JOURNAL OF BACTERIOLOGY, Nov. 1980, p. 481-4880021-9193/80/11-0481/08$02.00/0

Vol. 144, No. 2

Differential Effects of Ethanol and Hexanol on theEscherichia coli Cell Envelopet

L. 0. INGRAM* AND N. S. VREELANDDepartment ofMicrobiology and Cell Science, University ofFlorida, Gainesville, Florida 32611

Both ethanol and hexanol inhibited the growth of Escherichia coli, but theireffects on the organization and composition of the cell envelope were quitedifferent. Hexanol (7.8 x 10-3 mM) increased membrane fluidity, whereas ethanol(0.67 M) had little effect. During growth in the presence of ethanol, the proportionofunsaturated fatty acids increased. The opposite change was induced by hexanol.Unlike hexanol, growth in the presence of ethanol resulted in the production ofun-cross-linked peptidoglycan with subsequent lysis. Salt (0.3 M) protected cellsagainst ethanol-induced lysis but potentiated growth inhibition by hexanol.Mutants isolated for resistance to ethanol-induced lysis synthesized cross-linkedpeptidoglycan during growth in the presence of ethanol but remained sensitive tohexanol. A general hypothesis was presented to explain the differential effects ofethanol and hexanol. All alcohols are viewed as sinmilar in having both an apolarchain capable of interacting with hydrophobic environments and a hydroxylfunction capable of hydrogen bonding. The differential effects of short-chainalcohols may represent effects due to the high molar concentrations of hydrogenbonding groups with an apolar end within the environment. These may replacebound water in some cases. With longer-chain alcohols such as hexanol, theeffects of the acyl chain would dominate, and limitations of solubility and cellularintegrity would mask these hydroxyl effects.

Alcohols are amphipathic molecules that alterboth polar and apolar environments. Alcoholshave been shown to affect numerous biologicalprocesses, many of which are associated withthe cell membrane (12, 14, 15, 27, 41). The po-tency of alcohols in eliciting these changes isdirectly related to their lipid solubility, indicat-ing a hydrophobic site of action (24, 30, 33, 35).Ethanol is a major fermentation product of

Escherichia coli (5) and a compound to whichthis organism may have evolved an adaptiveresponse. The lipid composition of E. coli isextensively regulated (34, 36). In previous stud-ies in our laboratory, the changes in lipid com-position in E. coli which occur during growth inthe presence of ethanol and other alcohols havebeen examined (1, 16, 18). The effectiveness ofalcohols in inducing these changes is directlyrelated to chain length or hydrophobicity.Growth in the presence of alcohols of up to ninecarbons in length and phenethyl alcohol (31)results in the synthesis of lipids containing anincreased abundance of anionic phospholipidsrelative to phosphatidylethanolamine (18), andthese changes may be part of an adaptive re-

t Florida Agricultural Experiment Station publication no.2356.

sponse. Clark and Beard (7) have recently iso-lated ethanol-resistant mutants which overpro-duce anionic phospholipids. Isolated membranesfrom these mutants exhibited an increased re-sistance to disruption by ethanol (7). Althoughthe effects of all alcohols are similar with respectto phospholipid composition, their effects onfatty acid composition are not as simple. Alco-hols of up to four carbons in length induce largechanges in fatty acid composition which are theopposite of those induced by the presence oflonger-chain alcohols (16, 38). During growthwith the shorter-chain alcohols, cells synthesizeelevated levels of phospholipids containing twounsaturated fatty acids (1). During growth withthe longer-chain alcohols such as hexanol, cellssynthesize phospholipids containing one unsat-urated and one saturated fatty acid (1). Thesechanges in acyl chains have also been proposedas part of an adaptive response (16). Other cel-lular processes have been observed in whichlong-chain and short-chain alcohols differ intheir actions. For example, permease-mediatedfi-galactoside uptake is inhibited by short-chainalcohols (12, 19) and is stimulated by long-chainalcohols (19, 39). In this paper, we investigateddifferential effects of ethanol and hexanol on cellgrowth and the cell envelope of strain TB4.

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482 INGRAM AND VREELAND

MATERIALS AND METHODSStrains and growth conditions. E. coli K-12

strain TB4 is a fadE derivative of strain CSH2 (3) andrequires thiamine and tyrosine for growth. Cultureswere grown in Luria broth (29) containing glucose (2g/liter), yeast extract (5 g/liter), tryptone (10 g/liter),NaCl (5 g/liter), and 2 N NaOH (1.1 ml/liter), unlessstated otherwise. Flask cultures (50 ml of broth per250-ml flask) were incubated at 370C in a reciprocatingwater bath shaker. Growth was monitored by measur-ing optical density at 550 nm in a Bausch & LombSpectronic 70 spectrophotometer. Cell morphologieswere examined and photographed with a Zeiss phase-contrast microscope.Fluorescence depolarization. The fluorescence

depolarization of 1,6-diphenyl-1,3,5-hexatriene (Ald-rich Chemical Co., Milwaukee, Wis.) was used as acomparative measure of the fluidity of membranes(11). Initially, two different methods of membranepreparation were examined: ultrasonic disruption fol-lowed by gradient separation of inner and outer mem-branes as described by Ito et al. (22) and the freeze-thaw method of DiRienzo and Inouye (9). In ourhands, the freeze-thaw preparations which containedboth inner and outer membranes yielded much morereproducible values, and this method was used in thisstudy. Membranes were suspended in Tris-hydrochlo-ride buffer (0.01 M, pH 7.5) at a final concentration of100 ,ug of protein per ml. Protein was measured by themethod of Lowry et al. (26, 28). 1,6-Diphenyl-1,3,5-hexatriene was dissolved in tetrahydrofuran (2 mM).A microcrystalline dispersion of 1,6-diphenyl-1,3,5-hexatriene was produced by injecting 1 pl/ml intobuffer with a syringe. The membrane suspension andbuffer dispersion were mixed in equal volumes andincubated in the dark for 2 h in a reciprocating waterbath shaker to allow probe insertion. Samples weremeasured with an SLM series 4000 polarization fluo-rometer (excitation at 360 nm and emission above 470nm). This instrument simultaneously measures paral-lel and perpendicular emissions, averaging 10 discretemeasurementa per output reading. Polarization wascomputed as described by Chen and Bowman (4).Sample temperature was controlled with a Neslabthermoregulator and was monitored within the cu-vette with a thermocouple. Ethanol was added inincrements by syringe and was allowed to equilibratefor 2 min before measurement. For measurementswith hexanol, the membranes and probe were pre-pared as a 2x concentrate. Hexanol concentrationswere obtained by diluting this concentrate with bufferand a stock solution of hexanol (3.9 x 10-2 M) inbuffer.

Analysis of lipids. Celi were harvested at anoptical density (550 nm) of 0.4 (10' cells per ml),inactivated with trichloroacetic acid (5%), and ex-tracted in chloroform-methanol (2:1, vol/vol). Thefatty acid composition of bulk lipids was determinedby gas chromatography (16). Phospholipids were an-alyzed by thin-layer chromatography (18). Spots cor-responding to the major phospholipids were scrapedand counted in a Beckman scintillation counter.

.Analysis of peptidoglyean cross-linking. Ly-sozyme digestion products of heat-activated cells were

initially purified on an ECTEOLA-cellulose column(42). The purified digestion products (0.2 ml) wereseparated by using a Sephadex G-25 column (60 by 0.9cm) as described previously (21). Fractions were ana-lyzed for total reducing sugar by the method of Parkand Johnson (32) as modified by Ghuysen et al. (13)with N-acetylglucosamine as a standard.

RESULTSDifferential effects of ethanol and hex-

anol on growth. Figure 1 shows the effects ofethanol and hexanol on the growth of strain TB4at 370C. Increasing concentrations of alcoholprogressively inhibited initial growth as previ-ously reported (12, 16). Hexanol was a muchmore potent inhibitor of growth than wasethanol. After about 2.5 h of growth in thepresence of 0.51 and 0.67 M ethanol, growthceased and the optical density began to decrease.At this time, rounded and swollen cells wereobserved by phase-contrast microscopy. After 4h, extensive lysis and debris were observed. Nolysis was observed during growth in the presenceof hexanol.The possibility of autolysin activation was

investigated as a mechanism for ethanol-inducedlysis. Nongrowing cells were incubated for 6 and12 h at 370C in the presence and absence of 0.67M ethanol. Three types ofnongrowing cells wereexamined: exponential cells which were washedand suspended in 0.1M sodium phosphate buffer(pH 7.2), exponential cells to which sodium azide(1 mM) and chloramphenicol (50 ,ug/ml) wereadded, and stationary-phase cells. No decreasein turbidity or lysis was observed under theseconditions. Microscopically, cells retained theirrod-shaped morphology (data not shown).

In previous studies by Fried and Novick (12)with a mineral salts medium, ethanol-inducedlysis was not reported. Since Luria broth con-tains lower levels of electrolytes, the effects ofsalt on ethanol-induced lysis were investigated.Elevated concentrations of sodium chloride pro-tected the cells against lysis during growth inthe presence of ethanol. Sodium chloride at finalconcentrations of 0.2 and 0.3 M was adequatefor protection, whereas lower concentrationspermitted lysis. The presence ofsodium chloridedid not relieve the initial reduction in growthrate caused by ethanol. Higher concentrationsof sodium chloride also resulted in slowergrowth.Potassium chloride, potassium sulfate, and so-

dium chloride were equally effective in protect-ing cells against lysis, whereas neither equimolarnor equiosmolar concentrations of sucrose pro-vided complete protection against lysis. The fail-ure of sucrose to provide complete protection

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DIFFERENTIAL EFFECTS OF ALCOHOLS

2 0r A

0.50

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00.10 k

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FIG. 1. Effect ofalcohols on the growth ofstrain TB4. (A) Ethanol. Symrbols: *, none; 0, 0.34 M; A, 0.51 M;A, 0.67M; O:, 0.83 M. (B) Hexanol. Symrbols: 0, none; 0, 3.9 mM; A, 5.5 mM; 4, 7.8 mM; , 10mM; C, 1.2 mM.O.D., Optical density.

suggested that the effects of salts (0.3 M) may

not result from osmotic stabilization alone. Ifosmotic protection alone were responsible, theaddition of salt (0.3 M) at any time before lysisshould have been sufficient to block its occur-

rence. The addition of salt after 30 min ofgrowthafforded complete protection. However, the ad-dition of salt after 1 h of growth in the presence

of ethanol (0.67 M) provided no protection.These results suggest that cell damage occurs

during growth in the presence of ethanol, whichis not readily repaired after salt addition.The inclusion of sodium chloride (0.3 M) in-

creased the degree of growth inhibition causedby hexanol (compare Fig. 1B and 2). Similarresults were obtained with potassium chlorideand sodium sulfate.Effects of ethanol and hexanol on pepti-

doglycan cross-linking. The extent of cross-

linking of peptidoglycan was compared in cellsgrown with and without ethanol (Fig. 3). Mon-omer and dimer fragments released by lysozymedigestion were separated by gel filtration. Instrain TB4 grown without ethanol, the bulk ofthe peptidoglycan was in the cross-linked formand eluted near the void volume. After growthfor 2 h in medium supplemented with 0.51 Methanol, only the un-cross-linked monomer was

present. This form eluted with a Kay of 0.4, ingood agreement with previous results (21, 42).Material from cells grown in lower concentra-

tions of ethanol contained intermediate levels ofthe monomer and dimer peaks. After growth inthe presence of both 0.51 M ethanol and 0.3 Msodium chloride, partial cross-linking was re-stored. Salt alone (0.3 M) had no effect on cross-linking. Growth for 3 h in 6.3 mM hexanol didnot alter cross-linking (data not shown).

Differential effects of ethanol and hex-anol on membranes in vitro. We comparedthe effects of ethanol and hexanol on the polar-ization of 1,6-diphenyl-1,3,5-hexatriene in mem-branes from celLs grown under different condi-tions (Table 1). Cells grown in regular Luriabroth and those grown in Luria broth containing0.3 M NaCl differed in their initial polarization(Table 1). Membranes from cells grown withethanol appeared slightly more fluid, and mem-branes from cells grown with hexanol appearedslightly more rigid than their respective controlcells. However, this shift is, at best, near thelimits of detection in a comparison of severalbatches of membranes. The change in polariza-tion with ethanol and hexanol could be mea-sured much more accurately within each mem-brane preparation. For comparison, concentra-tions which cause a roughly equivalent inhibi-tion of growth were chosen. Hexanol (7.8 mM)caused a large decrease in polarization, whereasethanol had little effect on polarization. Mem-branes from cells grown with ethanol or hexanolremained sensitive to these agents.

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484 INGRAM AND VREELAND

Mutants resistant to ethanol-inducedlysis. Ten spontaneous mutants were isolatedby serial cultivation of strain TB4 in 0.83 Methanol. All of these grew equally well in thepresence of ethanol and did not round up and

2.0

0.50-

0

00 .1

0.02

0 1 2 3 4TIME (hr)

FIG. 2. Effect of hexanol on the growth of strainTB4 in Luria broth contazinig 0.3M NaCI. Symbols:0, no hexanol; 0, 3.9 mM hexanol; A, 5.5 mM hex-anol; A,7.8 mM hexanol; I 1.0mM hexanol; C, 12mM hexanol. O.D., Optical density.

30r

ENAzw

-J 20

w

0

z

0

w I0U)

w

-J

0

VO

5 15 2FRACTION NUMB

FIG. 3. Separation of monomerments from lysozyme digest of stra0, cels grown in the absence ofgrown for 2 h in 0.51 M ethanol;ethanol and 0.3M NaCI.

lyse. Figure 4A shows the growth of one of these,strain LI-1, in the presence of a series of concen-trations of ethanol. Although all mutants wereresistant to lysis, their growth rate was stillprogressively inhibited by increasing concentra-tions of ethanol. The level of resistance to lysisachieved by these mutants in Luria broth wasequivalent to that exhibited by the parent or-ganism during growth in Luria broth containing0.3 M sodium chloride (Fig. 4B). Strain LI-1 wasresistant to lysis induced by acetone, methanol,and dioxane, but did not exhibit increased re-sistance to hexanol or to hexanol plus salt (datanot shown). Unlike the parent, un-cross-linkedmonomer did not accumulate during the growthof strain LI-1 with ethanol (0.51 M).The alcohol-induced changes in the mem-

brane lipids of strain LI-1 were similar to thoseobs'erved with strain TB4. Both strains exhibitedan increase in phosphatidylglycerol (14 to 17.5%)when grown with ethanol (0.51 M). The propor-tion of unsaturated fatty acids also increased inboth strains (Table 2). Strain LI-1 exhibited alarger change than did strain TB4 when bothwere grown in regular Luria broth containingalcohol. However, in the presence of 0.3 M salt,the ethanol-induced changes in fatty acid com-position were identical. The polarization in iso-lated membranes from strain LI-1 was identicalto that in isolated membranes from strain TB4.The addition of ethanol (0.67 M) or hexanol (7.8mM) caused a change in polarization of -0.003and -0.013, respectively.

DISCUSSIONAlcohols and other amphipathic molecules

have long been used as antimicrobial agents toprevent the growth of bacteria (14, 15, 41). In E.coli, both ethanol and hexanol inhibit growth.Although these alcohols are chemically similar,their modes of action must differ in some way.Previous studies have shown that ethanol andhexanol affect acyl chain composition in oppo-site fashions (16). Additional differences in theeffects of these agents on the growth of strainTB4 were also found. During growth in Luriabroth, ethanol concentrations which caused anapproximately 50% inhibition ofgrowth rate also

Vt resulted in subsequent lysis. Other agents, which_ had been shown previously to induce fatty acid

changes similar to those caused by ethanol, also25 35 induced lysis. Hexanol did not induce lysis, al-IER though it was a potent inhibitor of growth.and dimer firag- Lysis by ethanol does not appear to be the

tin TB4. Symbols: result of autolysin activation or complete mem-ethanol; 0, cells brane disruption. Growth in the presence ofceUs grown with ethanol was required for lysis. The addition of

electrolytes protected cells against ethanol-in-

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TABLE 1. Effects of ethanol and hexanol on fluorescence depolarization in membranes from strain TB4aChange in polarization

Growth conditions PolarizationEthanol (0.67 M) Hfexanol (7.8 mM)

Luria broth 0.2761 ± 0.0006 (3) -0.0007 i 0.0004 (8) -0.0121 + 0.0021 (2)Luria broth plus hexanol (4 0.2771 ± 0.0047 (9) -0.0028 ± 0.0006 (4) -0.0110 NDb (1)mM)

Luria broth with 0.3 M NaCl 0.2889 ± 0.0021 (6) -0.0009 + 0.0002 (4) -0.0070 ND (1)Luria broth with 0.3 M NaCl 0.2828 ± 0.0008 (3) -0.0004 ± 0.0005 (2) -0.0117 ± 0.0007 12)

plus ethanol (0.67 M)a Values are reported ± standard deviation, with the number of experiments given within parentheses.b ND, Not determined.

Ec0U')

00

2.0

0 1 2 3 4 0 2 3. 4TIME (hr) TIME (hr)

FIG. 4. Effect of ethanol on the growth of strains LI-1 and TB4. (A) Strain LI-1 in Luria broth. Symbols:0, no ethanol; 0, 0.67M ethanol; A, 0.83 ethanol; 4 !.23Methanol. (B) Strain TB4 in Luria broth containing0.3M NaCl. Symbols: 0, no ethanol; 0, 0.67M ethanol; A, 0.83M ethanol; 4 1.23M ethanol. O.D., Opticaldensity.

TABLE 2. Effect of ethanol and NaCl on fatty acidcomposition

Fatty acid composition (%)Strain Additive

12:0 14:0 16:0 16:1+A17 18:1

TB4 None 3.5 4.0 31.5 33.7 27.3TB4 0.51 M ethanol 4.5 2.9 22.8 26.6 43.2TB4 0.3 M NaCl 4.3 3.8 32.0 33.5 26.4TB4 0.3 M NaCl plus 3.5 1.9 19.0 27.7 47.9

0.51Methanol

LI-1 None 4.4 4.1 31.4 33.9 26.2LI-i 0.51 M ethanol 3.4 2.0 18.8 27.4 48.3

a Combination of palmitoleic acid and ciu-9,10-methylene-hexadecanoic acid.

duced lysis. Although lysis per se is driven byinternal osmotic pressure, protection by electro-

lytes did not result from simple osmotic stabili-zation. During growth in Luria broth containingethanol, envelope damage occurred which wassubsequently shown to be prevented by increas-ing the electrolyte concentration of the medium.Possible damage to envelope proteins and pep-tidoglycan cross-linking were investigated. Lyticconcentrations of ethanol prevented the assem-bly of cross-linked peptidoglycan. In contrast,growth in the presence of hexanol did not inhibitthe cross-linking of peptidoglycan. The effects ofethanol on cross-linking were partially relievedby 0.3 M NaCl. Mutants, isolated for resistanceto ethanol, produced highly cross-linked pepti-doglycan during growth in the presence ofethanol. Thus, we conclude that ethanol-in-duced lysis results from the inhibition of pepti-

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486 INGRAM AND VREELAND

doglycan cross-linking by ethanol.The effects of ethanol on cross-linking are

similar to those reported for penicillins (2, 10,42). Low concentrations of penicillin have beenshown to selectively inhibit cell division, result-ing in the production of snake forms (20, 37).The snake forms previously observed by Friedand Novick (12) during the growth of E. coli inhigh concentrations of ethanol may also resultfrom the effect of ethanol on peptidoglycancross-linking. Ethanol may alter the synthesis oflipopolysaccharide or other aspects of peptido-glycan synthesis such as chain length or netsynthesis. Such changes may also be involved inethanol-induced lysis but have not been ad-dressed in this study.

Alcohols and other amphipathic moleculeshave been shown to alter a variety ofmembrane-associated processes (24, 27, 35). The potency ofalcohols and other agents in eliciting these ef-fects is directly related to their hydrophobicityor octanol-water partition coefficients (16, 35).Although this provides strong evidence for ahydrophobic site(s) of action, the specific site ortypes of interaction need not be identical. Manybiological processes are known to change in asimilar fashion in response to alcohols of chainlengths of 1 through 8 or 10 (35). For example,the ratio of anionic lipids to phosphatidyletha-nolamine increases in a similar fashion duringgrowthwith all alcohols in E. coli (18). However,some processes are differentially affected by al-cohols of different chain lengths. Previous stud-ies by Dagley et al. (8) reported differences inthe effects ofhexanol and ethanol on the growthof Enterobacter in minimal medium supple-mented with amino acids. In E. coli, alcohols ofdifferent chain lengths have a differential effecton lac permease activity. Ethanol and othersimple alcohols of up to four carbons in length(12, 19) inhibit permease activity, whereaslonger-chain alcohols such as hexanol (19, 39)stimulate permease activity. These two groupsof alcohols also have differential effects on acylchain composition in E. coli. During growth withethanoL the proportion of unsaturated fattyacids (16) and ofphospholipid species containingtwo unsaturated fatty acids (1) is increased. Theopposite effects occur during growth with hex-anol (1, 16). In the present study, numerousother differences in the effects of ethanol andhexanol were found. Hexanol increased mem-brane fluidity, whereas ethanol had little effectwhen compared at concentrations causing simi-lar growth inhibition. Growth in the presence ofethanol resulted in the production of un-cross-linked peptidoglycan and subsequent lysis. Incontrast, hexanol did not alter cross-linking orcause lysis. Ethanol-resistant mutants exhibited

no increase in resistance to hexanol. Electrolytesprotected cells against ethanol-induced lysis butpotentiated growth inhibition by hexanol. Thesedifferences in the biological activities of short-versus long-chain alcohols indicate that thesetwo groups differ in their specific interactionswith biological membranes.

In all cases, small, relatively polar compoundssuch as acetone, methanol, and dioxane causechanges similar to those caused by short-chainalcohols, whereas more hydrophobic moleculescause changes similar to those caused by hexanol(17). However, size alone does not appear to beof major importance. We observed that 1,6-hex-anediol induces lysis and increased synthesis ofunsaturated fatty acids. Simple alcohols consistof two parts: a hydrophobic tail and a hydroxylfunction. Two general properties are usually as-sociated with these parts: a disruptive effect onmembrane organization resulting from the inter-calation (6, 27) of the hydrophobic tail and adehydrating effect involving hydrogen bonding.The intercalation of all alcohols of up to eightcarbons in length has a qualitatively similareffect on membrane organization, a decrease inorder (6, 23, 43). Quantitatively, the effect ofhexanol on membrane fluidity is 10-fold that ofethanol when compared at concentrations caus-ing a similar inhibition of growth in E. coli.Although a change may be involved in some ofthe shared effects of alcohols, it cannot explainthe differential effects of alcohols. In the secondeffect involving hydrogen bonding and possiblewater replacement, short-chain alcohols and rel-atively polar compounds would be expected tobe much more active than long-chain alcohols.Short-chain alcohols such as ethanol are com-patible with both hydrophobic and hydrophilicenvironments as evidenced by their miscibilitywith both diethyl ether and water. All agentsthus far found to cause changes similar to thosecaused by ethanol in E. coli are capable ofhydrogen bonding. At physiologically employedconcentrations, short-chain alcohols and the rel-atively polar agents are present in high concen-trations in the aqueous environment, in compar-ison with the concentration ofhexanol, and oftenconstitute over 1% ofthe molecules present. Thishigh concentration ofshort-chain alcohols wouldenhance the opportunities for water replace-ment. All alcohols are thus viewed as similar inactivities. Intercalation is promoted by longerhydrocarbon chains, whereas dehydration, as aproperty of the hydroxyl group (or hydrogenbonding group), primarily reflects molar abun-dance. With the more potent longer-chain alco-hols, the biological effects would be expected toresult from intercalation into hydrophobic re-gions. Limitations of solubility and cell integrity

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may mask our observations of the hydroxyl ef-fects. With the relatively polar short-chain al-cohols, intercalation would also be expected aspredicted by the partition law. However, thesecond effect involving hydrogen bonding andwater replacement may represent the dominantaction, facilitating the differential of effects ofshort-chain alcohols. Both dehydration and in-tercalation activities could potentially alter avariety of membrane properties in much moresubtle ways than changes in bulk fluidity. Sitesfor these alterations include both polar and apo-lar associations in the membrane between lipids,between proteins, between lipids and proteins,and within proteins. In addition, the relativepermeability of biological membranes to theseagents would also allow interactions with solublecellular constituents.The assembly of cross-linked peptidoglycan in

E. coli was impaired during growth in the pres-ence ofethanol but was not inhibited by hexanol.The regulation of peptidoglycan cross-linking isnot completely understood (2, 5, 40), but in-volves both glycopeptide transpeptidase andcarboxypeptidase activities (2, 25, 40). Althoughthe proteins catalyzing these functions are mem-brane associated (2, 25), their substrates arepolar in nature and located within the nascentpeptidoglycan. Thus, these enzymes must spanthe interface of the membrane and protrude intothe aqueous environment within the periplasmicspace. Enzymes such as these, poised at thepolar-apolar membrane interface, may be partic-.ularly sensitive to the effects of relatively polaramphipathic molecules. Regions may exist atsuch boundaries where hydrogen bonding be-tween molecules such as ethanol may be ther-modynamically favorable. Although hexanolcould also participate in hydrogen bonding in-teractions, the low molar concentration of hex-anol, coupled with its probable alignment withacyl chains and possible steric hindrance, mayminimize this type of interaction. The lac per-mease must also be poised at the hydrophobicinterface. The differential effects of alcohols ofdifferent chain lengths on this activity may alsoreflect the balance between increased fluidityfrom intercalation and hydrogen bonding.

ACKNOWLEDGMET41S

We thank M. G. Pate and C. R. Press for their technicalassistance and C. E. Carty and L. C. Eaton for their criticalreading of this manuscript.

This investigation was supported by Public Health Servicegrant iROl GM 24059-01 from the National Institutes ofHealth and grant iROl AA 03816-01 from the National Insti-tute of Alcohol Abuse and Alcoholism. L.O.I. is the recipientof Career Development award 1 K02 00036-01 from the Na-tional Institute of Alcohol Abuse and Alcoholism.

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3. Buttke, T. M., and L 0. Ingram. 1978. Mechanism ofethanol-induced changes in lipid composition of Esch-erichia coli: inhibition of saturated fatty acid synthesisin vivo. Biochemistry 17:637-644.

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5. Chesbro, W., T. Evans, and R. Eifert. 1979. Very slowgrowth of Escherichia coli. J. Bacteriol. 139:625-638.

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7. Clark, D. P., and J. P. Beard. 1979. Altered phospholipidcomposition in mutants of Escherichia coli sensitive orresistant to organic solvents. J. Gen. Microbiol. 113:267-274.

8. Dagley, S., E. A. Dawes, and G. A. Morrison. 1950.Inhibition of growth ofAerobacter aerogenes: the modeof action of phenols, alcohols, acetone, and ethyl ace-tate. J. Bacteriol. 60:369-379.

9. DiHienzo, J. M., and M. Inouye. 1979. Lipid fluidity-dependent biosynthesis and assembly of the outermem-brane proteins of E. coli. Cell 17:155-161.

10. Duguid, J. P. 1946. The sensitivity of bacteria to theaction of penicillin. Edinburgh Med. J. 53:401-412.

11. Esko, J. D., J. R. Gilmore, and M. Glaser. 1977. Use ofa fluorescent probe to determine the viscosity of LMcell membranes with altered phospholipid composi-tions. Biochemistry 16:1881-1896.

12. Fried, V. A., and A. Novick. 1973. Organic solvents asprobes for the structure and function of the bacterialmembrane: effects of ethanol on the wild type and anethanol-resistant mutant of Escherichia coli K-12. J.Bacteriol. 114:239-248.

13. Ghuysen, J., D. J. Tipper, and J. L Strominger. 1966.Enzymes that degrade bacterial cell wals. MethodsEnzymol. 8:685-699.

14. Harold, F. M. 1970. Antimicrobial agents and membranefunction. Adv. Microb. Physiol. 4:45-104.

15. Hugo, W. B. 1967. The mode of action of antibacterialagents. J. Appl. Bacteriol. 30:17-50.

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