

may be mediated through a common mechanism.
















Table 13. Effect of incubation temperature on the
fatty acid products synthesized in vitr
fatty acid products synthesized in vitro


SFAb UFA PFA

Temperature cpm (%)c cpm (%) cpm (%) UFA/SFA


32 C 14,917 (19.3) 52,137 (67.5) 10,246 (13.2) 3.50


37C 15,229 (24.3) 38,881 (62.2) 8,443 (13.5) 2.55


420C 30,668 (36.6) 43,939 (52.4) 9,199 (11.0) 1.43


aFatty acid synthetase enzymes were purified by ammonium sulfate
fractionation. Enzymes were added to assay mixtures and incubated
at either 320C, 37C or 420C for 30 minutes. Fatty acids were
extracted and separated by argentation chromatography.

SFA, saturated fatty acids; UFA, unsaturated fatty acids; PFA, polar
fatty acids.

CExpressed as percent of total fatty acids synthesized at the indicated
temperature.
















DISCUSSION


It has previously been shown that the addition of ethanol to

E. coli caused a reduction in the proportion of palmitic acid (16:0)

esterified into membrane lipids (Ingram, 1976). This decrease in

palmitic acid was subsequently found to occur in both phosphatidyl

glycerol and phosphatidyl ethanolamine (Ingram, 1977a), the major

phospholipids present in E. coli (Cronan and Vagelos, 1972). The

experiments described in this dissertation represent an extension of

these previous reports, and were designed to determine the mechanisms

by which ethanol induced fatty acid changes. Fatty acid degradation,

fatty acid synthesis, and fatty acid utilization (incorporation into

phospholipids) were considered as possible sites for the interaction

with ethanol. The results presented in this dissertation provide

evidence that these changes in fatty acid composition result primarily

from the preferential inhibition of saturated fatty acid synthesis by

ethanol.

Several observations support this conclusion: (1) Mutants

deficient in fatty acid degradation showed an ethanol-induced change

in their fatty acid composition identical to that observed in wild-

type cells (Table 4). Thus the addition of ethanol does not lead

to reduced incorporation of palmitic acid into phospholipids as

a result of preferential degradation of the saturated fatty acids.

(2) The immediacy of the ethanol-induced changes in the fatty acid











composition of newly synthesized phospholipids, as well as their

equally rapid reversal upon ethanol removal, suggests that ethanol is

acting directly at the level of lipid synthesis (Table 2). (3) In an

unsaturated fatty acid auxotroph, the ratio of unsaturated to saturated

fatty acids incorporated into phospholipids was elevated in response

to ethanol. This elevation occurred regardless of the unsaturated

fatty acid supplement provided (Table 5). Thus the increase in the

proportion of unsaturated fatty acids did not result from a stimulation

of their synthesis by ethanol. (4) When fatty acid synthesis was

uncoupled from phospholipid synthesis, ethanol caused a dose-related

reduction in the proportion of saturated fatty acids synthesized

(Figures 2A and C). This indicates that ethanol is acting directly

at the level of fatty acid synthesis. In addition, the similarity

between the ratios of free fatty acids and esterified fatty acids

suggested that the effect of ethanol on fatty acid synthesis is

responsible for most, if not all, of the observed reduction in

esterified palmitic acid. (5) In the absence of endogenous fatty

acid synthesis, ethanol did not alter the ratios of fatty acids

incorporated into phospholipids (Table 6). Unlike temperature

adaptation, adaptation to ethanol is not mediated by altering the

selectivity of the acyltransferase enzymes. These in vivo

experiments demonstrate that the endogenous synthesis of saturated

fatty acids is essential for ethanol-induced fatty acid changes to

occur. The ability to eliminate several lipid synthesizing and

degradative processes without eradicating the ethanol-response is

in agreement with our conclusion that ethanol preferentially inhibits

saturated fatty acid synthesis.











This conclusion is further supported by the results of

our experiments with ethanol and the fatty acid synthetase enzymes

in vitro. In crude, cell-free extracts, the addition of ethanol

resulted in a preferential inhibition of saturated fatty acid

synthesis in a fashion analogous to the effects in vivo. This effect

could not be eliminated by the addition of excess magnesium and

calcium to the assay mixture (Table 10). The crude enzymes were

taken through two additional steps of purification to try and

determine the level of the ethanol interaction. The removal of most

of the membrane lipids by ultracentrifugation did not prevent the

ethanol-induced inhibition of saturated fatty acid synthesis

(Table 11). Thus the effects of ethanol in the cell membrane, per se,

are not involved in the ethanol-induced changes in fatty acid

composition. However, since some proteins are surrounded by a lipid

annulus that cannot be removed by simple ultracentrifugation (Fourcans

and Jain, 1974), the possibility of an ethanol effect at the level

of a lipid-protein association cannot be excluded.

The membrane-free preparation was further purified by ammonium

sulfate fractionation to remove excess protein. The addition of

ethanol to a reaction mixture catalyzed by this preparation also

resulted in the preferential inhibition of saturated fatty acid

synthesis (Table 11). Based upon this experiment, we conclude that

ethanol is acting directly at the level of the fatty acid synthetase

enzymes.

The specific enzyme(s) affected by ethanol are not known. In

vitro experiments aimed at elucidating the specific enzyme(s)











demonstrated that ethanol does not totally arrest saturated fatty

acid synthesis, nor does ethanol prevent the synthesis of long chain

saturated fatty acids. Rather, ethanol seems to be inhibiting the

rate of saturated fatty acid synthesis to a greater degree than it

inhibits the rate of unsaturated fatty acid synthesis, leading to

the observed increase in the UFA/SFA ratio. This effect could be

produced by an interaction of ethanol with at least two enzymes:

B-hydroydecanoyl dehydrase or B-ketoacyl-acyl ACP synthetase.

B-Hydroydecanoyl dehydrase catalyzes a double-bond formation in

the ten carbon intermediate in fatty acid synthesis in E. coli. This

enzyme is capable of forming an a,B trans double bond, a B,Y cis

double bond, or an isomerization between the two forms (Kass et al.,

1967). If a trans double bond is produced, the a,B trans decenoyl

ACP derivative will be reduced to decanoyl ACP followed by a

subsequent elongation to palmitic acid (16:0). However, when a cis

double bond is introduced it is not subsequently reduced, and the B,Y

cis decenoyl ACP is elongated to form palmitoleoyl ACP (16:1) and cis

vaccenoyl ACP (18:1). Therefore, if ethanol altered the relative

rates at which the enzyme introduced cis or trans double bonds, the

final proportions of fatty acids synthesized could be modified to

include less saturated fatty acids. Unfortunately, a reduction in

trans double bond formation might be expected to yield an increase in

cis bond formation, and hence an increase in unsaturated fatty acids.

Since the addition of ethanol also inhibited total fatty acid synthesis,

it could not be determined if such an increase in unsaturated fatty

acid synthesis was actually occurring.










An attractive alternative for the site of ethanol interaction is

the condensing enzyme responsible for saturated fatty acid synthesis

in E. coli. The presence of two condensing enzymes have recently been

demonstrated in E. coli, -ketoacyl-acyl ACP synthetase I, and B-

ketoacyl-acyl ACP synthetase II (D'Agnolo et al., 1975), and it is

believed that a third condensing enzyme may also exist (John E.

Cronan, Jr.; personal communication). 8-Ketoacyl-acyl ACP synthetase I

is primarily involved in unsaturated fatty acid synthesis, and the

absence of this enzyme has been demonstrated in fabB unsaturated fatty

acid auxotrophs (D'Agnolo et al., 1975). The type II synthetase is

absent from cvc fatty acid mutants (no cis-vaccenic acid) and is

proposed to be involved in elongating palmitoleoyl ACP to cis-

vaccenoyl ACP (D'Agnolo et al., 1975). However, both of these enzymes

display a broad preference for substrates, suggesting that either

type can also participate in saturated fatty synthesis.

Since strain K1060 (a fabB mutant deficient in B-ketoacyl-acyl

ACP synthetase I) underwent an ethanol-induced fatty acid change, it

is unlikely that ethanol is interacting with this condensing enzyme.

This leaves either the type II condensing enzyme or the putative

type IIIcondensing enzyme. In view of their broad substrate

specificities, it is likely that both (or all three) condensing

enzymes participate in forming the short-chained intermediates

prior to the ten carbon intermediate, and the enzymes may be specific

for saturated or unsaturated fatty acid synthesis only after the

formation of the B-hydroxydecanoyl ACP fatty acid. Thus an

inhibition of either the type II condensing enzyme or the type III











(if it exists) by ethanol would result in the reduced rate of total

fatty acid synthesis observed in vivo as well as in vitro.

Since the condensation reaction is the rate limiting reaction in fatty

acid synthesis (Bloch and Vance, 1977), an inhibition of this reaction

by ethanol would be expected to result in a corresponding decrease in

the rate of overall fatty acid synthesis. Further, since the type I

synthesis would already be expected to be working to its capacity, the

inhibition of saturated fatty acid synthesis would probably not lead

to an increase in the levels of unsaturated fatty acids. Therefore,

the condensing enzyme responsible for saturated fatty acid synthesis

in E. coli represents a logical site for the specific interaction of

ethanol.

It is interesting that both ethanol and temperature were able to

exert a direct effect on fatty acid synthesis in vitro. Our results

clearly indicate that neither ethanol nor temperature depends upon

the membrane, per se, to induce changes in de novo fatty acid synthesis.

However, it is very possible that both effects are mediated through

a lipid-protein interaction. Fatty acid synthesis is believed to occur

in close proximity with the plasma membrane (van den Bosch, et al.,

1970), which could facilitate an association between membrane lipids

and one or more of the fatty acid synthetase enzymes. Alternatively,

the lipid-protein site may actually reside at the level of an enzyme-

substrate interaction. This could explain our observation that

ethanol induced the synthesis of shorter-chained fatty acids in vitro

(Table 12). Both of the enzymes described above as potential sites for

the ethanol interaction (B-hydroxydecanoyl ACP dehydrase and 0-











ketoacyl-acyl ACP synthetase II) have also been proposed to control

temperature adaptation of the fatty acid composition in E. coli

(Brock et al., 1967; Gelmann and Cronan, 1972). Thus the similarity

in fatty acid changes induced by ethanol and temperature may actually

result from their interaction with a common enzyme in fatty acid

synthesis.
















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BIOGRAPHICAL SKETCH


Thomas Martin Buttke was born on December 5, 1952, in Bay Shore,

New York. The author graduated from Bay Shore High School in 1970,

and received the Bachelor of Arts degree from the University of North

Carolina at Wilmington in 1974. In September of 1974, he entered the

Department of Microbiology at the University of Florida as the

recipient of a Graduate Council Fellowship, and received the Master

of Science degree in microbiology in December, 1975. From September,

1977 to June, 1978, he was the recipient of a fellowship from the

Center of Gerontological Studies, and he is presently a candidate for

the Doctor of Philosophy degree in the Department of Microbiology and

Cell Science. The author is married to the former Mary Elizabeth

Thompson.










I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.




Lonnie 0. Ingram, Chairman
Associate Professor of Microbiology
and Cell Science


I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.




Charles M. Allen, Jr.
Associate Professor of Biochemistry


I certify that I have read this study and that in my opinion
it conforms to acceptable standards of scholarly presentation and
is fully adequate, in scope and quality, as a dissertati n for the
degree of Doctor of Philosophy.




/aes F. Preston III
Associate Professor of Microbiology
and Cell Science


This dissertation was submitted to the Graduate Faculty of
the Department of Microbiology and Cell Science in the College of
Arts and Sciences and to the Graduate Council, and was accepted
as partial fulfillment of the requirements for the degree of
Doctor of Philosophy.


June, 1978

Dean, Graduate School




Full Text

PAGE 1

MECHANISM OF ETHANOL-INDUCED CHANGES IN LIPID COMPOSITION OF ESCHERICHIA COLI : PREFERENTIAL INHIBITION OF SATURATED FATTY ACID SYNTHESIS By THOMAS MARTIN BUTTKE A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1978

PAGE 2

ACKNOWLEDGEMENTS Le ciiemin tet long da pJiojeJ: a la clo^z. Moliere The author would like to thank Dr. L.O. Ingram for his help and encouragement during the author's graduate career. Dr. Ingram's knowledge as well as his friendship has been invaluable and his willingness to share both is greatly appreciated. Thanks are also due to Drs. J.F. Preston and CM. Allen for donating their time and suggestions during the course of this research. The author is especially grateful to his wife, Mary, for providing companionship during late night experiments, and for typing this dissertation. Her encouragement and good humor have contributed immeasurably towards the completion of this research. Part of this work was performed while the author was the recipient of a fellowship from the Center for Gerontological Studies, and this support is gratefully acknowledged.

PAGE 3

TABLE OF CONTENTS Page ACKNOWLEDGMENTS ii LIST OF TABLES iv LIST OF FIGURES v ABBREVIATIONS USED vi ABSTRACT vii INTRODUCTION 1 EXPERIMENTAL PROCEDURE 4 RESULTS 13 DISCUSSION 47 REFERENCES 54 BIOGRAPHICAL SKETCH 57 iii

PAGE 4

LIST OF TABLES Table Page 1 Bacterial strains 5 Effect of ethanol on the fatty acid composition of newly synthesized phospholipids in strain TB4 14 3 Effect of incubation temperature on ethanolinduced changes in the fatty acid composition of newly synthesized phospholipids 17 4 Effect of fatty acid degradation on ethanolinduced changes in fatty acid composition 20 5 Effect of ethanol on the fatty acid composition of an unsaturated fatty acid auxotroph, strain K1060 25 6 Effect of ethanol on the incorporation of fatty acids into phospholipids 28 7 Effect of exogenous 16:0 on ethanol-induced changes in fatty acid composition in strain TB4 30 8 Effect of ethanol on the incorporation of exogenous 16:0 [ H] and endogenous 16:0 [ 14 C] into the phospholipids of strain TB4 32 9 Effect of ethanol on the incorporation of 17:0 into the phospholipids of an unsaturated fatty acid auxotroph, strain K1060 33 10 Effect of ethanol on the fatty acids synthesized in vitro by a crude fatty acid synthetase 35 11 Effect of purification of fatty acid synthetase on ethanol-induced fatty acid changes in vitro 37 12 Effect of ethanol on the chain lengths of the fatty acid products synthesized ±n vitro 44 13 Effect of incubation temperature on the fatty acid products synthesized in vitro 46

PAGE 5

LIST OF FIGURES Figure Page 1 Biosynthetic pathway for fatty acids in E. coli showing the location of the defects in fatty" acid synthesis mutants 7 2 Effect of ethanol on free fatty acid synthesis and total lipid synthesis in strains LW1 and LW3 23 3 Effect of ethanol on the rates of synthesis of saturated and unsaturated fatty acids in vitro 40 4 Argentation chromatography of fatty acid products synthesized ^in vitro 42

PAGE 6

ABBREVIATIONS USED UFA Unsaturated fatty acid SFA Saturated fatty acid PFA Polar fatty acid ACP Acyl carrier protein 16:0 Palmitic acid 16:1 Palmitoleic acid 17:0 Heptadecanoic acid 9 cisA -18:1 Oleic acid cisA -18:1 cis-Vaccenic acid A17 c_is-9,10-Methylene hexadecanoic acid 20:2 Eicosadienoic acid A19 cis11,12 Methylene octadecanoic acid MOPS Morpholinopropane sulfonic acid

PAGE 7

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MECHANISM OF ETHANOLINDUCED CHANGES IN LIPID COMPOSITION OF ESCHERICHIA COLI : PREFERENTIAL INHIBITION OF SATURATED FATTY ACID SYNTHESIS By Thomas Martin Buttke June 1978 Chairman: Lonnie 0. Ingram Major Department: Microbiology and Cell Science Experiments were performed to determine the mechanisms by which ethanol induces fatty acid changes in Escherichia coli . Mutants deficient in various aspects of lipid metabolism were used to determine the mechanisms of ethanol interaction in vivo . Under conditions which uncoupled fatty acid synthesis from phospholipid synthesis, ethanol decreased the amount of saturated fatty acids synthesized, but had little effect on their incorporation into phospholipids. In the absence of fatty acid degradation and unsaturated fatty acid synthesis, E. coli still demonstrated ethanol-induced fatty acid changes, while the inhibition of total fatty acid synthesis eliminated this response. Experiments with the fatty acid synthetase enzymes in vitro supported the in vivo results. Fatty acid synthetase preparations from three levels of purification demonstrated a preferential inhibition of saturated fatty acid synthesis by ethanol, similar to that observed

PAGE 8

in vivo. The fatty acid changes in vitro did not result from a total arrest of saturated fatty acid synthesis, nor were they due to a premature termination of saturated fatty acid elongation. These results indicate that ethanol-induced fatty acid changes in E. coli result from a preferential inhibition of the rate of saturated fatty acid synthesis.

PAGE 9

INTRODUCTION In a previous report Ingram demonstrated that Escherichia coli will alter its membrane fatty acid composition when grown in the presence of straight chain alcohols (Ingram, 1976). These alcoholinduced fatty acid changes are similar to those induced by changes in growth temperature (Marr and Ingraham, 1962). Short chain alcohols such as ethanol cause a decrease in the amounts of saturated fatty acids, similar to a shift down in growth temperature. Long chain alcohols such as hexanol induce changes analogous to a shift up in temperature, an increase in the proportion of saturated fatty acids (Ingram, 1976). Like temperature adaptation, these changes in fatty acid composition are independent of major changes in phospholipid composition (Ingram, 1977a). These results with alcohols have been extended to show that a wide variety of other lipophylic agents (organic solvents and food preservatives) also induce fatty acid changes in _E. coli (Ingram, 1976) . The diverse structures of these compounds as well as their lipophylic nature support the theory that these changes in fatty acid composition result from a general interaction with a hydrophobic site, rather than a result of specific catabolic processes (Ingram, 1977b). The intercalation of these agents into the membrane would be expected to alter membrane fluidity. The resulting changes in fatty acid composition suggest that the cell may be able to detect changes in membrane fluidity, per se, and compensate for the direct actions of drugs.

PAGE 10

In view of the importance of ethanol, ethanol-tolerance and withdrawal syndrome in society, the ability of ethanol to induce dramatic changes in the fatty acid composition is of particular interest. A membrane whose lipid composition has been altered in response to ethanol could be considered as being adapted to the presence of the alcohol (tolerance) , much as membrane lipid changes allow a membrane to adapt to growth temperature (Shaw and Ingraham, 1965) . The removal of ethanol would cause additional changes in membrane organization which would be compensated for by a return to the original composition (withdrawal). Thus, an understanding of the mechanisms responsible for ethanol-induced changes in fatty acid composition in E. coli could provide a model for the development of experiments in mammalian systems. The similarity of the changes in fatty acid composition induced by ethanol and temperature adaptation suggests that these processes may share a common mechanism. This regulation of fatty acid composition could be carried out at any of three levels: (1) fatty acid degradation; (2) fatty acid synthesis; and (3) phospholipid synthesis (Cronan and Vagelos, 1972). E. coli mutants blocked in one or more of these processes have proven useful in studying the control of thermal regulation of membrane lipid composition (Cronan and Gelmann, 1975) . These same mutants were used in this study to elucidate the mechanisms by which ethanol induces membrane fatty acid changes in E. coli . The results of these experiments indicate that the ethanol-induced changes in fatty acid composition ±n vivo result primarily from the inhibition of saturated fatty acid synthesis.

PAGE 11

Experiments were also conducted with the fatty acid synthesis enzymes ^n vitro to determine if the relative amounts of fatty acids synthesized ^ vitro were also affected by ethanol. In a fashion analogous to that observed in vivo , ethanol inhibited total fatty acid synthesis in vitro but with a preferentially greater inhibition of saturated fatty acid synthesis. Taken collectively, the results of both the jin vivo as well as the jin vitro experiments suggest that ethanol-induced fatty acid changes in IS. coli result from a decreased availability of saturated fatty acids for phospholipid synthesis. Further, the decreased availability of saturated fatty acids reflects a preferential inhibition of saturated fatty acid synthesis by ethanol.

PAGE 12

EXPERIMENTAL PROCEDURE Bacterial Strains . The E. coli K12 derivatives used in this study are listed in Table 1. Figure 1 illustrates the biosynthetic pathway for fatty acids in E. coli , showing the location of the defects in fatty acid synthesis. Strain TB4 is derived from strain CSH2 and is defective in fatty acid degradation ( fadE~ ) . Strain CSH2 was made proB by mutagenesis with ultraviolet light followed by penicillin selection (Miller, 1972). A proB fadE lysate was made from strain L43 by the method of Marsh and Duggan (1972) using the Plvir (Cold Spring Harbor Laboratory) , and this was used to introduce the fadE gene into strain CSH2. Potential fadE transductants were selected by their ability to grow without proline supplementation. Growth Conditions . Unless otherwise indicated, all bacterial strains were grown in a gyrotory shaker at 30 C. The standard medium used was E medium (Vogel and Bonner, 1956). Supplements differed for each strain. Strains CSH2, TB4 and L43 were grown with sodium succinate (0.2%), thiamine (0.001%), tryptophan (0.004%), and yeast extract (0.0005%). Strains LW1 and LW3 were supplemented with sodium succinate (0.2%), thiamine (0.001%) , casein hydrolysate (vitamin free) (0.05%), and sn-glycerol-3-phosphate (0.01%). Glycerol-3-phosphate deprivation was performed by filtration as described by Cronan e_t al . (1975). For strains L8 and L48, the media was supplemented with glycerol (1.0%), thiamine (0.001%), monosodium glutamate (0.1%),

PAGE 13

PL, 4_l

PAGE 14

Figure 1. Biosynthetic pathway for fatty acids in Escherichia coli showing the location of the defects in the fatty acid synthesis mutants.

PAGE 15

fabB ? CHjCO-S-CoA ACP > CHyCO-S-CoA A HCO Vc oA CHjC OS-AC P I HOOC-CHjCOACP. HOOO-CHrCO-S-ACP NADP CHjCHj-CHj-CO-S-ACP NADPH, * H CHwCsC-CO-S-ACP 3 H H CHjC-CHjCO-S-ACP fabE labD CHjOCHj-CO-S-ACP NADP FH CH5(CH 2 )§CHjOCHjCOS-ACP H HpO JL HgO . H H C Hj(C H 2 )gC=C-C HjC O-S-AC P • H H CHjfcH^C^OfcH^COSACP $H3(CH 2 )g C=0(CH 2 )gCO-S-ACP CHjtCHjjJgOCCO-S-ACP H NADP^J i^NADP CH 3 (C H 2 )gCH^CH 2 C O-S-ACP I CH^(CH 2 )^CO-S-ACP

PAGE 16

yeast extract (0.0001%), Brij 58 (0.4%), potassium palmitate (0.001%) and potassium oleate (0.001%). Cell growth was monitored by measuring the absorbance at 550 nm. using a Beckman model 25 spectrophotometer. Cultures were routinely used in the experiments o at a cell density of 2x10 cells/ml (0.36 absorbance units at 550 nm.) to insure that cells were in log phase. Cells grown for ^n vitro fatty acid synthesis studies were grown into late log phase (0.70 absorbance units at 550 nm.) in Medium 63 mineral salts (Miller, 1972) supplemented with glucose (0.2%), thiamine (0.001%), tryptophan (0.02%) and casamino acids (vitamin free; 0.1%). These cells were also grown in a gyrotory shaker, except the incubation temperature was 37 C. Preparation of Fatty Acid Synthetase Enzymes . Cells grown into late log phase were harvested by centrifugation and washed once with 10 mM phosphate buffer pH 7.0. The cells were disrupted by passage through a French pressure cell (12,500 psi) and centrifuged at 5,000 rpm for ten minutes to remove unbroken cells. The supernate contained the fatty acid synthesis enzymes (Lennarz e_t al . , 1962) and is called the "crude" enzyme preparation. A membrane free enzyme preparation was obtained by centrifuging the "crude" enzyme preparation at 100,000 x g for two hours to pellet the membranous material. The supernate which contained the fatty acid synthesis enzymes after this step was referred to as the "100 Kg" preparation. This preparation was further purified by ammonium sulfate fractionation as described by Lennarz et^ a_l. (1962) . Ammonium sulfate was added to the "100 Kg" preparation to 55% saturation and the precipitated protein was removed by centrifuging at 10,000 rpm for ten minutes. The

PAGE 17

supernate was brought to 75% saturation with additional ammonium sulfate and the precipitated fatty acid synthetase enzymes were harvested by centrifugation. The resultant pellet was resuspended in 10 mM phosphate buffer, pH 7.0, containing 1 mM dithiothreitol, and frozen at -20 C in 0.2 ml aliquots. In Vitro Fatty Acid Synthesis Assay . The conditions for assaying fatty acid synthesis ^n vitro were a modification of the procedure of Silbert (1976). Standard incubation mixtures contained MOPS buffer, pH 7.1 at 37 C (50 umoles) , dithiothreitol (1 pmole) , NADH (100 nmoles) , NADP (100 nmoles) , glucose-6-phosphate (1 pmole) , glucose-6-phosphate dehydrogenase (0.65 units), acetyl CoA (15 nmoles), 14 acyl carrier protein (25 yg) , and [2C] malonyl CoA (30 nmoles; 3.8 pCi/pmole) in a final volume of 0.48 ml. The reaction was started by the addition of 100 ug of enzyme protein, and the reaction mixtures were incubated at 37 C in screw-cap tubes sealed with Teflon-lined caps. The reaction was quenched by the addition of 1 ml of 15% K0H in methanol, and the samples were heated for two hours at 70 C. The samples were acidified with 0.6 ml of 6N HC1, carrier fatty acids were added, and the mixture was extracted two times with pentane. Combined pentane extracts were washed twice with water, added to preweighed screw-cap tubes, and the pentane was evaporated under nitrogen at 45 C until one gram of pentane remained. Aliquots of 100 pi were removed for measuring total lipid synthesis, and the remainder was taken to dryness prior to transesterif ication. The labeled methyl esters were separated into saturated, unsaturated and polar fatty acids by argentation chromatography.

PAGE 18

10 Analysis of Fatty Acids by Gas Chromatography . Cells were harvested by centrifugation, inactivated by the addition of 5% trichloroacetic acid, and extracted into chloroform-methanol as described by Kanfer and Kennedy (1973) . For the separation of neutral lipids (includes free fatty acids) from phospholipids, washed lipid extracts were chromatographed on unactivated silica gel G impregnated glass-fiber paper (Gelmann ITLC-SG, Fisher Scientific Co., Pittsburg, Pa.) using solvent 1 as described by Freeman and West (1966). Phospholipids were eluted with chloroform-methanol (2:1), and transesterif ied using 2% H.SO, in methanol, as described by Silbert et al . (1973a) . The methyl esters were extracted into pentane and concentrated under N„ prior to analysis. The conditions for gas chromatography have been described (Ingram, 1976). Analysis of Radioactive Lipids . The incorporation of labeled acetate into fatty acids was assayed essentially by the procedure of Cronan and Wulf f (1969) . One milliliter samples of cells were added 14 to tubes containing 10 uCi of [ C] acetate (61 mCi/mmole) and incubated at 37 C for ten minutes. The reaction was stopped by the addition of chloroform-methanol (1:2) and extracted overnight. The resulting lipid extract was separated into two phases by the addition of chloroform and water; radioactive lipids were present in the lower (chloroform) phase. An aliquot (0.2 ml) was removed from the chloroform phase and counted to determine total lipid synthesis. The remaining radioactive lipids were transesterif ied as described above, and the radioactive methyl esters were analyzed by argentation chromatography. When 14 the incorporation of [ C] acetate into free (non-esterif ied) fatty

PAGE 19

11 acids was measured, the lipid extract was separated into phospholipids and neutral lipids by thin layer chromatography using either the solvent system of Klein e± a_l. (1971) or the two step procedure described by Cronan e_t al . (1975) . Both the phospholipid and the neutral lipid fractions were eluted, transesterif ied and analyzed by argentation chromatography. Incorporation of exogenously supplied 14 3 3 [ C] or [ H] palmitic acid and [ H] oleic acids were performed in essentially the same fashion, except phospholipids were separated from neutral lipids by thin layer chromatography using the solvent diethyl ether-acetic acid (100:1). The phospholipid region was scraped into vials containing toluene scintillation solution (Omnifluor; New England Nuclear) and counted on a Beckman model 133 liquid scintillation counter. When the counter was adjusted for dual-label 3 14 counting, the efficiency was 58.2% for [ H] and 72.5% for [ C] . Argentation Chromatography . Thin layer chromatography plates coated with silica gel G were impregnated with 10% aqueous silver nitrate and activated as described by Cubero and Mangold (1965). The labeled methyl esters were applied to the argentation plate in pentane and developed twice in toluene at -17 C (Morris et al. , 1967). Radioactive regions were scraped into vials and counted. Reversed Phase Chromatography . The chain lengths of the saturated and unsaturated fatty acids synthesized Tn vitro were determined by reversed phase chromatography (Bergelson et^ al_. , 1964) . Regions of argentation plates corresponding to saturated and unsaturated fatty acids were scraped and the methyl esters were eluted with pentane. The methyl esters were applied in pentane to silica gel G plates impregnated

PAGE 20

12 with dodecane. Impregnation was achieved by ascending a 10% dodecane in light petroleum ether solution up a silica gel G plate and the petroleum ether evaporated. The solvent system used for development consisted of acetone-acetonitrile (1:1) saturated with dodecane (Bergelson et al. , 1964) . Labeled methyl esters were located by autoradiography using Kodak X-OMAT R film. Chemicals . Heptadecanoic acid was purchased from Applied Science Laboratories Inc. (State College, Pa.). All other unlabeled fatty acids were obtained from the Sigma Chemical Co. (St. Louis, Mo.). [1C] Acetate, [9,10 (n)Hj oleic acid, and [1C] palmitic acid 3 are products of Amersham Corp. (Arlington Heights, II.). I9,10(n)H] 14 Palmitic acid and [2C] malonyl CoA were purchased from New England Nuclear (Boston, Ma.). Unlabeled malonyl CoA, acetyl CoA and dodecane are the products of Sigma Chemical Co. Acyl carrier protein (ACP) was purified by the procedure of Majerus jrt al_. (1968) from one kilogram of E. coli K12 (Grain Processing Co., Muscatine, Iowa).

PAGE 21

RESULTS Effect of Ethanol on the Fatty Acid Composition of Newly Synthesized Phospholipids . Previous experiments have shown that growth in the presence of ethanol results in the synthesis of phospholipids rich in unsaturated fatty acids (Ingram, 1976). To determine if these changes represent a direct effect of ethanol on lipid synthesis (fatty acid synthesis and utilization) or a secondary effect mediated through such processes as enzyme induction or other alternatives in metabolism, we have examined the immediate effects of ethanol addition on newly synthesized lipids. Strain TB4 (fadE~) was pulse-labeled with [ C] acetate to measure the effects of various concentrations of ethanol on the incorporation of newly synthesized fatty acids into phospholipids. The addition of ethanol caused an immediate increase in the abundance of newly synthesized unsaturated fatty acids which were incorporated into phospholipids (Table 2). This resulted in a doserelated increase in the UFA/SFA ratios, and is analogous to the changes previously observed in the bulk lipid composition (Ingram, 1976) . High concentrations of ethanol (4% and 6%) strongly inhibited the synthesis and utilization of both unsaturated and saturated fatty acids, and this inhibition may be related to the "lipid poor" cells described earlier (Ingram, 1977). The ethanol-induced increase in the UFA/SFA ratio was immediately reversed by ethanol removal (washing), 13

PAGE 22

u Table 2. Effect of ethanol on the fatty acid composition of newly synthesized phospholipids in strain TB4 EtOH(%) 14 [ C] Acetate Incorporation (cpm) Total Saturated Unsaturated . c UFA/SFA Ratio 0°

PAGE 23

15 even after 16 hours of growth in the presence of 3% ethanol. The immediacy of action of ethanol on the UFA/SFA ratio of newly synthesized lipids coupled with its equally rapid reversibility provide evidence for the direct action of ethanol on lipid synthesis. Pulse-labeling experiments typically yielded UFA/SFA ratios which were higher than those of bulk lipids. This probably resulted from the incorporation of newly synthesized fatty acids into phosphatidyl glycerol. In E. coli this phospholipid is enriched in unsaturated fatty acids and turns over rapidly during normal growth. Thus, the pulse-labeling studies may reflect both the de novo synthesis as well as the turnover of phosphatidyl glycerol which would give rise to the increased UFA/SFA ratios observed. However, the UFA/SFA ratios following the initial addition of ethanol (Table 2) were much higher than expected based upon determinations of bulk lipid composition (Ingram, 1976). During growth in the presence of ethanol, the ratios observed in log phase cells pulsed with [ C] acetate approached that of bulk lipid, 7.1, 6.1, 5.8, and 4.4 after 0, 1, 2, and 16 hours, respectively. Thus the initial increase in the UFA/SFA ratio following the addition of ethanol represented an overcompensation in newly synthesized lipids which was subsequently decreased as the cells adjusted to the presence of ethanol. Effect of Incubation Temperature on Ethanol-Induced Changes in the Fatty Acid Composition of Newly Synthesized Lipids . E_. coli alters its membrane fatty acid composition in response to changes in growth temperature (Marr and Ingraham, 1962). A shift to a higher growth

PAGE 24

16 temperature leads to an increase in the proportion of palmitic acid while a shift to a lower growth temperature leads to an increase in the proportion of unsaturated fatty acids. These changes in lipid composition are regulated both at the level of fatty acid synthesis (Cronan, 1975; Okuyama et^ a_l . , 1977) and fatty acid utilization for phospholipid synthesis (Esfahani et al. , 1969; Sinensky, 1971). We have shown that the addition of ethanol causes changes in fatty acid composition similar to a reduction in growth temperature. To further explore this relationship, we have investigated the simultaneous effect of ethanol addition and a shift in incubation temperature on the fatty acid composition of newly synthesized lipids. Samples from an exponential culture of strain TB4 grown at 30 C were incubated at 23 C, 30 C and 37 C, and pulse-labeled with [ c] acetate in the presence and absence of 3% ethanol. Ester if ied fatty acids were separated by argentation chromatography, scraped and counted. Strain TB4 altered its fatty acid composition (UFA/SFA ratio) in response to temperature (Table 3) as previously reported (Marr and Ingraham, 1962) . This same trend was reflected among samples containing ethanol indicating that ethanol did not disrupt the normal thermoregulatory systems. The addition of ethanol resulted in an increase in the proportion of unsaturated fatty acids at all incubation temperatures. Thus the effects of ethanol and a shift down in temperature were roughly additive while the effects of ethanol and a shift up in temperature were opposing. The increases in the proportion of unsaturated fatty acids in response to ethanol addition and a shift to lower temperature were

PAGE 25

Table 3. Effect of incubation temperature on ethanolinduced changes in the fatty acid composition of newly synthesized phospholipids 17 Fatty Acids (cpm) Incubation Temperature Conditions Saturated 16:1 cis-A -18:1 UFA/SFA Ratio 23°C

PAGE 26

18 primarily the result of an absolute decrease in esterified saturated fatty acids. Unlike ethanol addition, a shift to lower temperature was also accompanied by an absolute increase in esterified unsaturated fatty acids. A shift to higher temperature caused an inhibition of acetate incorporation into both saturated and unsaturated fatty acids which was further inhibited by the inclusion of ethanol. The stimulation of lipid synthesis by a shift to lower temperature and its inhibition by a shift to higher temperature are difficult to explain. These changes may be related to changes in the levels of guanosine-5'-diphosphate-3' -diphosphate (ppGpp) . High levels of ppGpp inhibit both phospholipid synthesis (Merlie and Pizer, 1973) and fatty acid synthesis (Nunn and Cronan, 1976) . Recent studies by Gallant et_ al_. (1977) have shown that E. coli rapidly accumulates this nucleotide upon shifting to a higher temperature. Effect of Fatty Acid Degradation (fadE) on Ethanol-Induced Changes in Fatty Acid Composition . Since fatty acid synthesis is tightly coupled to phospholipid synthesis, the changes observed in UFA/SFA ratios could be due to the direct action of ethanol on the availability of fatty acids, or their utilization by the acyltransferase system, or both. E. coli possesses a multi-enzyme complex capable of fatty acid degradation (Binstock et al. , 1977). This enzyme complex may partially determine the fatty acid composition of membrane lipids by altering the relative amounts of fatty acid species available for incorporation into phospholipids. Thus, the ethanol-induced reduction in palmitic acid previously reported in E. coli (Ingram, 1976)

PAGE 27

19 could result from a preferential degradation of endogenously synthesized palmitic acid. To determine if this was indeed the case, the effects of ethanol on strain CSH2, proficient in fatty acid degradation ( fadE ), and strain TB4, defective in fatty acid degradation ( fadE ), were compared (Table 4). In both strains the proportion of palmitic acid (16:0) was reduced by 10%, with an equal increase in the proportion of cis-vaccenic acid during growth in the presence of ethanol. Neither strain showed a significant change in the level of palmitoleic acid (16:1). These results indicate that preferential fatty acid degradation is not responsible for ethanolinduced changes in fatty acid composition. Effect of Ethanol on Free Fatty Acid Synthesis . Although fatty acid degradation is not essential for ethanol-induced fatty acid changes (Table 4), the availability of fatty acids for phospholipid synthesis could also be modified by a direct effect of ethanol on fatty acid synthesis. In E. coli , fatty acid synthesis is normally tightly coupled with phospholipid synthesis (Cronan, 1974) and prevents an evaluation of the effect of ethanol on either of these systems independent of the other. In order to determine if ethanol directly affects fatty acid synthesis, we have used two mutants in which fatty acid synthesis can be uncoupled from phospholipid synthesis jin vivo by glycerol-3-phosphate starvation. The removal of exogenous glycerol-3phosphate from growing cells of strains LW1 and LW3 causes an inhibition of phospholipid synthesis and an accumulation of free (non-esterif ied) fatty acids (Cronan et_ al. , 1975) . These are easily labeled by pulsing

PAGE 28

20 1

PAGE 29

21 14 the cells with [ C] acetate during starvation. Subsequent analysis of the free fatty acids provides an in vivo method for examining the regulation of fatty acid synthesis, per se , independent of the acyltransf erase system. The addition of ethanol to strains LW1 and LW3 altered the proportions of saturated and unsaturated free fatty acids which accumulated (Figure 2A and C) . Although the synthesis of both saturated as well as unsaturated fatty acids was inhibited by ethanol 14 ([ C] acetate incorporation), 3% ethanol caused only a 23% decrease in the synthesis of unsaturated fatty acids while causing a 73% reduction in the synthesis of saturated fatty acids. Thus the ethanolinduced increase in the UFA/SFA ratio during the free fatty acid synthesis results from the preferential inhibition of saturated fatty acid synthesis. In order to determine whether ethanol affects the incorporation of fatty acids into newly synthesized phospholipids in strain LW1 and LW3, we also examined the fatty acid composition of the phospholipids. In the absence of exogenous glycerol-3-phosphate, a small amount of residual phospholipid synthesis continues in these strains (Cronan et al . , 1975) . The ratio of unsaturated to saturated fatty acids either in these residual phospholipids or in the phospholipids of cells supplemented with glycerol-3-phosphate can be compared with the ratios in the accumulated free fatty acids to determine the effect of ethanol on the selectivity of the phospholipid synthesizing enzymes. Figure 2 (B and D) demonstrates that phospholipids extracted from glycerol-3-phosphate supplemented cells as well as from glycerol-3-

PAGE 30

Figure 2. Effect of ethanol on free fatty acid synthesis and total lipid synthesis in strains LW1 and LW3. Exponential cultures grown with glycerol-3-phosphate supplementation were filtered, washed and resuspended in fresh media minus glycerol-3-phosphate. Cultures were split and one-half of each culture was supplemented with glycerol-3-phosphate. Growth was continued for 3 hours at which time one-milliliter samples were removed from the cultures and added to tubes containing 10 uCi of [1C] acetate (61 mCi/mmole) and various concentrations of ethanol. After ten minutes the reaction was stopped by the addition of chloroform-methanol (1:2). Lipids were extracted and analyzed by argentation chromatography as described in the "Experimental Procedure" section. A, Free fatty acid synthesis in strain LW3 starved for glycerol-3phosphate. B, Incorporation of fatty acids into phospholipids of strain LW3 starved for glycerol-3phosphate ( # ) and supplemented with glycerol-3phosphate ( O )• C, Free fatty acid synthesis in strain LW1 starved for glycerol-3-phosphate. D, Incorporation of fatty acids into phospholipids of strain LW1 starved for glycerol-3-phosphate ( • ) and supplemented with glycerol-3-phosphate ( O ) •

PAGE 31

23 I 2 ETHANOL (%) ETHANOL (%) ETHANOL (%) I 2 ETHANOL (%)

PAGE 32

24 phosphate starved cells contained reduced levels of saturated fatty acids as a result of ethanol addition. Further, the dose-related reductions of saturated fatty acids incorporated into phospholipids are very similar to the dose-dependent inhibition of de novo saturated fatty acid biosynthesis. This suggests that the ethanolinduced changes in the fatty acid composition of newly synthesized phospholipids arise prior to phospholipid synthesis and that ethanol may have little effect upon the selectivity of fatty acid utilization for phospholipids. Our results with strains LW1 and LW3 demonstrate that ethanol acts directly at the level of fatty acid synthesis, preferentially inhibiting the synthesis of saturated fatty acids. Effect of Ethanol on the Fatty Acid Composition of an Unsaturated Fatty Acid Auxotroph . Our results thus far demonstrate that the addition of ethanol causes a reduction in the proportion of saturated fatty acids available for incorporation into phospholipids. This reduction could reflect an increased availability of unsaturated fatty acids for phospholipid synthesis resulting from a stimulation of unsaturated fatty acid synthesis by ethanol. To test this, we used an unsaturated fatty acid auxotroph, strain K1060, which derives all of its esterified unsaturated fatty acids from exogenous supplements (Overath et al . , 1970). Strain K1060 was grown in the presence and absence of ethanol with several unsaturated fatty acid supplements (Table 5) . When supplemented with either palmitoleic acid (16:1) or oleic acid ( cisA -18:1), the addition of ethanol resulted in an increase in the

PAGE 33

25 C o O r-j 6 w o "+-| c cO xi u

PAGE 34

26 proportion of esterified unsaturated fatty acids. Further, this ethanol-induced change was not dependent upon specific unsaturated acyl chains. E. coli does not normally synthesize di-unsaturated fatty acids or fatty acids of chain lengths greater than 18 carbons. However, strain K1060 supplemented with eicosadienoic acid (20:2) still underwent significant changes in the proportion of unsaturated fatty acids in response to ethanol (Table 5) . These results demonstrate that ethanol does not act by stimulating unsaturated fatty acid synthesis, and that the regulation of unsaturated fatty acid synthesis is not essential for the ethanol response. Effect of Ethanol on the Incorporation of Exogenous Fatty Acids into Phospholipids in the Absence of de novo Fatty Acid Synthesis . We have shown that ethanol increases the UFA/SFA ratio produced during de novo fatty acid synthesis by preferentially inhibiting the synthesis of saturated fatty acids. The dose-dependent shift in this ratio is almost identical to that observed following incorporation into phospholipids, suggesting that the acyltransf erase system may not be affected directly by ethanol (Figure 2) . This differs from temperature adaptation of fatty acid composition, which has been shown by other workers to be regulated by both fatty acid synthesis (Cronan, 1975; Okuyama et_ al_. , 1977) and the selectivity of the acyltransf erase system (Esfahani et^ al. , 1969; Sinensky, 1971). However, it is possible that in vivo the selectivity of the acyltransferase enzymes is only a secondary means of regulating fatty acid composition. Cells which can regulate their fatty acid composition at the level of fatty acid synthesis may not require or exhibit additional changes in

PAGE 35

27 selectivity of incorporation of fatty acids into phospholipids (Okuyama et_ al . , 1977). Indeed, the similarity between synthesized fatty acids and phospholipid composition in E. coli in response to growth temperature (Cronan, 1975) would seem to support this theory. Thus the effect of ethanol on fatty acid synthesis may obscure any effect of the alcohol on the acyltransf erase enzymes. To test this possibility, the effect of ethanol on the incorporation of exogenous fatty acids into phospholipids jLn vivo was examined under conditions which minimize de novo fatty acid synthesis. Strains L8 and L48 synthesize fatty acids at one-fourth the normal rate when shifted to 37 C, and therefore require supplementation with both saturated and unsaturated fatty acids for phospholipid synthesis (Harder et^ al. , 1972). The results in Table 6 show that ethanol caused a slight inhibi9 tion of the incorporation of exogenous 16:0 and cis-A -18:1 fatty acids into phospholipids, but did not alter the ratio of their incorporation. These results demonstrate that ethanol has little direct effect upon the selectivity of the acyltransf erase enzymes. Effect of Exogenous Fatty Acids on Ethanol-Induced Changes in Fatty Acid Composition . Our results have shown that ethanol preferentially inhibits the synthesis of saturated fatty acids. The ethanol-induced reduction in the proportion of saturated fatty acids in cellular phospholipids could simply result from a reduced availability. To examine this possibility, we have determined the fatty acid composition of phospholipids from strain TBA grown with a series of concentrations of 16:0 in the presence and absence of ethanol.

PAGE 36

28 Table 6. Effect of ethanol on the incorporation of fatty acids into phospholipids Fatty Acids Incorporated No EtOH 3% EtOH Strain Total Saturated Unsaturated Total Saturated Unsaturated L8 298 216 82 266 193 73 L48 283 204 79 225 161 64 Cells were grown with palmitic acid and oleic acid supplements at 37 C, to a density of 10 cells/ml. The cells were harvested by centrifugation and resuspended in one-half volume of fresh media. Samples (1 ml) were removed and added to tubes containing 1 ml of media, 50 uCi of I9,10(n)H] oleic acid (2.2 Ci/mmole) and 1.3 yCi of [1C] palmitic acid (56 mCi/mmole) in the presence and absence of ethanol. Phospholipids were separated by thin layer chromatography and analyzed by liquid scintillation counting. picamoles of fatty acid/sample.

PAGE 37

29 Supplementation with exogenous palmitic acid increased the proportion of ester if ied 16:0 with decreases in both cis-A -18:1 and 16:1 fatty acids (Table 7). A similar pleiotropic effect of 16:0 was previously reported by Silbert et^ al. (1973a) . The increase in abundance of esterified 16:0 was nearly constant at all levels of supplementation. Further, supplementation with palmitic acid did not prevent the ethanol-induced decrease in esterified 16:0. This decrease in esterified palmitic acid was compensated for primarily by an increase in 16:1 fatty acid rather than cis-A -18:1, in contrast to the previous experiments without supplements. The results in Table 7 would seem to suggest that the ethanolinduced reduction in esterified 16:0 is not due solely to a limitation of 16:0 available to the cell. However, it is not clear to what extent the activated form of 16:0 is available to the acyltransf erase system. Thus the apparent failure of excess 16:0 to eliminate the ethanol-induced reduction in esterified palmitic acid could result from a limited ability to transport or activate exogenous 16:0, consistent with the lack of a dose-dependent response to 16:0 supplement. Alternatively, ethanol could directly inhibit the transport or activation of exogenously supplied 16:0. Either of these coupled with a high affinity of the acyltransf erase enzymes for 16:0 would lead to changes in fatty acid composition which still predominantly reflect the biosynthetic production of 16:0. To investigate these possibilities, we have examined the effect of ethanol on the contribution of de novo biosynthesis and of exogenously supplied 16:0 to

PAGE 38

30 Cl)

PAGE 39

31 esterified lipids in both pulse-label experiments and also in bulk lipid composition. Double label experiments were performed with strain TB4 using 14 [ C] acetate to label esterified palmitic acid derived from biosyn3 thesis and [ H] palmitic acid to label that derived from supplements (Table 8). In strain TB4, the incorporation of exogenously supplied 16:0 [ H] was slightly stimulated by the addition of ethanol while 14 the incorporation from biosynthesis [ C] was strongly inhibited. These results confirm that ethanol inhibits saturated fatty acid synthesis and demonstrate that ethanol does not decrease the availability of exogenously supplied 16:0 to the acyltransf erase enzymes. The results of the pulse-label experiments were further confirmed in a second organism, strain K1060, using exogenously 9 supplied 17:0 and cisA -18:1 fatty acids. Heptadecanoic acid (17:0) is used by E. coli as a 16:0 equivalent (Silbert et al., 1973b; Ingram et al. , 1977). In the absence of a direct effect of ethanol on the utilization of exogenous fatty acids for esterif ication, the ratio of esterified (14:0 + 16:0)/17:0 provides a measure of saturated fatty acid biosynthesis. These fatty acids are easily resolved by gas chromatography. As observed in pulse-label experiments, the utilization of saturated fatty acids derived from biosynthesis (16:0 + 14:0) and of that derived from exogenous supplements (17:0) did not follow the same trends in response to ethanol addition (Table 9). The addition of ethanol resulted in a dramatic increase in the proportion of exogenously supplied saturated fatty acid (17:0)

PAGE 40

32 Table 8. Effect of ethanol on the incorporation of exogenous 16:0 [^H] and endogenous 16:0 [1 4 C] into the phospholipids of strain TB4 a Palmitic Acid Incorporated into Phospholipids (cpm) Conditions Endogenous [ C] Exogenous [ H] Ratio No EtOH 2,500 18,200 7.3 3% EtOH 310 20,800 67.1 Samples from an exponential culture were pulsed for 10 minutes with 10 uCi of [114 C] acetate (61 mCi/mmole) and 10 yCi/ml of [9,10(n)H] palmitic acid (442.5 mCi/mmole) . Lipids were extracted and the phospholipids separated using thin layer chromatography. Ratio of the [ H] cpm to [ C] cpm.

PAGE 41

33 Table 9. Effect of ethanol on the incorporation of 17:0 into the phospholipids of an unsaturated fatty acid auxotroph, strain K1060 a Fatty Acid Composition (%) Conditions 14:0 16:0 17:0 cis-A 9 -18:l(A19) Ratio (14:0+16:0)/17 :0 No Ethanol 3% Ethanol 7.0 40.7 25.4 3.4 25.8 41.2 26.9 29.6 1.9 0.7 Cells were grown with both 17:0 and 18:1 fatty acids in the presence and absence of ethanol. Phospholipids were purified by thin layer chromatography and analyzed by gas chromatography. Results are expressed as a percentage of total fatty acids.

PAGE 42

34 and a simultaneous decrease in the proportion of saturated fatty acids derived from biosynthesis (16:0 + 14:0). Further, the ethanolinduced increase in the proportion of unsaturated fatty acid (18:1) was also reduced in strain K1060 as compared to strain TB4 grown with a saturated fatty acid supplement (Table 7) . This may be due to the more efficient transport or utilization of exogenous fatty acids by strain K1060. Spontaneous secondary mutations appear to be selected for in fatty acid auxotrophs (Fox e_t al_. , 1970) . Thus the exogenous supply of saturated fatty acids to this mutant partially compensates for the direct inhibition of saturated fatty acid synthesis by ethanol, These results provide further evidence that the ethanol-induced changes in fatty acid composition do not result from a direct effect of ethanol on the selectivity of the acyltransf erase system ^n vivo . Effect of Ethanol on Fatty Acid Synthesis in Vitro . If ethanol is acting at the level of fatty acid synthesis in E. coli , it should be possible to demonstrate an effect on the fatty acids synthesized in vitro . A crude preparation of fatty acid synthesizing enzymes was prepared from _E. coli , and the fatty acids synthesized In vitro were separated by argentation chromatography. The effect of ethanol on fatty acid synthesis in vitro is demonstrated in Table 10. Ethanol preferentially inhibited saturated fatty acid synthesis in vitro in a manner similar to the preferential inhibition observed in vivo . The addition of divalent cations (5 mM) could not prevent this inhibition (Table 10) , suggesting that ethanol does not disrupt an essential ion-protein interaction. Therefore, to determine the

PAGE 43

35 Table 10. Effect of ethanol on the fatty acids synthesized in vitro by a crude fatty acid synthetase

PAGE 44

36 mechanism by which ethanol affects fatty acid synthesis, the crude enzymes were taken through two additional steps of purification. Crude enzymes were centrifuged at 100,000 x g for two hours to remove most of the membrane fragments, and the resulting enzyme is referred to as the 100 Kg enzyme preparation. An aliquot of the 100 Kg preparation was further purified by ammonium sulfate fractionation as described by Lennarz et^ al. (1962) , and this enzyme represents the (NH,)„S0, preparation. The three different preparations were then assayed in the presence and absence of ethanol, and the products were analyzed by argentation chromatography. The results in Table 11 demonstrate that all three enzyme preparations were markedly affected by the presence of ethanol. In each case, the addition of ethanol resulted in a preferential inhibition of saturated fatty acid synthesis, resulting in an increased UFA/ SFA ratio. Thus ethanol seems to be exerting its effect directly at the level of the fatty acid synthesizing enzymes. It seemed possible that ethanol could be affecting saturated fatty acid synthesis in a manner which would lead to the arrest of further saturated fatty acid synthesis without affecting unsaturated fatty acid synthesis. To test this, ammonium sulfate enzyme preparations were incubated for increasing periods of time in the presence and absence of ethanol. The synthesized fatty acids were separated by argentation chromatography and the amounts of saturated and unsaturated fatty acids determined. Figure 3 demonstrates the effects of ethanol on fatty acid synthesis in vitro . Both saturated fatty acid synthesis as well as

PAGE 45

37 Table 11. Effect of purification of fatty acid synthetase on ethanol-induced fatty acid changes in vitro

PAGE 46

38 unsaturated fatty acid synthesis were inhibited by ethanol, but the inhibition of saturated fatty acid synthesis was greater. In the presence of 4% ethanol, the rate of unsaturated fatty acid synthesis was decreased by 30%, while the rate of saturated fatty acid synthesis was inhibited by 48%. In a duplicate experiment, unsaturated fatty acid synthesis was not inhibited, while saturated fatty acid synthesis was inhibited by 30%. Thus saturated fatty acid synthesis is not totally arrested, but rather it occurs at a slower rate than unsaturated fatty acid synthesis. In the presence of ethanol, short-chained saturated fatty acids (decanoic and dodecanoic) may be synthesized, but their subsequent elongation to myristic or palmitic acids could be inhibited. To examine this possibility, fatty acids were synthesized In vitro in the presence and absence of 4% ethanol. The products were separated into saturated, unsaturated and polar fatty acids by argentation chromatography (Figure 4) . Regions corresponding to these fatty acids were eluted with pentane and rechromatographed. Saturated and unsaturated fatty acids were separated on the basis of chain length by reversed phase chromatography; polar fatty acids were rechromatographed on argentation thin layer plates. The results of this experiment are shown in Table 12. In agreement with a previous report (Lennarz et al., 1962), the fatty acids synthesized In vitro were found to be of greater chain lengths than fatty acids synthesized in vivo. This is thought to result from an absence of phospholipid synthesis, which would normally utilize the palmitic, palmitoleic and vaccenic acids before they can

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Figure 3. Effect of ethanol on the rates of synthesis of saturated and unsaturated fatty acids jin vitro . Fatty acid synthetase enzymes purified by ammonium sulfate fractionation were added to the standard reaction mixture. The assay mixtures were incubated o at 37 C for various periods of time in the presence and absence of 4% ethanol. Labeled fatty acids were extracted and analyzed by argentation chromatography. Symbols: ( ), unsaturated fatty acid synthesis in the absence of ethanol; (A ), unsaturated fatty acid synthesis in the presence of ethanol; ( ), saturated fatty acid synthesis in the absence of ethanol; ( D ), saturated fatty acid synthesis in the presence of ethanol.

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40 10 20 TIME (min) 30

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Figure 4. Argentation chromatography of fatty acid products synthesized jln vitro . Fatty acid synthesis enzymes, purified by ammonium sulfate fractionation, were used to synthesize fatty acids iji vitro . Assay mixtures were incubated for 30 minutes in the presence and absence of 4% ethanol. Labeled fatty acids were extracted and analyzed by argentation chromatography. Regions corresponding to the radioactive fatty acids were located by autoradiography. Designated regions represent: SFA, Saturated fatty acids; UFA, Unsaturated fatty acids; PFA, Polar fatty acids.

PAGE 50

42 No EtOH 4% EtOH SFA cis-A 15 -22:l ,13 cisA -20:1 UFA cis-A 1:L -18:l cis-A -16:1 PFA

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43 be further elongated (Cronan et al . , 1975). In the absence of ethanol, large amounts of stearic, arachldic and eicosenoic fatty acids were synthesized due to the continued elongation of palmitic and vaccenic acids. In the presence of 4% ethanol there is a shift to shorter fatty acids, as demonstrated by the marked reduction observed in arachidic and eicosenoic acids. However, the high levels of palmitic and stearic acids synthesized as well as the absence of any shorter chain intermediates (lauric or myristic acids) demonstrate that ethanol is not preventing the complete elongation of saturated fatty acids. Since ethanol also causes a reduction in the chain lengths of the unsaturated fatty acids observed in vitro (Table 12) , the observed reduction in chain lengths may result from an interaction of ethanol with the condensing enzymes responsible for elongating the fatty acid intermediates. Despite the separation of the polar fatty acids (8-hydroxy intermediates) into three distinct regions by argentation chromatography, we were unable to identify these different polar acids. Although there was an increase in fraction C in the presence of ethanol (Table 12) , we were not able to demonstrate an accumulation of any specific intermediate. Thus the inhibition by ethanol of a specific enzyme cannot be demonstrated by the accumulation of a specific enzyme's precursor. These results suggest that the ethanol-induced reduction in saturated fatty acid synthesis is due to a greater inhibition of the rate of saturated fatty acid synthesis as compared to the rate of unsaturated fatty acid synthesis.

PAGE 52

44 c 0) a xi ,C 4-1 QJ P, h» i^vo m vD vo \D vD t-H vD m r-^ o 00 M * H H CTi r-~ r-^ .-i o\ en VO
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45 The fatty acid changes induced by alcohols are similar to the fatty acid changes induced by temperature shifts (Marr and Ingraham, 1962). Recent evidence by Cronan (1975) suggests that in vivo t fatty acid synthesis is one site of thermal control of phospholipid fatty acid composition. Therefore, it seemed appropriate to investigate the effects of incubation temperature on fatty acid synthesis ^n vitro . Enzymes purified by ammonium sulfate fractionation were used to synthesize fatty acids at three incubation temperatures, and the results of this experiment are given in Table 13. Increasing incubation temperature resulted in a corresponding increase in the relative amounts of saturated fatty acids synthesized in vitro . These results are in agreement with those reported by Okuyama et al. (1977) , and provide evidence that temperature adaptation of fatty acid composition does not depend solely upon interactions with the cell membrane per se. The effect of temperature is also exerted directly upon the fatty acid synthetase enzymes. These results with temperature suggest that the fatty acid changes induced by temperature shifts and the fatty acid changes induced by ethanol may be mediated through a common mechanism.

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46 Table 13. Effect of incubation temperature on the fatty acid products synthesized in vitro SFA b UFA PFA Temperature cpm (%) cpm (%) cpm (%) UFA/SFA 32°C 14,917 (19.3) 52,137 (67.5) 10,246 (13.2) 3.50 37 C 15,229 (24.3) 38,881 (62.2) 8,443 (13.5) 2.55 42°C 30,668 (36.6) 43,939 (52.4) 9,199 (11.0) 1.43 Fatty acid synthetase enzymes were purified by ammonium sulfate fractionation. Enzymes were added to assay mixtures and incubated at either 32 C, 37 C or 42 C for 30 minutes. Fatty acids were extracted and separated by argentation chromatography. SFA, saturated fatty acids; UFA, unsaturated fatty acids; PFA, polar fatty acids. c Expressed as percent of total fatty acids synthesized at the indicated temperature.

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DISCUSSION It has previously been shown that the addition of ethanol to E. coli caused a reduction in the proportion of palmitic acid (16:0) esterif ied into membrane lipids (Ingram, 1976) . This decrease in palmitic acid was subsequently found to occur in both phosphatidyl glycerol and phosphatidyl ethanolamine (Ingram, 1977a), the major phospholipids present in E. coli (Cronan and Vagelos, 1972). The experiments described in this dissertation represent an extension of these previous reports, and were designed to determine the mechanisms by which ethanol induced fatty acid changes. Fatty acid degradation, fatty acid synthesis, and fatty acid utilization (incorporation into phospholipids) were considered as possible sites for the interaction with ethanol. The results presented in this dissertation provide evidence that these changes in fatty acid composition result primarily from the preferential inhibition of saturated fatty acid synthesis by ethanol. Several observations support this conclusion: (1) Mutants deficient in fatty acid degradation showed an ethanol-induced change in their fatty acid composition identical to that observed in wildtype cells (Table 4). Thus the addition of ethanol does not lead to reduced incorporation of palmitic acid into phospholipids as a result of preferential degradation of the saturated fatty acids. (2) The immediacy of the ethanol-induced changes in the fatty acid hi

PAGE 56

48 composition of newly synthesized phospholipids, as well as their equally rapid reversal upon ethanol removal, suggests that ethanol is acting directly at the level of lipid synthesis (Table 2) . (3) In an unsaturated fatty acid auxotroph, the ratio of unsaturated to saturated fatty acids incorporated into phospholipids was elevated in response to ethanol. This elevation occurred regardless of the unsaturated fatty acid supplement provided (Table 5) . Thus the increase in the proportion of unsaturated fatty acids did not result from a stimulation of their synthesis by ethanol. (4) When fatty acid synthesis was uncoupled from phospholipid synthesis, ethanol caused a dose-related reduction in the proportion of saturated fatty acids synthesized (Figures 2A and C) . This indicates that ethanol is acting directly at the level of fatty acid synthesis. In addition, the similarity between the ratios of free fatty acids and esterified fatty acids suggested that the effect of ethanol on fatty acid synthesis is responsible for most, if not all, of the observed reduction in esterified palmitic acid. (5) In the absence of endogenous fatty acid synthesis, ethanol did not alter the ratios of fatty acids incorporated into phospholipids (Table 6) . Unlike temperature adaptation, adaptation to ethanol is not mediated by altering the selectivity of the acyltransf erase enzymes. These in vivo experiments demonstrate that the endogenous synthesis of saturated fatty acids is essential for ethanol-induced fatty acid changes to occur. The ability to eliminate several lipid synthesizing and degradative processes without eradicating the ethanol-response is in agreement with our conclusion that ethanol preferentially inhibits saturated fatty acid synthesis.

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49 This conclusion is further supported by the results of our experiments with ethanol and the fatty acid synthetase enzymes in vitro . In crude, cell-free extracts, the addition of ethanol resulted in a preferential inhibition of saturated fatty acid synthesis in a fashion analogous to the effects j_n vivo . This effect could not be eliminated by the addition of excess magnesium and calcium to the assay mixture (Table 10) . The crude enzymes were taken through two additional steps of purification to try and determine the level of the ethanol interaction. The removal of most of the membrane lipids by ultracentrifugation did not prevent the ethanol-induced inhibition of saturated fatty acid synthesis (Table. 11). Thus the effects of ethanol in the cell membrane, per se , are not involved in the ethanol-induced changes in fatty acid composition. However, since some proteins are surrounded by a lipid annulus that cannot be removed by simple ultracentrifugation (Fourcans and Jain, 1974) , the possibility of an ethanol effect at the level of a lipid-protein association cannot be excluded. The membrane-free preparation was further purified by ammonium sulfate fractionation to remove excess protein. The addition of ethanol to a reaction mixture catalyzed by this preparation also resulted in the preferential inhibition of saturated fatty acid synthesis (Table 11). Based upon this experiement, we conclude that ethanol is acting directly at the level of the fatty acid synthetase enzymes , The specific enzyme(s) affected by ethanol are not known. In vitro experiments aimed at elucidating the specific enzyme(s)

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50 demonstrated that ethanol does not totally arrest saturated fatty acid synthesis, nor does ethanol prevent the synthesis of long chain saturated fatty acids. Rather, ethanol seems to be inhibiting the rate of saturated fatty acid synthesis to a greater degree than it inhibits the rate of unsaturated fatty acid synthesis, leading to the observed increase in the UFA/SFA ratio. This effect could be produced by an interaction of ethanol with at least two enzymes: B-hydroydecanoyl dehydrase or 8-ketoacyl-acyl ACP synthetase. B-Hydroydecanoyl dehydrase catalyzes a double-bond formation in the ten carbon Intermediate in fatty acid synthesis in E. coli . This enzyme is capable of forming an 0,6 trans double bond, a £,Y cis double bond, or an isomerization between the two forms (Kass et al. , 1967). If a trans double bond is produced, the a, (3 trans decenoyl ACP derivative will be reduced to decanoyl ACP followed by a subsequent elongation to palmitic acid (16:0). However, when a cis double bond is introduced it is not subsequently reduced, and the (B,Y cis decenoyl ACP is elongated to form palmitoleoyl ACP (16:1) and cis vaccenoyl ACP (18:1). Therefore, if ethanol altered the relative rates at which the enzyme introduced cis or trans double bonds, the final proportions of fatty acids synthesized could be modified to include less saturated fatty acids. Unfortunately, a reduction in trans double bond formation might be expected to yield an increase in cis bond formation, and hence an increase in unsaturated fatty acids. Since the addition of ethanol also inhibited total fatty acid synthesis, it could not be determined if such an increase in unsaturated fatty acid synthesis was actually occurring.

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51 An attractive alternative for the site of ethanol interaction is the condensing enzyme responsible for saturated fatty acid synthesis in E. coli . The presence of two condensing enzymes have recently been demonstrated in E. coli , g-ketoacyl-acyl ACP synthetase I, and &ketoacyl-acyl ACP synthetase II (D'Agnolo et al., 1975), and it is believed that a third condensing enzyme may also exist (John E. Cronan, Jr.; personal communication). B-Ketoacyl-acyl ACP synthetase I is primarily involved in unsaturated fatty acid synthesis, and the absence of this enzyme has been demonstrated in fabB unsaturated fatty acid auxotrophs (D'Agnolo e_t al . , 1975). The type II synthetase is absent from cvc fatty acid mutants (no cis -vaccenic acid) and is proposed to be involved in elongating palmitoleoyl ACP to cisvaccenoyl ACP (D'Agnolo £t al. , 1975). However, both of these enzymes display a broad preference for substrates, suggesting that either type can also participate in saturated fatty synthesis. Since strain K1060 (a fabB mutant deficient in g-ketoacyl-acyl ACP synthetase I) underwent an ethanol-induced fatty acid change, it is unlikely that ethanol is interacting with this condensing enzyme. This leaves either the type II condensing enzyme or the putative type III condensing enzyme. In view of their broad substrate specificities, it is likely that both (or all three) condensing enzymes participate in forming the short-chained intermediates prior to the ten carbon intermediate, and the enzymes may be specific for saturated or unsaturated fatty acid synthesis only after the formation of the g-hydroxydecanoyl ACP fatty acid. Thus an inhibition of either the type II condensing enzyme or the type III

PAGE 60

52 (if it exists) by ethanol would result in the reduced rate of total fatty acid synthesis observed jln vivo as well as _in vitro . Since the condensation reaction is the rate limiting reaction in fatty acid synthesis (Bloch and Vance, 1977), an inhibition of this reaction by ethanol would be expected to result in a corresponding decrease in the rate of overall fatty acid synthesis. Further, since the type I synthesis would already be expected to be working to its capacity, the inhibition of saturated fatty acid synthesis would probably not lead to an increase in the levels of unsaturated fatty acids. Therefore, the condensing enzyme responsible for saturated fatty acid synthesis in E. coli represents a logical site for the specific interaction of ethanol. It is interesting that both ethanol and temperature were able to exert a direct effect on fatty acid synthesis ^n vitro . Our results clearly indicate that neither ethanol nor temperature depends upon the membrane, per se , to induce changes in de novo fatty acid synthesis. However, it is very possible that both effects are mediated through a lipid-protein interaction. Fatty acid synthesis is believed to occur in close proximity with the plasma membrane (van den Bosch, e_t al. , 1970), which could facilitate an association between membrane lipids and one or more of the fatty acid synthetase enzymes. Alternatively, the lipid-protein site may actually reside at the level of an enzymesubstrate interaction. This could explain our observation that ethanol induced the synthesis of shorter-chained fatty acids in vitro (Table 12) . Both of the enzymes described above as potential sites for the ethanol interaction (8-hydroxydecanoyl ACP dehydrase and 3-

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53 ketoacyl-acyl ACP synthetase II) have also been proposed to control temperature adaptation of the fatty acid composition in E. coli (Brocket al., 1967; Gelmann and Cronan, 1972). Thus the similarity in fatty acid changes induced by ethanol and temperature may actually result from their interaction with a common enzyme in fatty acid synthesis.

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REFERENCES Bergelson, L.D., Dyatlovitskaya, E.V. and Voronkova, V.V. (1964) J. Cromatog . 15, 191-199. Binstock, J.F., Praraanik, A., and Schultz, H. (1977) Proc . Nat . Acad . Sci . U.Si.A. 74, 492-495. Bloch, K. and Vance, D. (1977) Ann. Rev . Biochem . 46 , 263-298. Brock, D.J.H., Kass, L.R. and Bloch, K. (1967) J. Biol . Chem . 242 , 4432-4440. Chin, J.H., and Goldstein, D.B. (1977) Science 196 , 684-685. Cronan, J.E., Jr. (1974), Proc . Nat . Acad . Sci . U.S^.A. 71 , 3758-3762. Cronan, J.E., Jr. (1975), J. Biol . Chem . 250 , 7074-7077. Cronan, J.E., Jr. and Wulff, D.L. (1969), Virology 38, 241-246. Cronan, J.E., Jr. and Vagelos, P.R. (1972), Biochim . Biophys . Acta 265 , 25-60. Cronan, J.E., Jr., and Gelmann, E.P. (1975), Bact . Rev . 39.. 232-256. Cronan, J.E., Jr., Weisberg, L. and Allen, R.G. (1975), J_. Biol . Chem . 250 , 5835-5840. Cubero, J.M., and Mangold, H.K. (1965), Microchem . J. 9^, 227-236. D'Agnolo, G., Rosenfeld, I.S. and Vagelos, P.R. (1975), J. Biol . Chem . 250 , 5289-5294. Esfahani, M. , Barnes, E.M., and Walkil, S.J. (1969), Proc . Nat . Acad . Sci . U.SI.A. 64, 1057-1064. Fourcans, B. and Jain, M. (1974), Adv. Lipid Res . 12 , 147-226. Fox, C.F., Law, J.H., Tsukagoshi, N. , and Wilson, G. (1970) Proc . Nat . Acad . Sci . U.SI.A. 67_, 598-605. Freeman, C.P., and West, D. (1966), J. Lipid Res . ]_, 324-327. 54

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55 Gallant, J., Palmer, L. and Pao, C. (1977), Cell 11, 181-185. Gelmann, E.P. and Cronan, J.E., Jr. (1972), J_. Bacteriol . 112 , 381-387. Harder, M.E., Beacham, I.R., Cronan, J.E., Jr., Beachman, K., Honnegger, J.L., and Silbert, D.F. (1972), Proc . Nat . Acad . Sci. U.S.. A. J39, 3105-3109. Ingram, L.O. (1976), J. Bacteriol . 125 , 670-678. Ingram, L.O. (1977a), Can. J. Microbiol . 23_, 779-789. Ingram, L.O. (1977b), Appl . Environ . Microbiol . 13, 1233-1236. Ingram, L.O., Chevalier, L.S., Gabbay, E.J., Ley, K.D. , and Winters, K. , (1977), J. Bacteriol . 131 , 1023-1025. Kanfer, J., and Kennedy, E.P. (1973), J. Biol . Chem . 238 , 2919-2922. Kass, L.R., Brock, D.J.H., and Bloch, K. (1967) J. Biol . Chem . 242, 4418-4431. Klein, K. , Steinberg, R. , Fiethen, B., and Overath, P. (1971), Eur . J. Biochem . 19, 442-450. Lennarz, W.J., Light, R.J. and Bloch, K. (1962) Proc . Nat . Acad . Sci . IhS.A. 48, 840-846. Majerus, P.W., Alberts, A.W. and Vagelos, P.R. (1968) Biochemical Preparations , vol. XII, 56-65. Marr, A.G., and Ingraham, J.L. (1962), £. Bacteriol . 84 , 1260-1267. Marsh, N.J., and Duggan, D.E. (1972), J. Bacteriol . 109 , 730-740. Merlie, J. P., and Pizer, L.I. (1973), J. Bacteriol . 116 , 355-366. Miller, J.H. (1972), Experiments in Molecular Genetics , Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory, pp. 230-234. Morris, L.J., Wharry, D.M., and Hammond, E.W. (1967), J. Chroma tog . 31 , 69-76. Nunn, W.D. (1977), Biochemistry 16, 1077-1081. Nunn, W.D., and Cronan, J.E., Jr. (1976), J. Mol. Biol . 102 , 167-172. Okuyama, H. , Yamada, K. , Kameyama, Y. , Ikezawa, H. , Akamatsy, Y. and Nokuma, S. (1977) Biochemistry 16, 2668-2673.

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56 Overath, P., Schairer, H.U., and Stoffel, W. (1970), Proc . Nat . Acad . Sci . U.Si.A. 67, 606-612. Rando, R.R., and Bloch, K. (1968), J. Biol . Chem . 243, 5627-5634. Semple, K.S., and Silbert, D.F. (1975), J. Bacteriol . 121, 1036-1046. Shaw, M. , and Ingraham, J.L. (1965), J. Bacteriol . 90, 141-146. Silbert, D.F. (1976) Methods in Membrane Biology (E.D. Korn, ed.) vol. VI, 151-182, Plenum Press, N.Y. Silbert, D.F., Ulbright, T.M., and Honeggar, J.L. (1973a), Biochemistry 12, 164-171. Silbert, D.F., Ladenson, R.C., and Honegger, J.L. (1973b), Biochim . Biophys . Acta . 311 , 349-361. Silbert, D.F., Pohlman, T., and Chapman, A. (1976), J. Bacteriol . 126 , 1351-1354. Sinensky, M. (1971), J. Bacteriol . 106 , 449-455. van den Bosch, H., Williamson, J.R. and Vagelos, P.R. (1970), Nature 228 , 338-341. Vogel, H.J., and Bonner, D.M. (1956), J. Biol . Chem . 218 , 97-106. Volpe, J.J., and Vagelos, P.R. (1976), Phys. Rev . 56, 339-417.

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BIOGRAPHICAL SKETCH Thomas Martin Buttke was born on December 5, 1952, in Bay Shore, New York. The author graduated from Bay Shore High School in 1970, and received the Bachelor of Arts degree from the University of North Carolina at Wilmington in 1974. In September of 1974, he entered the Department of Microbiology at the University of Florida as the recipient of a Graduate Council Fellowship, and received the Master of Science degree in microbiology in December, 1975. From September, 1977 to June, 1978, he was the recipient of a fellowship from the Center of Gerontological Studies, and he is presently a candidate for the Doctor of Philosophy degree in the Department of Microbiology and Cell Science. The author is married to the former Mary Elizabeth Thompson. 57

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. i/ /9~ Lonnie 0. Ingram, Chairman Associate Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Charles M. Allen, Jr. (/ Associate Professor of Biochemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. tf'jy u^ 7 /^zJZy^ Tames F. Preston III 'Associate Professor of Microbiology and Cell Science This dissertation was submitted to the Graduate Faculty of the Department of Microbiology and Cell Science in the College of Arts and Sciences and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. June, 1978 Dean, Graduate School

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