Vol. 153, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1983, p. 232-2400021-9193/83/010232-09$02.00/0Copyright © 1983, American Society for Microbiology

Porin Channels in Escherichia coli: Studies with 13-Lactams inIntact Cells

HIROSHI NIKAIDO,1* EMIKO Y. ROSENBERG,' AND JOHN FOULDS2Department of Microbiology and Immunology, University of California, Berkeley, California 94720,1 and

National Institute ofArthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health,Bethesda, Maryland 202052

Received 18 June 1982/Accepted 8 October 1982

Wild-type Escherichia coli K-12 produces two porins, OmpF (protein la) andOmpC (protein lb). In mutants deficient in both of these "normal" porins,secondary mutants that produce a "new" porin, protein PhoE (protein E), areselected for. We determined the properties of the channels produced by each ofthese porins by measuring the rates of diffusion of various cephalosporins throughthe outer membrane in strains producing only one porin species. We found that allporin channels retarded the diffusion of more hydrophobic cephalosporins andthat with monoanionic cephalosporins a 10-fold increase in the octanol-waterpartition coefficient of the solute produced a 5- to 6-fold decrease in the rate ofpenetration. Electrical charges of the solutes had different effects on differentchannels. Thus, with the normal porins (i.e., OmpF and OmpC proteins)additional negative charge drastically reduced the penetration rate through thechannels, whereas additional positive charge significantly accelerated the penetra-tion. In contrast, diffusion through the PhoE channel was unaffected by thepresence of an additional negative charge. We hypothesize that the relativeexclusion of hydrophobic and negatively charged solutes by normal porinchannels is of ecological advantage to E. coli, which must exclude hydrophobicand anionic bile salts in its natural habitat. The properties of the PhoE porin arealso consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol.143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157,1980) that its biosynthesis is derepressed by phosphate starvation; the channelmay thus act as an emergency pore primarily for the uptake of phosphate andphosphorylated compounds.

Gram-negative bacteria, including Escherich-ia coli, are covered by outer membranes, so thatnutrients as well as antibiotics must first pene-trate through these membranes before theyreach their final destination or target. Fraction-ation and in vitro reconstitution of active princi-ples, as well as the use of mutants deficient inthe presumptive active component, have led usto conclude that most nutrients and most antibi-otics penetrate the outer membrane of E. coliand related organisms through water-filled chan-nels produced by a class of proteins calledporins (4, 15, 19). In a previous study, we haveshown that the rates of penetration of hydrophil-ic, uncharged solutes through the E. coli channelwere very strongly dependent on the size of thesolute, an observation that led to the estimationof the pore diameter at about 1.2 nm (21). In afurther attempt to characterize the properties ofthe porin channel, we examined the effect ofsolute hydrophobicity and charge on penetrationrates through the channel. For this purpose, we

needed a group of closely related compoundsdiffering in hydrophobicity and charge and alsomethods for measuring the diffusion rates ofthese compounds across the outer membrane.Both of these conditions were fulfilled by semi-synthetic P-lactam compounds, as thousands ofthese compounds have been synthesized (17, 29)and measurement of their permeation rates re-cently became feasible (30, 39).Another point emphasized in the present

study is the comparison of the properties ofchannels produced by different porins. E. coli K-12 normally produces two species of porin, laand lb, coded by ompF and ompC genes, re-spectively (2, 3, 7, 32, 38). (These proteins willbe called OmpF and OmpC, respectively.) Fur-thermore, the relative expression of these twoproteins is regulated by environmental condi-tions (3, 16), and this observation suggested thatthe properties of OmpF and OmpC channelsmight be different. In addition, some mutantsdeficient in normal porins generate "second-site

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PORIN CHANNELS IN E. COLI 233

suppressor mutants" in which a normally re-pressed "new" porin becomes derepressed (10,25); the channel produced by such a new porin,protein E (also called Ic and e, recently shown tobe coded for by gene phoE [36], and thereforecalled PhoE in this paper), present in nmpAmutants, has been included in this comparison.

MATERIALS AND METHODS

Organisms. All strains used were derivatives of E.coli JF568 (K-12 aroA357 ilv-277 metB65 his-53 purE41cyc-l xyl-14 lacY29 rpsL77 tsx63) and produced onlyone species of porin. Thus, JF701 (JF568 ompC264)produced only the OmpF porin, JF703 (JF568ompF254) produced only the OmpC porin, and JF694(JF568 ompC264 ompF254 nmpAI) produced only thePhoE porin (8). In addition, an R-factor specifying theproduction of a TEM-type, periplasmic ,-lactamase,R471a (12), was transferred to these strains by conjuga-tion from YC215 (E. coli K-12 lac gal mtlxyl arafR471a,a gift of M. Yoshikawa). R471a was chosen because itcaused the production of very high levels of 1-lacta-mase activity, more than an order of magnitude higherthan that obtained by the incorporation of such plas-mids as Ri (12). The stock cultures were maintained at-70°C, and preculture was performed in the presenceof 100 jig of ampicillin per ml, to prevent the loss of theplasmid. The extracts of the three strains containedabout equal levels of ,-lactamase; this indicates thatwe did not get preferential loss of plasmids from any ofthese strains.

Cephalosporins. Cephaloridine, cefazolin, cefaman-dole, cephalothin, cephaloglycin, and cephaloram, aswell as the experimental compound 7-[2-(2-benzo-thienyl)acetamido]cephalosporanic acid (benzothien-ylcephalosporin), were gifts of Lee F. Ellis, Eli Lilly &Co., Indianapolis, Ind. Cephacetrile and cephapirinwere donated by W. Zimmermann, CIBA-Geigy, Ba-sel, Switzerland, and F. Leitner, Bristol Laboratories,Syracuse, N.Y., respectively. SCE-20 and cefsulodinwere gifts from H. Nomura, Takeda PharmaceuticalCo., Osaka, Japan. The structures of these compoundsare shown in Fig. 1.Assay for hydrolysis of cephalosporins. Because the

absorption of cephalosporins in the vicinity of 260 nmis altered drastically by the cleavage of the ,B-lactamring (9, 18), continuous recording of optical density inthis range provided a convenient and accurate methodfor following the hydrolysis of cephalosporins. How-ever, when hydrolysis by intact cells was measured,the extensive scattering of light by these cells contrib-uted very strongly to the background, making theassay nearly impossible. We circumvented this diffi-culty by using cuvettes of 1-mm light path. Thisallowed us to use 10-fold-higher concentrations of bothcells and cephalosporins than would have been possi-ble with the standard, 10-mm cuvette. Since the ratesof hydrolysis by intact cells are largely limited bydiffusion rates through the outer membrane, which inturn are more or less proportional to the externalconcentration of cephalosporins, under these condi-tions the rate of hydrolysis is accelerated by a factor of10 x 10, or 100. Although we lose photometric sensi-tivity by a factor of 10, the overall effect of using the 1-mm cells is an approximately 10-fold gain in the

sensitivity of the assay, which enabled us to followaccurately the hydrolysis of slowly degraded cephalo-sporins by intact cells.The practical arrangement for the experiments was

as follows. Cells of bacterial strains carrying the Rplasmid were grown and washed as described previ-ously (20). A portion of the cell suspension (5 mg [dryweight] ml-') was sonicated as described before (20),and the sonic extract was used for the determination ofVmax (and in preliminary experiments also the Kin) ofthe periplasmic P-lactamase. Intact cells (0.15 mg [dryweight]) or extracts were added to an assay mediumcontaining 10 mM sodium phosphate buffer (pH 6.0)-S5mM MgCl2-1 mM cephalosporin (final volume, 0.5ml). The suspension was then mixed and rapidlytransferred to a cuvette with a 1-mm light path, and theoptical density was recorded at 260 nm with a Perkin-Elmer-Hitachi model 124 spectrophotometer. Assayswere performed at 25°C, and the diffusion rates of I-lactams across the outer membrane were calculatedessentially according to the method of Zimmermannand Rosselet (39). Km values of R471a ,-lactamasewere as follows: cephacetrile (1,350 ,uM), cefazolin(410 ,uM), cefamandole (780 ,uM), cephalothin (310,M), cephaloram (280 1±M), benzothienylcephalo-sporin (1,540 VtM), cephaloridine (930 ,uM), cephalo-glycin (680 ,uM), SCE-20 (780 p.M), and cefsulodin(540 FtM).An example of calculation of the permeability coeffi-

cient (P) is given below. When the sonic extract from0.15 mg (dry weight) of cells of JF701 (R471a) wasincubated in a 0.5-ml assay mixture containing 1 mMcephalothin, the optical density at 260 nm decreasedwith an initial rate of 0.109 min-1. In a separateexperiment, when the hydrolysis of various cephalo-sporin derivatives that were nominally 1 mM wasfollowed to completion, the total change in opticaldensity at 260 nm was found to be between 0.6 and 0.8.In view of the uncertainty regarding the purity of someof these compounds, we assumed that the completehydrolysis of a 1 mM solution of any cephalosporinwould decrease the optical density at 260 nm of thesolution by 0.8 in a cell of 1-mm light path (9). Thus,the rate of hydrolysis by an extract from 1 mg of cells(VeXt) is (0.109 . 0.8 - 0.15) x 0.5 x 103 = 454 nmolmin-'. Since the Km of the R417a ,-lactamase forcephalothin is 310 ,uM, from the Michaelis-Mentenrelationship, Vmax = Vext (1,000 + Km)/1,000 = 595nmol min-'. When intact cells (0.15 mg [dry weight])were incubated similarly with 1 mM cephalothin, therate of decrease of optical density at 260 nm was 0.027min-1, corresponding to the rate of hydrolysis of 113nmol min-' mg-'. When we substitute this rate(Vceis), the Vmax value obtained above, and the Kminto the Michaelis-Menten equation, we obtain a sub-strate concentration in the periplasmic space (Cp) of 73,uM (or 73 nmol cm-3). According to Fick's first law,Vcejjs = P * A * (CO- Cp), where A and C0 representthe area of cell surface per unit weight and theconcentration of the 3-lactam in the external medium,respectively. From the values of V,e,l, A (132 cm2mg-'; reference 33), C0 (1,000 nmol cm-3), and Cp, Pcan be estimated as (113 nmol min-1 mg-' 132 cm2mg-1) + (1,000 - 73 nmol cm3) = 9.2 x 10-4 cmmin-1 or 1.5 x 10-5 cm s-i.

Determination of the octanol-water partition coeffi-cient. Partition coefficients of the unionized forms (Pa)

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234 NIKAIDO, ROSENBERG, AND FOULDS

?I HR,-C-N>,S

COO.

RR R2

CEPHACETRILE N*C-CH2- -CH2OCOCH3

NMI~CEFAZOLIN I N-CH-C

CEFAMANDOLE -H- -CH2-SJ1OH

-CHtOCOCH3-7

CEPHALORAM 0CC27BN HIENYL- tI

CEPHALOSPORIN I_CEPHALORIDINE 1JCH2-

CEPHALOGLYCIN CH-NH

CEPHAPIRIN H*N3-S-CH2-SCE-20 H-

sotSO3CEFSULODIN Cm-

sO3

-CH2OGOCH3

-CH2OCOCH3

-CH

-CH20COCH3

-CH2OCOCH3

-CH20COCH3

_CHi2cO3CNH2FIG. 1. Structures of cephalosporins used in this

study.

of monobasic cephalosporins were determined as de-scribed by Tsuji et al. (37). Briefly, 1 mM solutions ofcephalosporin were made in 0.1 M glycine-HCl buffer,pH 2.0 or 3.0, or in 0.1 M sodium citrate buffer, pH4.0, and after vigorous shaking with an equal volumeof 1-octanol at 25°C, the concentrations of the drug inthe organic and aqueous phases were determined byspectrophotometry. The values of the apparent parti-tion coefficients (Ppp) obtained in this manner weremultiplied by ([H+] + Ka)/Ka, where [H+] and K.denote the hydrogen ion concentration in the aqueousphase and the dissociation constant of the cephalospo-rin, respectively, and the product was plotted against[H+]/Ka. The data points form a straight line, and theslope of the line corresponds to Pu (37). Ka wasobtained from the literature (37) for cephalothin andcefazolin. For other compounds, the exact pKa valueswere unknown. However, the nature of side chainsproduced very little difference in pKa values of cepha-losporins (9), and we found that an error of 0.5 pH unitin the assumed pKa value produced only an error ofabout 0.05 in the log Pu value obtained. Thus, weassumed the pKa of 2.4 for all other monobasic cepha-losporins. The values of log Pu obtained for cepha-cetrile, cefazolin, cefamandole, cephalothin, cepha-loram, and benzothienylcephalosporin were -0.45,-0.24, 0.50, 1.09, 1.31, and 1.68, respectively. The Pufor cefazolin was calculated by using Papp valuesobtained only at pH 4.0 and 3.0, because at pH 2.0 the

J. BACTERIOL.

effect of the protonation of the side chain (pKa = 1.7;see reference 37) became apparent.

Calculations of P. from chemical structure. Theexperimental determination of P, was very difficult orsometimes impossible with cephalosporins containingmore than one charged groups. Furthermore, in someof the comparisons, P, values for hypothetical com-pounds were required (see Results). The Pu, valueswere therefore calculated by a procedure based on theadditivity of hydrophobic interactions (11, 26). As anexample, the P, value for cephaloglycin was calculat-ed as follows. Since cephaloglycin contains a phenylgroup and a CHNH2 group instead of the thienylgroup-plus-CH2 group in the cephalothin, from the"fragmental constants" of phenyl group (1.90), CH(0.24), NH2 (-1.38), thienyl group (1.59), and CH2(0.53) (26) and the measured P, of cephalothin (1.09;see above), we can calculate the P, of cephaloglycin tobe 1.09 - 1.59 - 0.53 + 1.90 + 0.24 - 1.38 = -0.27.The P, values of other compounds were calculatedsimilarly by using the measured P, of cephalothin asthe starting point. Calculated P, values for cephaloramand benzothienylcephalosporin were 1.40, and 1.86,respectively, and were reasonably close to the experi-mentally determined values of 1.31 and 1.68; thisagreement suggested the general reliability of thesecalculations.

Determination of critical miceilar concentrations. Theshift in the absorption maximum of rhodamine 6G wasused as evidence for micelle formation (6). The cepha-losporins were dissolved in 10 mM sodium phosphatebuffer, pH 6.0, and the absorption spectra were re-corded at room temperature with a Perkin-Elmer-Hitachi model 124 spectrophotometer.

RESULTSExperimental approach. We have used the

Zimmermann-Rosselet method (39) of determin-ing the permeability of the outer membrane fromthe rates of hydrolysis of P-lactams by intactcells. Since the intrinsic ,B-lactamase activity ofE. coli K-12 strains is very weak, we had tointroduce an R-factor containing a ,-lactamasegene. R471a was chosen as an R-factor thatproduces very high levels of TEM-type 1-lacta-mase (12).Because the host cell has been modified by the

introduction of the R-factor, it was important toknow that the pores in the outer membrane werenot altered by this process. We believe that thisis a correct assumption because of the following.(i) The protein composition of the outer mem-brane was not altered noticeably by the intro-duction of the R-factor (Fig. 2). (ii) The introduc-tion of the R-factor into an E. coli mutantproducing greatly diminished amounts of porin(4) did not change the diffusion rates of sugarsacross the outer membrane (Nikaido and Rosen-berg, unpublished data), a result indicating thatthe R-factor does not produce additional non-specific channels. (iii) The strains containingR471a maintained the same sensitivity level tovarious antibiotics, such as tetracycline, chlor-

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PORIN CHANNELS IN E. COLI 235

__. ...

A B C D E F

FIG. 2. Sodium dodecyl sulfate-poly;slab gel electrophoresis of outer membranOuter membranes were prepared from varingrown in L broth, as described previousl)samples containing 15 ,ug of protein eachlyzed by electrophoresis as described by Iet al. (14). Lane A, JF-701 (containing OmpJF703 (containing OmpC only); C, JF694 (PhoE only); D, JF701 (R471a); E, JF703 (R4,JF694 (R471,). Molecular weight standardsylase b, 92,500; bovine serum albumin, 66,24min, 45,000; carbonic anhydrase, 31,000trypsin inhibitor, 21,500; and lysozyme, 14applied to lane G. Scanning of this Coomstained gel indicated that the amount ofOmin JF703 (R471a) was 70% of the amountprotein present in JF701 (R471a). Although aan apparent molecular weight of 50,000 (prphage lambda receptor) is more intense in 1in lane E, this difference was not reproducitexperiments.

amphenicol, and streptomycin (data nc(iv) The permeability characteristics diin cells containing R471a in this papersimilar to those determined by the usepurified from cells not containing thedescribed in the accompanying paperThe major problems in the determ.

cephalosporin hydrolysis by intact cthat the rates were frequently veryeven small amounts of P-lactamase relithe medium introduced large errors inlation of permeability coefficients, espislowly penetrating drugs. As describithe first problem was solved by thspectrophotometric cuvettes of 1-mm I

and the second was solved by growingmedium containing 5 mM MgSO4 anout the assay also in the presence of 5 1

Effect of hydrophobicity on rates of penetra-tion. When a series of monoanionic cephalospo-rins were used, we found large differences intheir rates of penetration through the outermembrane. Furthermore, the rates appeared tobe strictly dependent on the hydrophobicity ofthe molecule, a 10-fold increase in P,. producingabout a 5- to 6-fold decrease in the permeabilitycoefficient (Fig. 3). The dependence on hydro-phobicity was similar among OmpF, OmpC, andPhoE porins, but cells containing only theOmpC porin showed consistently about a 5-fold-lower permeability than those containing theOmpF porin. This cannot be entirely due to thedifference in the number of porin molecules percell, because differences in the porin contents ofJF701 and JF703 were not large (Fig. 2).

Effect of positive charge on rates of penetration.Zimmermann and Rosselet (39) noted that ceph-aloridine diffused across the outer membranemuch faster than expected from its apparentpartition coefficient. Since cephaloridine is zwit-

,e roteins. terionic, unlike the other cephalosporins theyOus strains tested, this result suggests that the additionaly (33), and positive charge may accelerate the diffusionwere ana- process. However, the apparent partition coeffi-Lugtenberg cient is influenced greatly by charges, and thus itIF only); B, does not accurately reflect the hydrophobicity of(containing the molecule. We tried to separate the effect of71a); and F,(phosphor-00; ovalbu- . ,); soybean,400) werelassie blue- ApC protein o_4 Bof OmpF

i band withrobably thelane B than Cble in other *

t shown). fetermined w\i0are very oof porins _R-factor, _ _(22). Eination of wells were c

slow andeased intothe calcu- .ecially for -0.5 0 0.5 1.0 1.5ed above, HYDROPHOBICITY [log PU(OCTANOL)]ie use of FIG. 3. Permeability coefficients of E. coli outerlight path, membrane to various monoanionic cephalosporins.D cells in a Symbols: A, JF694 (R471a) containing PhoE porin; *,d carrying JF701 (R471a) containing OmpF porin; 0, JF703 (R471a)mM Mg2+ containing OmpC porin. For other details, see text.

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236 NIKAIDO, ROSENBERG, AND FOULDS

charges from that of hydrophobicity by using P,i.e., the partition coefficient of the unchargedform, for correlation between the penetrationrates and properties of the solute. In the case ofcephaloridine, the experimental determinationof PU is impossible because it is zwitterionic andbecause the positive charge is carried by thequaternary nitrogen atom. We therefore had tocalculate the approximate value of its P,. from itsstructure, as described in Materials and Meth-ods. With its Pu value known, reference to Fig. 3enabled us to predict the permeability coeffi-cients for the monoanionic analog of cephalori-dine. When the predicted permeability coeffi-cients for the hypothetical monoanionic analogwere compared with the observed coefficientsfor the zwitterionic cephaloridine, it was clearthat the positive charge indeed resulted in a>100-fold acceleration of penetration throughOmpF and OmpC channels, but the diffusionthrough the PhoE channel was affected muchless (Table 1).

Similar comparison between the measuredpermeability coefficients of the zwitterioniccompounds cephaloglycin and cephapirin andthose predicted for their hypothetical monoan-ionic analogs again showed the accelerating ef-fect of the positive charge in OmpF and OmpCchannels (Table 1). The magnitude of the effect,however, was much less than with cephalori-dine. This may be because the positive charge of

cephaloridine is sterically quite close and there-fore may partially cover its negative charge, aswell as the possibility that only a fraction of thecephaloglycin and cephapirin molecules wasprotonated either at the entrance of or within theporin channel.The effect of an added negative charge is also

seen in Table 1. Here compound SCE-20, whichhas an additional negative charge, is seen topenetrate through "normal" porin channelsmade of the OmpF and OmpC proteins muchmore slowly than its hypothetical monoanionicanalog, but the additional charge did not pro-duce a noticeable difference in diffusion ratesthrough the channel produced by the PhoEprotein.

All comparisons described above involved thepredictive calculations of the permeability ofhypothetical analogs, and there may be doubtsabout the reliability and accuracy of such calcu-lations. In one situation, however, we couldcompare the measured permeability coefficientsof two real compounds. This comparison (Table2) involves cefsulodin and cephaloridine, and itis seen that the former compound penetrates 20to 30 times more slowly than the latter throughthe normal porin channels. A major part of thisdifference is almost certainly due to the presenceof the additional negative charge on cefsulodin(see Fig. 1). Although there are two other differ-ences between the two agents, the benzene and

TABLE 1. Effect of additional positive or negative charges on permeability of t3-lactams through porinchannels

Permeability coefficients (10-' cm s-1) in cells pro-,B-Lactam Electrical ducing only porin:-Latamcharge OmpF OmpC PhoE

Cephaloridine (A) +- 52.6 4.5 2.5Monobasic analog (B)a - 0.08 0.01 0.08(Ratio A/B) (658) (450) (31)

Cephaloglycin (A) +-b 9.8 1.0 1.0Monobasic analog (B) - 4.7 0.8 7.5(Ratio A/B) (2.1) (1.3) (0.1)

Cephapirin (A) +-C 19.4 1.2 1.3Monobasic analog (B) 3.2 0.6 5.0(Ratio A/B) (6.1) (2.0) (0.3)

SCE-20 (A) -- 0.29 0.04 1.6Monobasic analog (B) 1.3 0.12 1.7(Ratio A/B) (0.2) (0.3) (0.9)

a The expected permeability coefficients for the hypothetical monobasic analogs were calculated as follows.First, the log Pu value for the 1-lactam with an identical structure but with only one possible negative charge atthe 4-carboxyl position was calculated from the structure as described in the text. The permeability coefficientswere then predicted from the lines of Fig. 3, on the basis of this value of log Pu,,.

b From the reported pKa (6.8) of the side-chain amino group (36), about 88% of this group should be protonatedunder the conditions of assay, i.e., at pH 6.0.

c From the pKa (5.94) of an analog, 4-methylthiopyridine (13), of the side-chain portion of the molecule, asubstantial fraction of cephapirin molecules is expected to be in the zwitterionic form.

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PORIN CHANNELS IN E. COLI 237

TABLE 2. Permeability coefficients of cefsulodinand cephaloridine through porin channels

Permeability coefficientsElectrical (10- cm s-1) in cells

,-Lactam charge producing only porin:

OmpF OmpC PhoE

Cephaloridine (A) + - 52.6 4.5 2.5Cefsulodin (B) + - - 1.8 0.2 3.2(Ratio A/B) (29.2) (22.5) (0.8)

the thiophene rings are of about the same sizeand hydrophobicity (11, 26) and the -CONH2group present on cefsulodin should significantlyaccelerate, rather than retard, the diffusion bydecreasing log P,, by as much as 1.1 (26). Indeed,we observed some acceleration of the penetra-

tion of cefsulodin through the PhoE porin chan-nel (Table 2). These results then confirm theconclusions obtained by the use of predictedpermeability values of hypothetical compounds.

DISCUSSION

In this study we have examined the permeabil-ity coefficients of various cephalosporins to in-crease our knowledge of the properties of theporin channel in the outer membrane of E. coli.For this approach, we had to be certain that thecephalosporins were passing through the porinchannel, i.e., not through the lipid bilayer regionof the membrane or channels produced by otherproteins. We feel that this condition was satis-fied in our system on the basis of the followinglines of evidence. (i) With the usual biologicalmembranes, hydrophobic molecules, especiallynonelectrolytes, can easily traverse the mem-brane through the bilayer continuum of themembrane. However, the lipopolysaccharide-phospholipid region of the outer membrane of E.coli and Salmonella typhimurium is unusuallyimpermeable and indeed did not allow the pene-tration of even an extremely hydrophobic 1B-lactam, presumably due to the asymmetric ar-

rangement of the lipopolysaccharide in themembrane (19). Furthermore, the ubiquitousnegative charge present in all cephalosporinmolecules (Fig. 1) should act as a deterrentagainst diffusion through this pathway. (ii) Mu-tants with trace levels of porin showed only 5 to10% of the permeability of the wild-type cells tovarious ,B-lactams, including 6-aminopenicil-lanic acid (4), cephaloridine (23), cephacetrile,cefazolin, and cephalothin (Nikaido, unpub-lished data). (iii) Liposomes containing only thepurified porin proteins showed permeabilityproperties (22) similar to those observed withintact cells in this study.Permeability coefficients of monoanionic

cephalosporins, or thus their rates of diffusion

through porin channels, showed a monotonousdependence on the degree of hydrophobicity ofthe solute (Fig. 3). It is not clear why hydropho-bic molecules penetrate more slowly through theporin channels, but the following explanationseems plausible. Since the porin channels arevery narrow (equivalent to a hollow cylinder ofabout 1.2 nm in diameter [21]), it is expected thatwater molecules in the channel are rather strong-ly bound to groups on the walls of the channelthrough hydrogen bonds. When hydrophilic sol-utes, which possess many hydrogen bond-form-ing groups on their surface, come into the chan-nel, the hydrogen bonds between watermolecules and groups on the wall are broken,but they are now replaced by new hydrogenbonds between the solutes and the wall, so thatthe net energy requirement will be minimal. Incontrast, hydrophobic solutes will break manyhydrogen bonds in the channel without replacingthem with new hydrogen bonds; thus, the pene-tration of hydrophobic solutes would be anenergetically unfavorable process. An alterna-tive explanation is that the more hydrophobiccephalosporins tend to form micelles, which aretoo large for penetration through the porin chan-nel. However, our assay (see Materials andMethods) has shown that none of the cephalo-sporins used was in micellar form at the concen-tration used (1 mM) at the pH of assay (6.0) (datanot shown).The cephalosporins used in the experiment of

Fig. 3 had molecular weights ranging from 338 to451. Since the size of the solutes had a verystrong influence on their rates of penetrationthrough the porin channels (21), it was ratherunexpected to find that the penetration rateswere determined mostly by hydrophobicity andwere affected much less by the molecularweights of the agents (Fig. 3). However, buildingof space-filling models showed that the sizes ofmost cephalosporins varied much less than ex-pected from the difference in molecular weights.The observation that cefamandole and cefazolintended to have lower diffusion rates than thosepredicted from their hydrophobicity could be areflection of their somewhat larger sizes (Fig. 1and 3).The channels made of OmpF, OmpC, and

PhoE porins behaved similarly toward the hy-drophobicity of the solute (Fig. 3). However, theabsolute values of permeability coefficients inOmpF-containing cells was always several timeshigher than those in OmpC-containing cells.This difference cannot be ascribed to the differ-ence in copy numbers of these porins, as sodiumdodecyl sulfate-polyacrylamide gels of the outermembranes of OmpF- and OmpC-containingstrains showed that the levels of these porinswere not so different in these cells (Fig. 2).

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238 NIKAIDO, ROSENBERG, AND FOULDS

Although it is possible that fractions of channelsthat are in "open" conformation (31) differ inthese proteins, a simpler explanation of thisdifference in the magnitude of permeability coef-ficients is the slight difference in the equivalentdiameter of OmpF and OmpC channels, as dis-cussed in the accompanying article (22).The monotonic relationship between the hy-

drophobicity and the permeability coefficient(Fig. 3) allowed us to predict the permeability ofany hypothetical monoanionic cephalosporins,and this method was used to study the effect ofelectrical charges on penetration rates. The re-sults showed that with channels produced by thenormal E. coli porins OmpF and OmpC, theadditional positive charge accelerated the diffu-sion process, whereas the presence of the addi-tional negative charge significantly retarded thepenetration rates. These channels thus appear toprefer cations. An alternative explanation is thatthe Donnan potential (interior negative) acrossthe outer membrane (34) is pulling the cations in.However, the channel itself seems to have somediscriminatory power, as indicated by the fol-lowing observations. (i) In reconstitution studieswith black lipid film, it was also found that the E.coli porin channel preferred alkali metal cationsto the chloride anions (5). (ii) Retardation ofnegatively charged molecules was also con-firmed in liposome systems (22). (iii) In spite ofthe Donnan potential, the PhoE porin channelseems to favor negatively charged solutes (seebelow).The channels made of PhoE porin, whose

production is normally repressed in wild-type E.coli, were very different from those of OmpFand OmpC channels. Here the presence of anadditional positive charge produced either sig-nificant retardation or only a small acceleration(Table 2). The addition of the second negativecharge did not result in a significant retardation(Tables 1 and 2). Thus, the PhoE channel ap-pears to be a channel that tolerates, and evenprefers, multivalent anions. This conclusion is inagreement with the recent observation that theexpression of PhoE porin occurs under phos-phate starvation conditions (1, 35); probably thePhoE porin is an anion-preferring channel per-mitting the rapid diffusion of a wide range ofnegatively charged solutes.

After the first version of this paper was com-pleted, a paper by Overbeeke and Lugtenberg(24) was published. Their results are generallyconsistent with ours as regards the perference ofthe PhoE channel for negatively charged sol-utes. However, these workers also noted that2.5 mM Na2HPO4 added to intact cells at pH 7.0reduced the diffusion rate of cefsulodin throughthe PhoE channel by 40% (24). Because we used10 mM sodium phosphate buffer in our assay,

there was a possibility that the diffusion throughthe PhoE channel could have been inhibited bythe phosphate ions in the buffer. The assayswere therefore repeated with PhoE-, OmpC-,and OmpF-containing cells in 10 mM sodiumphosphate buffer, pH 6.0, as well as in 10 mMbis-(2-hydroxyethyl)amino-tris(hydroxymethyl)methane-hydrochloride buffer, pH 6.0. With theOmpC- and OmpF-containing cells, we did notobserve any inhibition by phosphate buffer withseveral substrates. With the PhoE-containingcells, the diffusion rates were decreased in phos-phate buffer, but only by 8% for cephaloridineand 15% for cefsulodin. (The values are aver-ages of four independent experiments.) What-ever the reasons for the discrepancy, theseresults suggest that our conclusions on the prop-erties of the pores remain essentially valid.

Since only E. coli and S. typhimurium (andprobably their close relatives), among the gram-negative species so far surveyed (19), have beenfound to contain the narrow porin channels thatexclude hydrophobic and anionic molecules, weshould briefly consider the possible physiologi-cal significance of the channels with these prop-erties. We feel that one of the most seriouschallenges E. coli faces in the upper intestinaltract is the presence of high concentrations ofbile salts, which are powerful detergents and forthis reason inhibit the growth of most nonentericbacteria on selective media such as McConkeyand deoxycholate agars. The E. coli porin chan-nels may have evolved in such a way as toexclude bile salts, which are hydrophobic, rath-er bulky, and negatively charged.Although the main aim of this study was to

understand the properties of the porin channel,some comments would be appropriate as regardsthe permeation of P-lactams in gram-negativebacteria, since these agents cross the outermembrane through porin channels and sincegreat efforts have been made to produce com-pounds with faster rates of penetration. Ourobservations can be summarized as follows. (i)Some of the ,B-lactams tested, for example,cephaloridine and cephacetrile, penetratethrough the channels quite rapidly, and theirpermeability coefficients (1 x 10-4 to 5 x 10-4cm s 1 through the OmpF porn channel) can befavorably compared with those of uncharged,very hydrophilic molecules of about the samesize, e.g., about 2 x 10-4 cm s-' for lactose (21).It would be difficult to produce further dramaticimprovement on penetration rates, unless a netpositive charge is introduced. (ii) The negativeeffect of hydrophobicity confirms earlier resultsfrom several other laboratories. For example, itwas shown repeatedly that the more hydropho-bic f-lactams tended to be less effective againstE. coli (17, 29), although the correlations were

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PORIN CHANNELS IN E. COLI 239

less than perfect as modification of the drug alsoalters its affinity to the target sites. Zimmermannand Rosselet (39) showed, for the first time, thecorrelation between hydrophobicity of the I-lactams and their penetration rates, although theeffect of electrical charges was not separatedfrom the effect of hydrophobicity. (iii) Since ourvalues of permeability are expressed in perme-ability coefficients rather than in arbitrary unitsused by other workers, we can calculate tj12, thetime necessary for the intracellular concentra-tion of P-lactam (C,) to reach 50%o of the extra-cellular concentration (CO). Fick's first lawstates, d(Ci * V)/dt = P * A * (CO - C,), where Vis the volume of cells. Rearrangement, integra-tion, and substituting Ci = 0 at t = 0 lead to: t =V ln(C0/(Co - Ci))/(P * A). At t = t112, Ci =CJ2, and therefore, t1/2 = In2 - V/(P * A). Sincewe know that A = 132 cm2 for cells of 1 mg ofdry weight (33), i.e., about 4 mg of wet weight orabout 4 x 10-3 cm3 cell volume, tj12 can beobtained for any value of P. This calculationshows that for P-lactams of the most permeablegroup (P 10-4 cm s-1), tl2 is about 0.1 s, andeven for very poorly penetrating ,B-lactams (P =

10-6 cm s ; see Fig. 3) t1/2 is only about 20 s.Thus, the P-lactams penetrate surprisingly rap-idly through E. coli outer membranes. The im-pressions of slow penetration created by thedifference in minimal inhibitory concentrationsbetween normal and "superpermeable" strains(27), for example, must therefore be due to therapid hydrolysis, or binding, of ,B-lactam mole-cules that have crossed the outer membranebarrier. We should thus keep in mind that someof the "permeability assays" of the outer mem-brane may be measuring the balance betweenpenetration and hydrolysis rather than the actualpenetration rates. In fact, one of these assaysindicates that both cephaloridine and cepha-lothin diffuse "unhindered" through the E. coliouter membrane and that cefamandole is muchless permeable than caphalothin (28). Theseresults are incompatible with our conclusionsand, in our analysis, illustrate the basic flaw inthe design of these assays. The magnitudes ofthe tj,2 values also suggest that P-lactams thatare very resistant to enzymatic hydrolysis canbe quite effective toward gram-negative bacte-ria, even if their penetration through the outermembrane is rather slow. Some of the morerecently developed ,-lactams indeed appear tofall into this category (Nikaido, manuscript inpreparation).

ACKNOWLEDGMENTS

This study was supported in part by Public Health Serviceresearch grant AI-09644 from the National Institute of Allergyand Infectious Diseases and by research grant BC-20 from theAmerican Cancer Society.

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