Molec. gen. Genet. I58, 2 3 - 3 3 (1977) © by Springer-Verlag 1977

Pleiotropic Transport Mutants of Escherichia coli Lack Porin, a Major Outer Membrane Protein* Patrick Bavoil and Hiroshi Nikaido Department of Bacteriology and Immunology, University of California, Berkeley, California 94720, U.S.A.

Kaspar von Meyenburg** Department of Bacteriology, University of California, Davis, California 95616, U.S.A.

Summary. Four "pleiotropic transport" mutants of Escherichia coli B/r with decreased affinity for the uptake of most nutrients were found to lack a major outer membrane protein of 36,500 daltons ("porin") previously shown to produce transmembrane diffusion channels in in vitro reconstitution experiments. Consequent decrease in outer membrane permeability was confirmed by measuring the transmembrane diffusion rate of 6-aminopenicillanic acid. Quantitative considerations on the porin-dependent permeability of the outer membrane show that (a) there may be very large differences in the actual rates of penetration, even among the "permeable" substances and (b) the numbers of porin molecules present in wild type cells is several orders of magnitude higher than that necessary for the uptake of rapidly diffusing substrates such as glucose from ordinary culture media. The absence of porin and the pleiotropic transport defect were always cotransduced, and the mutation was mapped at 73.7 rain between aroB and malT by Pl transduction. When "revertants" able to grow on low concentrations of lactose were selected, in addition to true revertants "suppressor" strains with increased amounts of non-porin membrane proteins were isolated.

Salmonella typhimurium outer membranes to small, hydrophilic molecules is dependent on the presence of a group of major outer membrane proteins of molecular weight around 35,000, called "porins" (Nakae, 1976a, b). The "pore-forming" function of porins was further confirmed by the observation of Nikaido et al. (1977) that the passive permeability, measured by the rate of diffusion of cephaloridine through the outer membrane, was substantially decreased in intact cells of porin-deficient mutants of S. typhimurium. In 1971, one of us isolated and characterized a class of pleiotropic transport mutants of E. coli B/r (von Meyenburg, 1971). These mutants have been shown to exhibit decreased affinity for the uptake of almost all substrates tested: sugars, amino acids, uracil, and even inorganic anions (von Meyenburg, 1971). The pleiotropic transport defect (kmt-) is here shown to be due to the absence of the porin, the 36.5 K t major outer membrane protein from the cell envelopes of these mutants. This paper also describes the mapping of the mutations, as well as the analysis of revertants that have regained the ability to grow on low concentrations of substrates. Materials and Methods

Introduction In vitro reconstitution experiments have shown that the passive permeability of the Escherichia coli and * This paper corresponds to paper XVI of the series dealing with the bacterial outer membrane from the laboratory of H.N. The preceding paper in the series is Nikaido, Bavoil, and Hirota, J. Bacteriol., in press ** On leave from University Institute of Microbiology, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark For offprints contact : H. Nikaido

Bacterial Strains and Growth Conditions. The strains used are listed in Table 1. The k m t - strains were maintained at - 8 0 ° C in 20% glycerol. Frequently the usual stab and slant cultures of these strains became overgrown by revertants that could utilize the low concentrations of nutrients remaining in such media. Growth conditions, measurement of growth and of half-saturation constant for growth on different substrates ("Km") were as described previously (yon Meyenburg, 1971). L broth was made according to Bertani (1951), but glucose was omitted.

Throughout this paper, we refer to various proteins by their apparent molecular weights inferred from their mobility in SDSacrylamide gels. In our notation, the apparent molecular weight x 10- 3 is followed by the ietter K, for the sake of brevity

24 Genetic Studies. P1 transduction and other genetic manipulations were performed according to Miller (1972). Plbc, a B-modified Plc for the transductions from E. coli B/r into K-12, was selected from a lysate of Plkc by repeated growth on strain CM7. Preparation of Membrane Fractions. Total membranes (cell envelopes) were prepared from exponential phase cultures grown in L broth at 37° C (or at 30° C with CM30, 31, 32 and their derivatives), according to the methods of Ames et al. (1974). Outer and inner membrane fractions were prepared from similar cultures as described by Smit et al. (1975). Protein Pattern of the Membranes. This was examined by SDS polyacrylamide slab gel electrophoresis as described by Ames et al. (1973). Samples were heated at 100° C for 2 rain in the "sample buffer" containing 2% SDS, and 9% gels were routinely used. Isoelectric Focusing. Proteins from the outer membrane fractions were analyzed by isoelectric focusing in polyacrylamide slab gel, essentially according to the method of O'Farrell (1975) as modified by Ames and Nikaido (1976). Diffusion Rate of fi-Lactams Across the Outer Membrane. This was calculated from the rates of hydrolysis of fl-lactams by intact cells of strains carrying the R1 plasmid, according to the method of Zimmermann and Rosselet (1977). The procedure used closely followed the modification used by Nikaido et al. (1977), but incorporated two further modifications. Firstly, 5 mM MgCI2 was added to the assay mixture itself in addition to the wash buffer and growth medium. Secondly, since in mutants with extensive alterations of the outer membrane cephaloridine was found occasionally to induce damages in the cell walls during the assay, a substrate of fl-lactamase with practically no antibiotic activity, 6-aminopenicillanic acid (purchased from Sigma), was used instead of cephaloridine. Under these conditions, the amount of fl-lactamase found in the medium does not increase during the assay, so that the rates of hydrolysis by intact cells can be properly corrected by subtracting the rates of hydrolysis by cell-free supernatants (Nikaido, Bavoil, and Hirota, J. Bact., in press). The R1 plasmid was transferred from YC219 (E. coli K-12 F- lac gal ara mtl xyl/R1) (a gift from Dr. M. Yoshikawa) by conjugation and selection for Mtl +, ampicillin-resistant clones. The membrane protein pattern of the transconjugants was found to be indistinguishable from that of the origianl plasmid-non-containing strains, by SDS polyacrylamide gel electrophoresis.

P. Bavoil et al. : Transport in Porin-Deficient Mutants of E. coli 1971), and of nucleoside m o n o p h o s p h a t e s [determined by the substrate gradient plate test (see von Meyenburg, 1971) using either 5 ' - d T M P as the source o f thymine or 2 ' ( 3 ' ) - U M P or 5 ' - U M P as the source o f phosphate] was impaired in these mutants. The increase in transport K m for m o s t substrates was in the range o f 50- to 300-fold. Uridine uptake was not affected, while only a slight increase in the apparent transport Km of uracil (fourfold) and glycerol (tenfold) was found. (For a detailed analysis o f the m u t a n t CM7, see von Meyenburg, 1971.) Utilization o f maltose in the E. coli B/r derivatives was very slow, but substrate gradient test showed significant differences between C M 6 and C M 7 ; thus maltose uptake also seemed to be affected in CM7. The maximal rates o f transport o f substrates were not affected in the mutants, and the mutants grew as fast as the parental strain C M 6 if a sufficiently high, i.e. saturating, concentrations o f c a r b o n sources were provided. The four mutants, t h o u g h very similar with respect to their pleiotropic transport defect, differed f r o m each other in other phenotypic characters. C M 7 was EDTA-sensitive, and lysis occurred readily when E D T A was added to a growing culture at a concentration high enough to bind all divalent cations. The growth o f CM30, CM31, and C M 3 2 was not severely affected by E D T A , but these m u t a n t s were temperature-sensitive and eventually lysed if grown at 42 ° C in L broth. All of them grew well at 30 ° C, but in the intermediate temperature range the degree o f heatsensitivity was quite different between the m u t a n t s and increased in the order C M 3 2 < C M 3 1 < C M 3 0 . The average cell volume of these heat-sensitive strains as determined by a modified Coulter particle analyzer (Schleif, 1967) was twice that of the parental strain C M 6 or the m u t a n t CM7, although the cell size distribution o f the latter was slightly b r o a d e r than the one of CM6.

Results Phenotype o f the Pleiotropie Transport Mutants. F o u r pleiotropic transport mutants, C M 7 ( = C P 3 6 7 ) , CM30, CM31, and C M 3 2 (Table 1), had been isolated after mutagenesis with nitrosoguanidine o f strain CM6, an E. coli B/r (Table 1), and selection for decreased affinity for glucose u p t a k e (i.e. increased Km for growth) by penicillin enrichment (von Meyenburg, 1971). The apparent Km values of the four mutants for glucose, determined by measurements o f growth rates at limiting glucose concentrations, were increased approximately 500-fold. The uptake not only o f glucose but also o f most other carbohydrates, as well as o f organic acids, a m i n o acids, and inorganic anions sulfate and phosphate (von Meyenburg,

fi-Lactam Diffusion R a t e through the Outer Membrane. Each o f the " t r a n s p o r t " processes measured in the preceding section actually consists o f two distinct stages. T r a n s p o r t substrates must first diffuse t h r o u g h the outer membrane, which is a significant permeability barrier (Decad and Nikaido, 1976 ; Nikaido, 1976), before they get transported t h r o u g h the inner, cytoplasmic membrane. The operation o f the first stage can be examined, for the diffusion of such small, hydrophilic molecules as the nutrients mentioned above, by measuring the rate o f diffusion o f fl-lactam antibiotics ( Z i m m e r m a n n and Rosselet, 1977). This involves first the introduction of an R plasmid that codes for a powerful fi-lactamase located in the periplasmic space, so that the rate o f hydrolysis of fi-lac-

P. Bavoil et al. : Transport in Porin-Deficient Mutants of E. coli

25

Table 1. E. coli strains used Strain

Line

Genotype

Source/Reference

CM6 CM7 CM30 CM31 CM32 CM23

B/r B/r B/r B/r B/r B/r

F thyA drm tonA mal F thyA drm tonA real kmt-7 F thyA drm tonA real kmt-30 F - thyA drm tonA real kmt-31 F - thyA drm tonA real kmt-32 F'13/thyA drm tonA mal kmt-7 IacZ

=CP366. von Meyenburg, 1971 - C P 3 6 7 . from CM6. von Meyenburg, 1971

HB45 AB2847 HfrG6 HfrG6AMD2 HfrG6AMD3 HfrG6AMD18 PA505 PA505MAA 101 PA505MAA 108 W680

B/r K-12 K-12 K-12 K-12 K-12 K-12 K-12 K-12 K-12

F met thr leu his pro trp argA lac gal rbs real strA F aroB351 tsx-354 mal-354 supE42? 2 - 2 g Hfr his Hfr his A(bioH malA glpR glpD glg asd) Hfr his d(rnalA glpR glpD glg ascO Hfr his A(bioH malA glpR glpD) F argH metA pro his malA strA F - argH metA strA A(malA glpR) F argH metA strA A(bioH malA glpR) F - thi pyrD glt galK trp his strA

tams by intact cells becomes limited by the rate of influx of the substrates across the outer membrane. With the modifications described in Methods, the rates of hydrolysis of 170 I-tM 6-aminopenicillanic acid at 25 ° C by intact cells of CM6/R1 and CM7/R1 were found to be 0.29 and 0.02 nmol/s/mg (dry weight) cells, after correction for the amounts hydrolyzed by the "leaked out" enzyme in the medium. The permeability coefficients calculated as described previously (Nikaido et al., 1977) were 13.9 x 10-6 and 1.0 x 10-6 cm/s, respectively. The permeability of the outer membrane of CM7 to 6-aminopenicillanic acid, and presumably also to other small, hydrophilic molecules, is thus reduced to less than 10% of that in the parental strain, CM6.

Analysis of Membrane Proteins. The pleiotropy of the mutations in these strains had indicated that they are transport-defective due to an alteration in the organization of the cytoplasmic membrane or of the cell envelope as a whole (von Meyenburg, 1971). Our finding of an impaired access of 6-aminopenicillanic acid to the/Mactamase in the periplasmic space (see above) now points to an alteration in the outer membrane (see Nikaido et al., 1977). SDS polyacrylamide gel electrophoresis of total membrane preparations of the mutants indeed revealed that all four mutants lacked one of the major outer membrane proteins, namely a protein of the molecular weight 36.5 K (Fig. 1). This protein is identical to the one isolated and characterized by Rosenbusch (1974) from E. coli B, and named "matrix protein"; synonyms are "protein 1" (Schnaitman, 1974), "protein I " (Hindennach and Henning, 1975), or "porin", the last designation being based on the finding that this protein confers permeability

] from CM6. von Meyenburg, 197I from CM7 via penicillin enrichment and F' transfer Boyer (1966) B. Bachmann

]

I

M. Schwartz (see Hatfield et al., 1969; [ Nagel de Zwaig et ai., 1973)

I

U, Henning

to reconstituted membrane vesicles (Nakae, 1976b). The outer membrane preparations of the transport mutants (see e.g. CM32 in Fig. 5) contained at most 1 - 3 % of the amount of 36.5 K protein present in the parental strain CM6, on the basis of the densitometric scanning of the stained SDS acrylamide gels. Besides this very obvious change in the protein composition of the outer membrane of all the mutants, mutants CM7 and CM32 both in addition lacked a minor protein with an apparent molecular weight of about 70 K in the envelope fraction (Fig. 1). Slab gel analysis of isolated outer and inner membranes showed, however, that this protein was associated with the inner, cytoplasmic membrane. The parental strain CM6 had a lower level of the 36.5 K protein when compared either with a K-12 strain or another B/r line (HB 45) (Fig. 1). Densitometric scanning of the gels showed that CM6 contained about one-sixth to one-third as much porin as the latter strains, per unit cell mass, although the apparently compensatory increase in the 33 K protein makes the difference appear more dramatic (Fig. 1). The common mutational change, i.e. the absence of porin, was quite readily observed even when total cell extracts were electrophoresed (not shown). This indicates that the 36.5 K protein does not accumulate in the cytoplasm; thus if the mutation prevents the modification or translocation of the protein, the unincorporated protein might get degraded rapidly. In all mutants, a 84 K protein, located in the outer membrane (see also Fig. 5), was significantly increased (Fig. 1). This protein remains to be identified, but on the basis of its size it could be one of the iron-chelator-complex transport proteins (Hancock et al., 1976; Wayne et al., 1977), possibly derepressed

26

P. Bavoil et al. : Transport in Porin-Deficient Mutants of E. coli tO ~I"

~0

D--

0 cO

co

cO

0 ro

-to

e~ to

cO

"I-

cO

cO

~

cO

CO

cO

cO

cO

cO

6

S

d

-6

~;

~

~

.E

.-

-:,

8 4 K--~

70 K---~

36.5 K --~ 33K ~.

Fig. 1. Protein composition of total membrane fractions of kmtl mutants and some kmt + strains. This figure is a composite of three slab gels. CM6 is the parent strain, and CM7 (and its derivative CM8), CM30, CM31, and CM32 are its kmt mutants. Another B/r line, HB45, and a K-12 strain, W680, are also included for comparision

because of the intracellular iron deficiency (see Ichihara and Mizushima, 1977; Pugsley and Reeves, 1977). Genetic Mapping of the Pleiotropic Transport Mutation. Mapping by conjugational transfer had placed the mutation giving the pleiotropic transport defect in CM7, termed k m t - , between rpsL (strA) and metB (yon Meyenburg, 1971), approximately 75% linked to rpsL (unpublished observations). Electrophoretic analysis of the total cell extracts of recombinants from crosses between CM23 (CM7 lac-/F'lae) and HB45 showed that all recombinants that had received the rpsL + allele of the donor with the transport defect ( k m t - ) were also lacking the porin (not shown), a result indicating a close relationship between the k m t - mutation and the absence of the 36.5 K protein. P1 transductions were made from the four k m t mutants into E. coli K-12 carrying various combina-

tions of "malA ", cysG, and aroB mutations as selective markers. In selecting for the wild type allele of any one of the three markers on minimal medium plates containing 0.2% glucose or maltose, we observed two types oftransductants, a faster and a slower growing ones (large versus small colonies) if P l b c was grown on any one of the four k m t - mutants. In contrast, all colonies were large when the Pibc lysates were grown on CM6. In a typical transduction with P l b c grown on any one of the four mutants into strain AB2847, 40% of the transductants selected for the aroB + marker were slow growers. A m o n g the aroB + transductants, 22% were also mal +, a result indicating that the mal354 mutation in strain AB2847 is a mutation in the " m a l A " gene cluster (Hatfield et al., 1969). For reasons discussed below, we believe it to be a malT mutation. Conversely, the fact that unselected mal + transductants were obtained at a high frequency suggests

P. Bavoil et al. : Transport in Porin-Deficient Mutants of E. coli

that the real- phenotype in the E. coli B/r donors is not due to mutations in the " m a l A " but rather due to one(s) in the " m a l B " region (see Hofnung, 1974). Of the aroB+mal + transductants, 95% or more were of the slow growing type. (The actual difference in growth rate between the slow and fast growing transductants on AB minimal medium with 0.2% glucose was 12-15%, the actual doubling times being 67 and 75 min, respectively.) Determination of the K m for glucose, lactose, and histidine uptake in different classes of transductants (see Methods) showed that the slower growing aroB + transductants always had an increased Krn for growth on glucose or lactose, or for histidine uptake (Table 2). This indicates that they have acquired the k e m t - mutation from the various donors. Electrophoretic analysis of membranes of AB2847 aroB ÷ transductants (Fig. 2) showed that the phenotype of slow growth, which seemed to be closely linked or identical with the pleiotropic increase in transport Kin, also coincided with the absence of both of the two species of K-12 porins, i.e. proteins 0-9 and 0-8 of Uemura and Mizushima (1975), b and c of Lugtenberg etal. (1976), and Ia and Ib of Schmitges and Henning (1976). We have tested ten fast growing and ten slow growing aroB÷malT354 transductants, as well as ten slow growing aroB +real + transductants, obtained by using Plbc grown on either of the four transport mutants. Fast growing transductants (kmt +) always have retained the porins in amounts typical of the K-12 recipient, while it was absent in all the slow growing transductants tested. The k m t - mutations which result in the pleiotropic transport defect and the absence of porin thus are genetically located between aroB and malT354. Furthermore, the analysis of aroB + transductants of AB2847 transduced with Plbc grown on CM6 indicates that the low level of expression of porin in E. coli B/r CM6 (Fig. 1 b) is genetically

27

determined by a site between aroB and malT (see Fig. 2b), as is the total absence of porin in the mutants. Presumably because of K-specific restriction the cotransduction frequencies between aroB and m a l t were somewhat lower (namely 20-25%) in the Plbc transductions from E. coli B/r (mutants or wild type) into E. coli K-12 than the published values of 53% obtained in crosses between K-12 strains (Anderson and Oxender, 1977). We therefore carried out a number of backcrosses using Plkc grown on the AB2847 aroB +kmt-7 malT + transductants as donors. Strain AB2847 again served as the recipient in selections for aroB ÷. The cotransduction frequency between aroB and m a l t in such crosses was 56-59%; cotransduction between aroB and the slow growth phenotype, indicative of the k m t - , was 77%. Again more than 95% of the aroB +real + transductants were of the slow type. The kmt mutations therefore are located at 73.7 rain on the current genetic map of E. coli K-12 (Bachmann et al., 1976), between aroB and malT, and approximately 75% cotransducible with either of the two markers. We have tested various " m a l A " deletion strains (Table 1) for the presence of porin. All strains tested, even those in which the deletion extended from " m a l A " beyond the bioH locus (e.g. MAA108, AMD2, and AMD 18), did contain normal amounts of porins as judged by the SDS acrylamide gel electrophoresis (not shown). Thus the kmt gene is most likely located between aroB and bioH (Fig. 3). With respect to the mal-354 mutation, we can conclude that it is in the m a l t locus, since the slab gel analysis showed that a 50 K protein, presumably corresponding to the )r-receptor or lamB protein (L. Randall, personal communication), was not expressed in AB2847 or real- transductants but became strongly expressed in real ÷ transductants (Fig. 2 b, c). Furthermore, the former strains were resistant to phage 2, to which the latter strains have become sensitive. The

Table 2. Km for growth on glucose or lactose, and for histidine transport, in various classes of AB2847 aroB + transductants Genotype of transductants"

(aroB +) kmt + malT354 b (aroB + kmt +) malT354 (aroB + kmt-7) malT354 (aroB + kmt-32) malT354

Donor used in transduction

Growth on 0.2% glucose minimal agar

Km (gM) Glucose

Lactose

Histidine

CM7, CM32 CM6 CM7 CM32

fast fast slow slow

3 3 500 500

40 40 3000 3000

0.1 ND ° 0.3 ND

Gene symbols in parenthesis indicate that those genes were derived from donor strain b Three strains with this genotype were tested for transport K m values. For each of the other genotypes, one representative transductant was used ° Not determined

28

P. Bavoil et al. : Transport in Porin-Deficient M u t a n t s of E. coli

b-

b-

CO

50K

-~

36.5 K

J'

33 K

-~

113

eJ 03

oroB + Tronsductonts

•~

./k,

eJ nn


Vutil.A- 1. Co 1

For 1 mg (dry weight) cells of S. typhimurium, A was 131 cm 2 (Smith et al., 1975). Assuming a similar surface-to-weight ratio in E. coli, we get 1.78(nmol • s- i. mg- l) P(CM 6) > 131 (cm 2. mg- 1). 1 (nmol • cm 3)- 1.36 x 10 2cm/s. At such very low concentrations of Co, C~ is centainly not insignificant in comparison with Co, and the true permeability coefficient is probably much larger than this number. Thus even if CM7 contained only 1% of the porin molecules found in CM6, the permeability coefficient of CM7 would be larger than 1.36 × 10 2 × 10-2 = 1.36 x 10-4.Onecancomparethis expected value with the actual permeability coefficient of CM7, calculated in a way similar to that described above, on the basis of 90 min doubling time at the "Km" of 7 x 1 0 - 4 M (von Meyenburg, 1971). Thus 2 1.78 =1.95 x 10 5cm/s. 131 x 700 Thus 1%, or even 0.1% residual level of porin is seen to be sufficient to explain the rapid growth of CM7 in the presence of high concentrations of glucose. Similar conclusion is reached when calculations (not shown) are done for other carbon sources. These calculations show us two additional, important points. (a) The permeability of the CM6 outer membrane toward glucose (higher than 1.36x 10 -z cm/s) was at least a thousandfold higher than that toward6-aminopenicillanicacid(1.39 x 10- 5cm/s), yetwe have seen that the porin channels are responsible at least for the major part of permeability for both of these substances. Although this quantitative difference is likely to reflect differences in some non-specific parameters (size, charge, or number of hydroxyl groups, for example) that may be important in determining the diffusion rates, at present we cannot rule out P(CMT)_~

2 In this case Co is high, and C~ is very likely to be negligible in comparison with Co. Thus the permeabilitycoefficientobtained is probably close to the correct one

the possibility that porin channels have some degree of specificity toward certain class of substances. The equilibrium-type assays of permeability showed the lack of specificity in reconstituted porin-containing membranes (Nakae, 1976a); the assays used, however, could not have detected such quantitative differences of diffusion rates as measured in the present study. (b) If one considers E. coli cells growing in ordinary culture media containing high concentrations of nutrients, the number of porin molecules present is in an enormous excess over what is needed for a fast enough uptake of nutrients. In order to allow growth at maximal rate with 0.2% glucose, approximately 0.01% of the number present in CM6 will suffice. [This explains why workers have failed to detect differences in growth phenotype in known "porin-deficient" mutants of E. coli K-12 (Henning and Haller, 1975) and S. typhimurium LT2 (Nurminen et al., 1976), that all produce significant amounts of residual porin]. Presumably a large number is necessary for successful competition in the presence of lower concentrations of nutrients, a situation found in Nature as well as in maintenance cultures in the laboratory, and/or for the efficient transport of less favorable substrates. Although very few residual porin molecules can thus explain the transport function in k m t - mutants, we cannot at present exclude the alternative possibility that the residual diffusion process takes place via other, non-porin, channels. Indeed two lines of evidence suggest that this possibility should be taken seriously. Firstly, increases in non-porin proteins can reverse the phenotype of kmt mutants (Results). These "suppressor" mutants are currently under study, but obviously the simplest interpretation of these results is that the diffusion is mediated by these non-porin proteins forming channels or acting as carriers. Secondly, the K-12 transductants that have received the k m t - allele seem to exhibit less severe transport defect than the B/r mutants, i.e. they show less dramatic increases in transport K m (Table 2). The most striking case is the transport of histidine: in the B/r line, the loss of porin due to the presence of the k m t - allele increased the Km about 30-fold to 3 laM (yon Meyenburg, 1971), but there was only a threefold increase in the K-12 line, from 0.1 to 0.3~tM. As far as one can judge from the slab gel electrophoresis, the loss of porins in kmt transductants seems as complete as in the B/r mutants. It thus seems probable that K-12 contains non-porin proteins that can produce alternative channels; such proteins would either be missing or exist in much smaller numbers in the outer membrane of E. coli B/r. We are currently studying the identity and functions of the presumed non-porin channels (or carriers).

P. Bavoil et al. : Transport in Porin-Deficient Mutants of E. coli Acknowledgement. We thank Dr. J.F. Lutkenhaus for making his manuscript available to us before publication, and Mr. H. Lew for photographing the gels. K. v. M. also thanks Drs. Christoph Beck and Sydney Kustu for their hospitality and many stimulating discussions. This work was supported by a travel grant from the Danish Natural Science Research Council to K, v. M., and by research grants from U.S. Public Health Sevice (AI-09644) and American Cancer Society (BC-20) to H.N.

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Communicated by W. Arber

Received September 9. )977

Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein.

Molec. gen. Genet. I58, 2 3 - 3 3 (1977) © by Springer-Verlag 1977 Pleiotropic Transport Mutants of Escherichia coli Lack Porin, a Major Outer Membra...
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