THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 8, Issue of April 25. pp. 3735-3742, 1981 Printed m U.S.A.

Transport of Long and Medium Chain Fatty Acids by Escherichia coli K12*

(Received for publication, September 18, 1980)

Stanley R. MaloyS, Charles L. Ginsburgh, Robert W. Simons, and William D. Nun@ From the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 9271 7

Kinetic, metabolic, and physical parameters of long and medium chain fatty acid transport by Escherichia coli K12 were determined. Uptake of long chain fatty acids (Cll-C18:l) mediated by the fadL gene involves concentrative transport. Evidence for this is as follows: (i) characteristic Kt and V,, values were obtained for long chain fatty acids, (ii) long chain fatty acid trans- port was inhibited by energy inhibitors, (iii) long chain fatty acids were concentrated 10-fold inside the cell against a concentration gradient, (iv) efflux of trans- ported long chain fatty acids did not occur, and (v) an energy of activation of 11.72 kcal mol” and &IO of 2.3 were obtained for long chain fatty acid transport. The fadL gene product shows some activity with medium chain fatty acids (C7-Clo) as well. Medium chain fatty acids also appear to enter the cell by simple diffusion since: (i) medium chain fatty acid transport by fadL strains is not saturable under our assay conditions, (ii) fadL strains do not concentrate medium chain fatty acids against a concentration gradient, and (iii) medium chain fatty acids are available for efflux in fadL strains. Physical parameters of long and medium chain fatty acid transport are also reported. These results present evidence for separate mechanisms of long and medium chain fatty acid transport in E. coli.

Wild type Escherichia coli K12 can utilize exogenous long chain fatty acids ( C l l - C l ~ l ) as a sole carbon and energy source for growth (1-7). The synthesis of at least five key enzymes involved in fatty acid degradation (fad) are coordinately in- duced during growth in media containing long chain fatty acids (2, 4). Medium chain fatty acids (C7-Clo) can serve as substrates for the fad enzymes, but cannot induce the synthe- sis of the fad enzymes. Therefore, only fatty acids longer than decanoic acid (Clo) can be used as a sole carbon source by wild type strains. Mutants constitutive for the synthesis of the fad enzymes (fadR) can be selected by growth on Cl0 fatty acid as a sole carbon source (2, 8). Such strains lack a func- tional repressor protein which normally prevents induction of the fad enzymes in the absence of long chain fatty acids (8,9). Strains bearing a fadR lesion are capable of growth on, and oxidation of, medium (C;-CIo) and long chain (CII-CIH) fatty acids.

Several studies suggest that E. coli K12 has a transport

* This work was supported by Public Healtb Service Grant GM22466-1A from the National Institute of General Medicine and from the National Science Foundation, PCM-7727140. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

f Supported by Training Grant GM07311 from the National Insti- tute of General Medicine.

5 Established Investigator of the American Heart Association.

mechanism for long and medium chain fatty acids. Klein et al. (2) reported that fadR mutants transport fatty acids with a chain length greater than Ca. Transport of fatty acids by fadR’ strains was found to be inducible by cis-A’-octadecenoic acid (C,,,,) (2). The uptake of fatty acids was shown to be strictly dependent upon the fadD gene product, acyl-CoA synthetase (fatty acid: CoA ligase (AMP forming)). The d m - ble mutant fadR fadD , which synthesizes constitutive levels of aff fad enzymes except acyl-CoA synthetase, cannot (i) utilize fatty acids of any chain length as a sole carbon source, (ii) accumulate exogenous fatty acids into the cytosol, or (iii) incorporate exogenous fatty acids into membrane phospholip- ids (2). In addition, no efflux of free fatty acids was observed after their uptake by fadD‘ strains. These results led Klein et al. (2) to propose that acyl-CoA synthetase is required for a group translocation step in the transport of exogenous fatty acids into E . coli. They termed this proposed transport mech- anism “vectorial acylation.”

Transport of the long chain fatty acid C l ~ : l was shown to be a saturable process (2), indicating a carrier-mediated mecha- nism. Frerman and Bennett (1) showed that C1, I transport was inhibited by arsenate, 4-pentenoate, and respiratory in- hibitors, suggesting a requirement for intracellular energy and coenzyme A. Strains with lesions in any of the fad enzymes showed a diminished rate of Clx:I transport, indicating that fatty acid transport is tightly coupled to further catabolism (1, 2). Furthermore, following uptake, neither free fatty acid nor oleyl-CoA was detectable in the cells (1,2). Thus, Frerman and Bennett (1) proposed that fatty acid transport is the rate- limiting step in catabolism.

Salanitro and Wegener (6) presented growth studies sug- gesting the presence of two physiologically distinct uptake systems for fatty acids of chain length C6-G and CIO-CM. Recently, another gene required for long chain fatty acid transport, fadL, has been mapped and partially characterized (3, 4) . These studies have shown that E. coli K12 requires a functional fudL gene in order to (i) optimally transport CW- CIS, , fatty acids into the cell, (ii) optimally grow on and oxidize CIO-CIS,~ fatty acids, and (iii) incorporate efficiently CI~-CI~ : I fatty acids into membrane phospholipids (4). The results of these studies suggest that the fadL gene product is required for transport of fatty acids of chain length greater than CW. Growth of fadR strains on the medium chain fatty acids C;- CIO does not require a functional fadL gene (3, 4). Thus, medium chain fatty acids appear to be transported by a different mechanism than long chain fatty acids.

In this paper, the pathways of long and medium chain fatty acid transport in E. coli K12 are further characterized. The kinetics of transport and oxidation of C6-CIH., fatty acids are presented, and physical and metabolic parameters of long versus medium chain fatty acid transport compared. A sepa- rate mechanism for the entry of medium chain fatty acids is proposed.

3735

3736 Fatty Acid Transport

EXPERIMENTAL PROCEDURES

Bacteria, Media, and Growth Conditions-All strains used in this study were derivatives of E . coli K12 and are described in Table I. Bacteria were routinely incubated in a New Brunswick gyrotory water bath shaker a t 37 "C. Bacteria were usually grown in M9 minimal medium (10) supplemented with 5 pgiml of thiamin and 5 mg/ml of Srij 58 (M9-Brij). Carbon sources were sterilized separately and added to the culture medium prior to inoculation. Fatty acids were suspended in 10% Brij 58, neutralized with KOH, sterilized, and added at a final concentration of 5 mM. Acetate was provided at 25 mM and all other carbon sources were provided a t 12.5 m~ unless specified otherwise. When indicated, trypticase peptone (BBL) was provided a t 1.0% final concentration from a 10% filter-sterilized solution. Bac- terial growth was monitored at 540 nm in a Klett-Summerson color- imeter.

Genetic Techniqaes-The bacteriophage Pluir was used in all transductions. Preparation of phage lysates, transductions, and mu- tagenesis were performed according to Miller (10). Strain LS7051 is a fadL fudRZ3:TnlO mutant and strain LS7050 is an isogenic fadL' fadRl3:TnZO derivative. These strains were obtained as follows. Wild type K12 was mutagenized with N-methyl-N'-nitronitrosoguanidine and mutants unable to grow on oleate (eis-A'-octadecenoic acid) were enriched for by a tritium suicide technique with tritiated oleate of high specific activity as a substrate (11). Some of these Ole- isolates were able to mutate such that decanoate could be used as the sole carbon source (Dec'), indicating that all of the enzymes required for &oxidation of fatty acids were functional. One such isolate, designated LS7049, was purified and characterized as a fudL mutant and the defect was mapped a t 50 min on the E. coli linkage map (3). Strain LS7051 is a fadRl3::TnZO derivative obtained by transducing LS7049 with phage Pluir grown on strain RS3040 and selecting for tetracycline resistance (Tc'). Strain LS7050 was obtained by transducing LS7051 with Plvir grown on strain K12 and selecting for growth on oleate as a sole carbon source (Ole').

Since 4-pentenoate is transported into E . coli by the acetoacetate: acetyl-coA transferase system (12), atoC (But') derivatives were required to study inhibition of fatty acid transport by 4-pentenoate. Spontaneous But' derivatives of LS7050 and LS7051 were obtained by plating on 10 mM butyrate. These strains were characterized as utoC mutants and designated LS7060 and LS7061, respectively.

Strain LS7071 is a spontaneous Dee' derivative of LS7049. LS7070

TABLE I Bacterial strains used

Strain Genotype

K12 Prototrophic LS7049 fadL LS7050 fudRZ3::Tn10h LS7051 fadl, fadRI3::TnlO LS7060 atoC fudRZ3:TnlO LS7061 fudL uloC fadRl3:TnlO LS7070 fadR LS7071 fadL fadR LS7072 fadR fudD zea::TnlO LS7073 fudL fadR fadD zea::TnlO LS7074 fudR fad5::TnlO LS7075 fadL fadR fad5::TnlO LS7076 fudR fadE zaf:TnlO LS7077 fadL fadR fudE zat:TnlO HS3040 fadRl3::TnlO K1 fad5 K19 fadE62 K27 fadD86 JF568 proC24 aroA357 ilu-277

metB6.5 his-52 cyc-1 xyl-14 lucY64 rpsL97 tsx-6.3

JF701 JF568 ompC JF703 JF568 ompF uroA +

JF694 JF568 ompC ompF nmpA +

Source

J . Lederberg via CGSC" This work This work This work This work This work This work This work This work This work This work This work This work This work R. Simons et ul. (8) P. Overath via CGSC P. Overath via CGSC P. Overath via CGSC J. Foulds via CGSC

J . Foulds via CGSC J. Foulds via CGSC J. Foulds via CGSC

" CGSC strains obtained from B. Bachmann, Coli Genetic Stock Center, Yale University, New Haven, Conn.

Transposon insertions are designated as previously described (14, 28). When an insertion is not within a known gene it is given a three- letter symbol starting with z, and the second and third letters indicate the approximate map location in minutes (eg. zaf corresponds to 5 min, and zbb corresponds to 11 min).

was obtained by transducing LS7071 with Pluir grown on K12 and selecting for Ole' transductants. To obtain fad- strains in an isogenic fadL and fudL' background, the transposon TnZO was inserted into the E. coli chromosome near the genes of interest and Tc' was used as a selective marker to move the fad- mutations into LS7071 and LS7070 (13). The fadD derivatives of LS7071 and LS7070 were obtained as follows: strain K27 (fudD) was transduced to Fad'Tc' with a Pluir phage stock grown on a random pool of K13 colonies, each individually Tc' due to the insertion of TnlO in a different region of the chromosome (8, 14). A Pluir phage stock of this strain was used to transduce K27 to Tc'. Both Fad' and Fad- transductants were obtained. A Fad-Tc' transductant was isolated and a Ploir phage stock was prepared from it. This phage stock was then used to transduce strains LS7071 and LS7070, and a Fad Tc' (fudD zea:: TnZO) transductant of each was isolated. These strains were desig- nated LS7073 (fadD zea::TnZO fadR fadL) and LS7072 (fudD zea:: TnZOfadR), respectively. Strains LS7077 (fadE za/?TnlOfadRfudL) and LS7076 (fadE zuf:TnlO fudR) were obtained as described above by insertion of TnlO near the fndE mutation in strain K19. Strains LS7075 (fud5::Tn5 fadR fudL) and LS7074 (fadkTn5 fudR), which have a polar mutation in the fadABC operon (5), were obtained by a localized mutagenesis by insertion of Tn5 near the metE gene as described (8), except kanamycin resistance (Kn') was used as a selective marker.

Assay of Fatty Acid Transport-Bacteria were generally grown overnight in M9-Brij containing 5 mM oleate and 1.08 of trypticase peptone, subcultured into the same medium and grown to a density of approximately 6.0 X loH cells/ml. Cells were then harvested by centrifugation a t 10,ooO X g for 5 min a t 4 "C, washed twice with M9- Brij, and briefly held on ice until use. Prior to assay of uptake, cells were resuspended to approximately 1.2 X 10q cells/ml in M9-Brij containing 100 pg/ml of chloramphenicol (Sigma) in 50-ml Erlen- meyer flasks, and starved for a carbon source for 15 min in a 25 O C water bath with a shake rate of 200 rpm. Cells were then diluted 2- fold (to yield 6.0 X 10" cells/ml) into another 50-ml Erlenmeyer flask containing the potassium salt of the l-''C-labeled fatty acid in M9- Brij and 100 pg/ml of chloramphenicol. The specific activity of fatty acids used ranged from 0.75 to 3.0 pCi/pmol depending upon the study. Unless stated otherwise, triplicate 0.5-nd samples were re- moved at 0.25 and 2.25 min and rapidly pipetted onto Sartorius membrane filters (type SM11307, 0.2 pm pore size) covered with 2.5 ml of M9-Brij. The filtered cells were then washed twice with 2.5 ml of M9-Brij. Filtration and washing were accomplished a t room tem- perature. Filters were air-dried and counted in ACS liquid scintillation fluid (AmershamiSearle) in a liquid scintillation counter. Correction for fatty acids adsorbed to the cell surface and filter was made by subtracting a 0.25-min value from a 2.25-min value for each sample. The loss of l-'4C-labeled fatty acids as "CO, under the conditions of this assay was negligible (see "Results"). Inclusion of carbon source levels of succinate or dextrose during the transport assay did not affect the rate of fatty acid transport observed (data not shown).

For studies involving inhibitors, pH variation, or temperature shifts, cells were starved for 10 min as described above followed by 5 min of preincubation under the conditions of transport. Studies on arsenate inhibition were performed in a medium identical to M9-Brij except with 25 mM Tris-HC1 buffer substituted for the phosphate buffer to provide a phosphate-free medium. Transport assays per- formed in this phosphate-free medium showed uptake similar to that observed in M9 medium.

Assay of P-Oxidation-Fatty acid oxidation was monitored by determining the amount of '"CO, formed from l-'4C-labeled fatty acids in oiuo. Cells were grown, washed, and incubated as described for fatty acid transport. For assay of "COP released, cells were incubated with the 1-'"C-labeled fatty acid in rubber-stoppered 25-ml Erlenmeyer flasks containing center wells (Kontes). After 60 min, 0.2 ml of ethano1amine:ethanol (1: 1 v/v) was added to the center wells to trap evolved CO, and 0.5 ml of 5 N HrSO, was added to the cell suspensions. Flasks were allowed to continue shaking for 60 min to ensure complete evolutioh and trapping of COL. The center wells were then removed, the bottoms were wiped, and the center wells were placed directly in scintillation vials. Liquid scintillation fluid (ACS) was added and the samples were counted in a liquid scintillation counter after dark adaptation. Values for control flasks lacking cells were subtracted as background for each condition assayed.

Enzyme Assays-Acyl-CoA synthetase was assayed by a modifi- cation of the procedure of Kamiryo et al. (15). Crude extracts were prepared by disruption of bacteria in a French press as previously described (13) and the lysate was centrifuged at 800 X g for 20 min at

Fatty Acid Transport 3737

4°C. The reaction mixture contained 1.25 pmol of ATP, 4 pnol of MgC12, 1 p o l of EDTA, 10 pmol of NaF, 1.5 mg of Brij 58, 20 pl of crude extract (100 to 200 pg of protein), and 5 to 500 nmol of l-I4C- labeled fatty acid (1 nCi/nmol) in 0.5 ml250 m~ Tris-HC1 buffer (pH 7.5). The reaction mixtures were preincubated 10 min in a gyrotory water bath shaker at 25’C with a shake rate of 200 rpm. The reaction was initiated by addition of 0.25 pnol of coenzyme A and reincubated as above. After 10 min, the reaction was stopped by adding 2.5 ml of isopropyl alcoho1heptane:l M H2S04 (4020:l) and vortexing. Then, 0.5 ml of Hz0 and 3 ml of heptane were added, the samples were vortexed for 10 s , and the organic phase was removed. The aqueous phase was extracted twice with 3 ml of heptane as above. A 1.0-ml aliquot of the aqueous phase was added to ACS liquid scintillation fluid and counted. Values for controls lacking coenzyme A were subtracted as background for each condition assayed. Other fad enzymes were assayed as previously described (4).

Intracellular Concentration of Fatty Acids-Intracellular water volume was determined as described by Maloney et al. (16). An intracellular water volume of 8.71 pl/mg of protein was obtained. This value was used to calculate the nanomoles of fatty acid accumulated per pl of cellular water during kinetic experiments.

Materials-All fatty acids were used without further purification. All unlabeled fatty acids except hexanoate were purchased from Sigma Chemical Co., St. Louis, MO. Hexanoic acid was purchased

‘ 6 r ” - 7 c 14

“- I O

- E 8

6

4

2

5 10 15 20 i

tlme (minutes)

FIG. 1. Uptake and 8-oxidation of long and medium chain fatty acids by strain LS7050 V i L * f i R ) . Transport and p- oxidation of l-I4C-fatty acids determined as described under “Exper- imental Procedures,” with an initial concentration of 104 PM fatty acid. CIX-~ accumulated inside the cell (0); sum of CIX-I accumulated and Clel lost as I4COz due to p-oxidation (0); CIO accumulated inside the cell (0); sum of CIO accumulated inside the cell and Cln lost as I4COz due to p-oxidation (m).

A B

fadL fadR 1.1 8 ”

4

2 ,i ob [;i I w

I00 200 300 400 500

lpdR ( 0 1

2 :-.../I: I om [g I002

100 200 300 400 500

lpdR ( 0 1

2 :-.../I: I om [g I002

100 200 300 400 500 [olelc acld] pM [decanoic o c ~ d ] pM

FIG. 2. Kinetics of transport and 8-oxidation of long and medium chain fatty acids. Rate of transport of fatty acids (panels A, B, and C ) were determined as described under “Experimental Procedures.” Transport of Cia I (panel A ) and CIO (panels B and C ) by strains LS7050 (fadL’ fadR) (0) and LS7051 (fadL fadR) (0) are shown. Lineweaver-Burk plots of these data are shown in the insets.

from Calbiochem, La Jolla, CA. All of the I4C-labeled fatty acids, except [l-14C]hexadecanoic acid and cis-As-octadecenoic acid, were purchased from Dhom Products, Hollywood, CA. [l-“’C]Hexadeca- noic acid (palmitate) and cis-A’-octadecenoic acid (oleate) were pur- chased from New England Nuclear, Boston, MA. All other chemicals used were of reagent grade.

RESULTS

Kinetics of Fatty Acid Transport-The kinetics of long and medium chain fatty acid transport were studied in strains LS7051 and LS7050 to further characterize the role of the fadL gene product in fatty acid transport. Both strains are constitutive for the synthesis of the fad enzymes (fadR).

The rate of uptake of long and medium chain fatty acids was linear for the fwst 2.5 min (Fig. 1). During this time, negligible loss of “ C 0 2 from 1-l4C-fatty acids was observed. After 2.5 min, loss of I4CO2 due to @-oxidation became signifi- cant, causing a decrease in the rate of accumulation of labeled fatty acid inside the cell. However, the rate obtained by adding the sum of the label lost as 14C02 and l-l4C-fatty acid accu- mulated inside the cell from 2.5 to 20 min is equivalent to the rate of accumulation by 2.5 min (Fig. 1). Therefore, in our experiments, the net uptake of fatty acids was measured between 0.25 and 2.25 min after addition of the fatty acid. When lipids were extracted immediately after uptake of [l-

described (4), it was determined that neither fatty acid was incorporated to a significant extent into phospholipids (data not shown).

As previously reported (3, 4), the fadL gene product is required for transport of long chain (>Cll)fatty acids (Fig. 2 A ) . A representative Michaelis-Menten curve for the trans- port of the long chain fatty acid C l ~ : l by LS7050 (fadL+ fadR) is shown in Fig. 2 4 . Transport of each long chain fatty acid studied (Cll-Cl%l) occurred by a saturable process with char- acteristic Kt and Vmax values (Table 11). Strain LS7051 (fadL fadR) failed to transport fatty acids with a chain length ?Cl1 at any concentration tested.

Both fadL’ fadR and fadL fadR strains can grow on medium chain fatty acids at equivalent rates (4). Therefore, it was of considerable interest to determine the specificity and possible mechanism of medium chain fatty acid transport by E. coli. Transport of C7-Cl0 by LS7050 (fadLC fadR) showed saturation kinetics with K, values considerably higher than for long chain fatty acids (Table 11). A representative Michae- lis-Menten curve for the transport of the medium chain fatty acid CIO by strain LS7050 (fadL’ fadR) is shown in Fig. 2B. Significant substrate inhibition of fatty acid transport was observed a t concentrations greater than 300 PM of all fatty acids with a chain length sC12. However, no substrate inhi-

14 Cldecanoate or [l-’4C]oleate and separated as previously

C D I fadL fodR 1.1 I

500 1000 1500 2000 2X J IO

[deconac acld] pM [ fatly ocld] pM

Velocity ( u ) is expressed as nmol min” mg of protein”, and S is the concentration of fatty acids in p ~ . Panel D shows the kinetics of p- oxidation determined from the rate of loss of I4CO2 from l-I4C-fatty acids as described under “Experimental Procedures.” Rate of p-oxi- dation of Cl~ . l (0) and CIO (0) by strain LS7050 (fadL’ fadR) and rate of p-oxidation of C1, by strain LS7051 (fadL fadR) (0) are shown.

3738 Fatty Acid Transport

TABLE I1 Kinetic parameters of fatty acid transport

fadR). Kinetics of fatty acid transport shown for strain LS7050 ( f ad . +

Fatty acid Kt Vm".

F M nmol min" rngprotern" C6 C; 152h 8.34 C" 122 3.50 CS 21 1 6.74 ClO 264 5.50 CII 243 4.70 CIP 76 9.40 c14 41 7.93 CiG 67 5.79 CI81 132 8.76

- _"

- indicates no detectable transport observed. Kinetic constants for medium chain fatty acid transport by the

fadL+ strain corrected for nonsaturable transport component.

bition was seen with longer chain fatty acids (>C,,) a t 25 "C. The substrate inhibition observed could possibly be due to (i) micelle formation at higher concentrations of fatty acids, or (ii) fatty acid toxicity. It seems unlikely that substrate inhi- bition is due to micelle formation since substrate inhibition is most predominant with medium chain fatty acids, while longer chain length fatty acids have lower critical micelle concentra- tions (17). However, the results are consistent with a fatty acid toxicity effect. High concentrations of medium chain fatty acids have been shown to inhibit bacterial growth and metabolism and cause cell lysis (18-20). Fatty acid toxicity is greater with shorter chain length fatty acids and increases at higher temperatures (18-20). At 25 "C, we observed greater substrate inhibition with shorter chain length fatty acids ( S I * ) (data not shown). Furthermore, in our studies we have observed substrate inhibition of C I ~ - ~ transport when assayed at 30 "C (data not shown). These results are complementary to the results of Frerman and Bennett (1) .

Strain LS7051 (fadL fadR) failed to show saturation kinet- ics for transport of medium chain fatty acids, although sub- strate inhibition was observed a t higher fatty acid concentra- tions. A representative Michaelis-Menten curve for the trans- port of the medium chain fatty acid Cl0 by strain LS7051 is shown in Fig. 2C. Double reciprocal plots of this transport data pass through the origin (Fig. 2C). Such plot,s, which place more emphasis on the lower, more physiological substrate concentrations rather than the higher concentrations, strongly suggest that medium chain fatty acid uptake occurs by a diffusional transport mechanism in fadL strains (21).

Intracellular Accumulation of Fatty Acid-In order to further characterize the mechanism of long and medium chain fatty acid transport, the relative accumulation of fatty acids inside the cell was determined. Strain LS7050 (fadL' fadR) concentrated C1%, inside the cell 8- to 10-fold over the exoge- nous concentration (Table 111). These results suggest that long chain fatty acid transport by fa&' strains occurs by either a group translocation or active transport mechanism allowing concentration of fatty acids against a concentration gradient. LS7050 concentrated C,O 2- to 3-fold over the exter- nal concentration (Table 111). However, strain LS7051 (fadL fadR) failed to show a significantly greater internal concen- tration of Clo than that in the external medium (Table 111). These results suggest that approximately 67% of the CIO transported by the fadL' strain is due to the fudL gene product. This may account for the apparent saturation of Cl0 transport observed in the fadL' but not the fudL strain.

Efflux of Fatty Acids-If E. coli transported fatty acids by an active transport or group translocation mechanism, then

incorporated fatty acids would be retained in the cytosol after washing with unlabeled fatty acids. However, if fatty acids were transported by a diffusional process, then efflux of incor- porated fatty acids in exchange for unlabeled substrate could occur. Thus, the relative efflux of long and medium chain fatty acids upon washing with unlabeled fatty acid was stud- ied. Less than 3% of the CIH:I incorporated by the fadL+ strain could be removed from the cytosol by unlabeled or Clo immediately following transport (Table IV). However, 28% of the Clo incorporated by the fudL' strain and 84% of the Clo incorporated by the fadL strain was lost upon washing with unlabeled Clo (Table 4). Washing with Cln:, had a similar effect on efflux of incorporated G o by the fadL' strain. These results suggest that Clo transported by the fadl-mediated mechanism is not available for exchange with exogenous fatty acid. Furthermore, these results imply that over 55% of the Clo transported by the fudL' strain enters via the fadL'- mediated mechanism.

On the other hand, if the cells were allowed to transport labeled fatty acids, then rapidly chilled, pelleted, and washed prior to resuspension in the unlabeled fatty acid as described by Klein et ul. (2), no efflux of or Clo fatty acid was observed in either strain (data not shown). This may be due to the complete conversion of labeled fatty acids to metabolic intermediates prior to washing with unlabeled fatty acids.

Kinetics of Fatty Acid Degradation-The kinetics of fatty acid degradation under the conditions used in the transport studies was determined in order to compare transport with further degradation. The kinetics of ,&oxidation of fatty acids in vivo are shown in Fig. 2 0 . &Oxidation of Cls:1 by the fudL+ strain showed saturation kinetics with a K, of 185 p~ and

TABLE I11 Net concentration of external fatty acids into the cytosol

Fatty acid External concen- Internal concentration

LS7050 LS7051 tration

PM p M1'

ClX I 50 536 100 925 250 2747 500 5100

h - - - -

c I" 50 147 48 100 294 74 250 592 215 500 1307 482

Net concentration of fatty acids into the cytosol calculated as

No net transport of CIS I was observed in strain LS7051. described under "Experimental Procedures."

TABLE IV Efflux of accumulated fatty acids

Bacteria were allowed to transport fatty acids for 2 min as described under "Experimental Procedures" and aliquots were removed and directly placed in an equal concentration of M9-Brij or M9-Brij + 5 mM fatty acid. These samples were washed once with the same solution, followed by one wash with M9-Brij.

Fatty acid Wash conditions Per cent efflux

LS7050 LS7051

CIR 1 M9-Brij 0" M9-Brij-C1" I 2.47 M9-Brij-Clo M9-Brij 0 0 MS-Brij-CIH 1 30.88 4.73 M9-Brii-Crn 27.67 83.83

6 - -

0 ClO

-

~~ ~

'' No efflux of incorporated fatty acid was observed following mul- tiple washes with M9-Brij.

No transport of C],, was observed in the fadL strain.

Fatty Acid Transport 3739

Vmax of 6.25 nmol min" mg of protein". /3-Oxidation of Clo by the fadL+ strain showed kinetics suggesting both a saturable and nonsaturable component (Fig. 20). The K,,, of the satur- able component was 357 p~ and the Vmax was 8.00 nmol min-I mg of protein". The fadL strain did not show saturation kinetics for the @-oxidation of Clo at concentrations less than 2 mM under these conditions (Fig. 2 0 ) . The kinetics of /3- oxidation of Clo by the fadL strain are similar to those observed for fatty acid transport, suggesting that under the conditions used, transport is the rate limiting step in fatty acid degradation.

The kinetics of acyl-CoA synthetase, the f i s t enzyme of the /3-oxidation pathway, was also determined in fadL' and fadL strains under conditions similar to the transport assay. The K, and V,,, values of acyl-CoA synthetase for CIX:I and Cl0 in fadL' and fadL strains were comparable. The acyl-CoA syn- thetase in strain LS7050 (fadL' fadR) had a K,,, of 87.3 p~ and a Vmax of 0.63 nmol min" mg of protein" for Cl8.1 and a K,,, of 133.5 p~ and V,,, of 0.65 nmol min" mg of protein" for Clo. This enzyme in strain LS7051 (fadL fadR) had a K , of 88.4 PM and v,,, of 0.60 nmol min" mg of protein" for Clal and a K, of 78.0 ~ L M and V,,, of 0.75 nmol mir"' mg of protein" for Clo. In each case, the K,,, and V,,, values were considerably less than those observed for transport in uiuo; however, it should be noted that acyl-CoA synthetase was assayed in uitro. Furthermore, the K,,, of acyl-CoA synthetase in crude extracts was observed to vary over 20-fold with different growth and assay conditions (data not shown).

Transport of Fatty Acids by fad- Mutants-Transport of long and medium chain fatty acids was studied in mutants defective for enzymes required for /3-oxidation of fatty acids. Mutants with lesions in fadD (acyl-CoA synthetase), fadE (acyl-CoA dehydrogenase associated flavoprotein), and polar mutations in the fadABC operon (enoyl-CoA hydratase, 3- hydroxyacyl-CoA dehydrogenase, thiolase) were studied. In agreement with the results of Klein et al. ( 2 ) , both fadD and fadE mutants failed to transport or @-oxidize ClR:, or Clo at any concentration studied (Table V). Klein et al. (2) have proposed that the lack of transport in fad- strains indicates there is an absolute linkage between transport and /3-oxida- tion. However, fadABC mutants, although somewhat leaky for /?-oxidation (Table V), showed significant rates of transport of both Clx and Clo (Table V) despite the absence of fadABC enzymatic activities' in in uitro enzyme assays ( 2 ) . I t is not clear why, if fatty acid transport is strictly coupled to p- oxidation, fadABC mutants continue to transport fatty acids at rates significantly greater than the rates they /3-oxidize them.

Effects of Metabolic Inhibitors on Fatty Acid Transport a n d /3-Oxidation-The energy requirements for fatty acid transport and the linkage between transport and further me- tabolism was studied with metabolic inhibitors. Under the conditions used, arsenate failed to inhibit long or medium chain fatty acid transport although further /3-oxidation was inhibited 40 to 50% (Table VI). The electron transport inhib- itors, 2,4-dinitrophenol, and cyanide inhibited both transport and /3-oxidation of long and medium chain fatty acids. How- ever, in each case, Clo transport by the fadL strain showed less inhibition than Clo or CIR transport and oxidation by the fadL' strain (Table VI). The inhibitor 4-pentenoate fully inhibited both Cl0 and fatty acid transport and oxidation in both fadL atoC and fudL' atoC strains. 4-Pentenoate inhibits growth of fadD mutants, but growth inhibition fails to occur in fadE mutants.2 Thus, it seems possible that 4-

S. R. Maloy, and C. L. Ginshurgh, unpublished results. W. D. Nunn, unpublished observations.

TABLE V Fatty acid transport and P-oxidation by fad- mutants

Rate of 8-oxida- Rate of trans- tion" p o d

Clll ClHl Clll CI"l

Strain Genotype

LS7070 fadR 3.66 3.94 3.72 3.48 LS7071 fadL fadR 1.09 <0.01 2.33 ~ 0 . 0 1 LS7072 fadD fadL fadR <0.01 <0.01 tO.O1 <0.01 LS7073 fadD fadR 10.01 t0.01 tO.O1 <0.01 LS7074 fadABCfadL fadR 0.04 tO.01 0.61 <0.01 LS7075 fadABC fadR 0.08 0.27 1.56 1.25 LS7076 fadE fadL fadR <0.01 t0.01 tO.O1 <0.01 LS7077 fadE fadR <0.01 <0.01 t0.01 tO.O1

Rate of /3-oxidation of 250 PM l-I4C-fatty acid under conditions of the fatty acid transport assay as described under "Experimental Procedures." Values are expressed as nmol min-l mg of protein".

'Rate of fatty acid transport as described under "Experimental Procedures" expressed as nmol min" mg of protein I .

TABLE VI Effects of metabolic inhibitors on fatty acid transport and

metabolism B Inhibition of transport B Inhibition of 8-oxidation

Inhibitor" LS7050 LS7051 LS7050 LS7051

c,,, ClVl Clii ClO cis, Clll

None 0 0 0 0 0 0 Arsenate 0 0 0 42 58 38 Dinitrophenol 99 99 97 99 99 99 Cyanide 97 91 62 99 99 98 4-Pentonoate' 99 99 99 99 99 99

" Inhibitors present at concentrations causing maximal inhibition of fatty acid transport and oxidation as follows: arsenate, 100 mM; dinitrophenol, 1 mM; cyanide, 100 mM; 4-pentenoate, 2.5 mM.

'Isogenic But+ derivatives of LS7050 and LS7051 were used to determine 4-pentenoate inhibition. These strains were LS7060 ( fadR a t 0 0 and LS7061 ( fadL fadR atoC).

pentenoate may indirectly inhibit transport of fatty acids by rapidly inhibiting /3-oxidation with concomitant accumulation of a pool of this inhibitory fatty acid, rather than by tying up the cellular coenzyme A as previously proposed (12).

Transport of Fatty Acids by Porin Mutants-The outer membrane of Gram-negative enteric bacteria is believed to act as a barrier to penetration of most hydrophobic agents (22). Hydrophilic molecules, on the other hand, are believed to pass through the outer membrane either by (i) a carrier- mediated process or (ii) simple diffusion via nonspecific pro- tein pores formed by the outer membrane porins. Thus, the mechanism of transport of fatty acids across the outer mem- brane was of interest. Transport and /3-oxidation of CIH:, and CIO were studied in mutants defective in one or both of the major outer membrane porins of E. coli K12 (ompC and ompF) and the isogenic parental strain with functional porins (23). In the mutant defective for both ompC and ompF, another porin, the nmpA gene product, is derepressed allowing diffusion of some substances through the outer membrane. Both ompC and ompF mutants transport CIH I at approxi- mately one-third the rate of the isogenic omp+ strain (Table VII). The ompC ompF double mutant transported and oxi- dized ClX., at a lower rate than the single mutants despite the presence of the nmpA gene product, suggesting that the nmpA gene product fails to suppress the ompC and ompF mutations for CISI transport. On the other hand, although the ompC mutation decreased Clo transport to about one-third of the wild type value, the ompF mutation did not decrease C1,, transport (Table VII). The ompC ompF nmpA+ mutant trans- ported Clo at levels intermediate between the ompC and ompF mutant. These data suggest that fatty acids must diffuse

3740 Fatty Acid Transport

TABLE VI1 Fatty acid transport and 8-oxidation by mutants defective in outer

membrane porins Rate of /3-oxida- Rate of trans-

tion" port" Strain Genotype

Cm Cxnl CIO C,n,

JF568 parent loo loo 100 100 JF701 ompC 33 30 53 24 JF703 ompF 82 41 74 68 JF694 ompC ompF nmpA' 70 18 43 45

Expressed relative to the rates of transport and P-oxidation of 250 ~ L M l-I4C-fatty acid by strain JF568. These values were as follows: Clo 8-oxidation, 1.70 nmol min" mg of protein"; CIR I @-oxidation, 2.15 nmol min" mg protein", Cl0 transport, 1.55 nmol min" mg of protein-'; CIH.I transport, 1.78 nmol min" mg of protein".

l-

6t 5

i 12

pH FIG. 3. Effect of pH on the transport of long and medium

chain fatty acids. Bacteria were equilibrated for 5 min at the indicated pH, then added to flasks containing 250 ~ L M l-14C-fatty acid at the same pH, and the rate of transport determined as described under "Experimental Procedures." The rate of transport of CIH I fatty acid (A) and Cl0 fatty acid (B) by strain LS7050 (fa&' fadR) (0) and strain LS7051 (fadL fadR) (m) are shown.

across the outer membrane via porins prior to transport across the inner membrane.

Physical Parameters of Long and Medium Chain Fatty Acid Transport-The temperature, pH, and detergent de- pendence of long and medium chain fatty acid transport was studied. Both long and medium chain fatty acids showed maximal rates of uptake at 42 "C (data not shown). A QI0 of 2.3 was calculated for C I ~ I transport while CIO transport showed a Q10 of 1.27. From Arrhenius plots of the temperature dependence data, an activation energy of 11.72 kcal mol" was calculated for Clkl transport while Cia transport had an acti- vation energy of 8.20 kcal mol" in the fudL' and 4.56 kcal mol" in the fadL strain.

The pH dependence of long and medium chain fatty acid transport was studied in order to determine whether the dissociated or undissociated form of the fatty acids was trans- ported. The maximal rate of transport of both long and medium chain fatty acids by the fadL+ strain occurred above pH 5.0 (Fig. 3). Since both long and medium chain fatty acids have a pK, of 4.8 (24), this suggests that both long and

medium chain fatty acids are mainly transported in the dis- sociated form by the fadL+ strain. The rate of uptake of the undissociated form of medium chain fatty acids by fadL and fadL+ strains were equivalent.

The detergent dependence of long and medium chain fatty acid transport was studied in order to determine whether fatty acids are transported in the free state or as micelles. When present in solution below the critical micelle concentration, fatty acids are present as monomers, while above the CMC,:' the monomer concentration equals the CMC and any excess fatty acid exists in micelles. In the presence of detergents, mixed micelles of detergent and fatty acid are formed (25). Both long and medium chain fatty acids were transported at maximal rates at concentrations below the CMC of the added detergent (data not shown). This was observed in the presence of Brij 58 (CMC = 8.21 ifrg/ml) (26) as well as with several other detergents, including Triton X-100 (CMC = 14.88 pg/ m l ) (26) and Tween 80 (CMC = 1.3 ifrg/ml) (26). Since the maximal rate of fatty acid transport occurred under conditions where micelles would not be expected to form, this suggests that long and medium chain fatty acids are transported in the free form and not as micelles.

DISCUSSION

Several observations demonstrate that long chain fatty acids enter the cell by a concentrative transport process requiring the fadL gene product: (i) the transport of long chain fatty acids (CII-&..) into fudL' strains is a saturable process (Fig. 2); (ii) energy inhibitors which dissipate the electrochemical gradient inhibited transport of long chain fatty acids (Table VI); (iii) long chain fatty acids were concen- trated 8- to 10-fold inside the cell against a concentration gradient (Table 111); (iv) no efflux of transported long chain fatty acids occurred when cells were washed with unlabeled fatty acids; and (v) both the energy of activation and Q10 of long chain fatty acid transport are representative of enzyme- mediated processes. On the other hand, long chain fatty acids were not transported by fadL strains (Fig. 2 A ) . These results c o n f i previous findings (3, 4) and strongly suggest that the fadL gene product is required for the active transport of long chain fatty acids.

Since both fadL and fadL+ derivatives of fadR strains can grow on medium chain (C~-CIO) fatty acids, the transport of medium chain fatty acids was examined. The concentrative transport mechanism for long chain fatty acids in fudL+ strains was found to have overlapping specificity for medium chain fatty acids (Table 11). However, medium chain fatty acids were apparently taken up by a separate diffusional process in both fadL' and fadL strains. At higher concentra- tions of medium chain fatty acids, diffusion exceeded fadL+- mediated transport. The evidence for diffusion of medium chain fatty acids is as follows: (i) fadL strains did not concen- trate medium chain fatty acids against a concentration gra- dient (Table 111); (ii) medium chain fatty acid efflux was stimulated by washing with unlabeled medium chain fatty acids (Table IV); and (iii) kinetic analysis of medium chain fatty acid transport by fadL strains indicates a nonsaturable process within the range of concentrations studied (Fig. 2C). However, it should be noted that saturation of medium chain fatty acid transport may have been obscured by the toxicity of high concentrations of medium chain fatty acids.

Medium chain fatty acid uptake by fadL+ strains charac- teristically showed Michelis-Menten curves that suggested the presence of both a saturable and nonsaturable component (Fig. 2). The fadL+ strains concentrated medium chain fatty acids 2- to 3-fold inside the cell in contrast to no net concen-

The abbreviation used is: CMC, critical micelle concentration.

Fatty Acid Transport 3741

tration of medium chain fatty acids by fudL strains (Table 111). Finally, fudL’ strains only showed about 28% efflux of medium chain fatty acids when washed with unlabeled me- dium chain fatty acids in contrast to over 80% efflux in fadL strains (Table IV). These results suggest that the fudL gene product, although absolutely required for long chain fatty acid transport has some overlapping specificity for medium chain fatty acids. However, the fudL gene product is not required for the transport of medium chain fatty acids. These results support the previous findings of Nunn et ul. (4).

Kinetics of P-oxidation of both long and medium chain fatty acids suggest that the entry of fatty acids into the cell is the rate-limiting step in metabolism at concentrations less than 2 mM. Thus, at these concentrations, P-oxidation of medium chain fatty acids appears to be dependent upon the rate of diffusion of fatty acid into the cell in fudL strains (Fig. 2 0 ) . However, in mutants with a fudD or fudE lesion, both long and medium chain fatty acid transport is abolished (Table V). This implies that fatty acid transport is tightly coupled to further metabolism as previously suggested (1, 2 ) . Further- more, several energy inhibitors block both fatty acid transport and metabolism (Table VI), although it is not known which process is primarily affected. However, it seems noteworthy that the metabolic inhibitor cyanide caused about 30% less inhibition of medium chain fatty acid transport than long chain fatty acid transport. This may reflect an energy require- ment for active transport of long chain fatty acids, while metabolic inhibitors only act at the level of further metabolism of medium chain fatty acids. This hypothesis is also consistent with the lower energy of activation and lower 810 observed for medium chain fatty acid transport. Furthermore, the observed energy of activation for transport of medium chain fatty acids by the fudL strain (4.56 kcal mol”) is in close agreement with the theoretical energy of activation (4.64 kcal mol”) calcu- lated for a diffusion-mediated process in a dilute aqueous solution (authors’ calculation^).^

Further studies were performed to characterize the physical parameters of long and medium chain fatty acid transport. Although fatty acids normally were present as mixed micelles with nonionic detergents, both long and medium chain fatty acids appear to be transported primarily as the dissociated acid (Fig. 3). Medium chain fatty acids, on the other hand, show significant permeability in the undissociated state in both fudL and fudL+ strains (Fig. 3), although there is a marked stimulation of medium chain fatty acid transport a t pH values above the pK, of the fatty acid (Fig. 3). This suggests that fudL-mediated fatty acid transport utilizes the dissociated acid, whereas medium chain fatty acids may also diffuse through the cytoplasmic membrane as the undisso- ciated lipophile. This trend agrees with the results of other workers which demonstrated greater toxicity of undissociated medium chain fatty acids than the dissociated acids (18-20). However, it should be noted that this could also be due to effects of pH on other cellular properties, such as the ioniza- tion state of membrane components, specific enzymes, or micelle formation.

Preliminary studies suggest that the fudL gene product is an inner membrane protein. Evidence for this is as follows: (i) spheroplasts of fudL mutants fail to P-oxidize CIH in the presence of bovine serum albumin, while fudL’ strains P- oxidize CIHl under these conditions,s and (ii) two dimensional gel electrophoresis indicate the absence of an inner membrane protein in fudL fudR strains compared to isogenic fudL’ fudR strains.6 However, the outer membrane of Gram-negative

R. C. Warner, personal communications. S. R. Maloy and R. W. Simons, unpublished observations. C. L. Ginsburgh and W. D. Nunn, unpublished observations.

enteric bacteria is believed to constitute a barrier to hydro- phobic agents. Thus, the mechanism of transport of fatty acids across the outer membrane was of interest. Defects in outer membrane porins (ornp) were found to decrease both long and medium chain fatty acid transport (Table VII). The greater rate of transport of fatty acids by ornp’ strains suggests that fatty acids may cross the outer membrane via porins. This suggestion is consistent with previous studies on medium chain fatty acid toxicity in mutants defective for outer mem- brane biosynthesis (2) which suggest that the dissociated form of fatty acids are unable to pass directly through the wild type outer membrane of E . coli. This may further suggest that the fudL gene product is an inner membrane transport protein.

The results presented in this paper provide further evidence for separate mechanisms of long and medium chain fatty acid transport in E. coli. Uptake of long chain fatty acids appears to occur by an “active transport” mechanism mediated by a specific “carrier protein,” the fudL gene product. Although this component has some affinity for medium chain fatty acids, medium chain fatty acids also appear to enter the cell by a diffusional mechanism. This is analogous to fatty acid transport by mitochondria, where long chain fatty acids con- jugated to carnitine are transported by acylcarnitine transfer- ase, while medium chain fatty acids cross the membrane barrier without the aid of the carnitine system (27 ) .

Acknowledgments-We thank Barbara Bachmann for her gener- ous gifts of strains. We are grateful to Robert C. Warner, Rowland H. Davis, Grover C. Stephens, and Paula Hennen for helpful discussions and comments on the manuscript. We gratefully acknowledge the assistance of Ken Kemeda in the development of the acyl-CoA synthetase assay.

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