Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10675-10679, October 1994 Biophysics Structural and functional alterations of a colicin-resistant mutant of OmpF porin from (bacteroi sentlty/coe N/porin channel/x-ray analysis) DENIS JEANTEUR*, TILMAN SCHIRMERt, DIDIER FOUREL0, VALERIE SIMONETt, GABRIELE RUMMEL§, CHRISTINE WIDMER§, JURG P. ROSENBUSCH§, FRANC PATTUS*¶, AND JEAN-MARIE PAGtSfII *European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, D-69012 Heidelberg, Germany; Departments of tStructural Biology and *Microbiology, Biozentrum, University of Basel, CH4056, Basel, Switzerland; and *Unit6 Propre de Recherche 9027, Centre de Biochimie et de Biologie Moleculaire, Centre National de la Recherche Scientifique, 31 Chemin Joseph-Aiguier, B.P. 71, Marseille Cedex 20, France Communicated by Eugene P. Kennedy, July 7, 1994

ABSTRACT A strain of Escherichia coli, selected on the residues that protrude from the barrel wall near the threefold basis of Its resistance to colicin N, reveals distinct structural molecular axis, faces two acidic side chains located on L3. and functional alterations in unspecific OmpF porin. A single This establishes a strong electrostatic field parallel to the mutation [Gly-119 -- Asp (G119D)] was identified in the membrane plane (11). Of the four colicin N-resistant point internal loop L3 that contributes critically to the formation of mutations that have recently been isolated and characterized the constriction inside the lumen of the pore. X-ray structure in OmpF porin (7), three are located on the external loops, analysis to a resolution of 3.0 A reveals a locally altered presumably impairing binding ofthe toxin. The fourth is aGly peptide backbone, with the side chain of residue Asp-119 Asp substitution at position 119 (G119D), located in loop protruding into the channe, causing the area of the constric- L3, far from the external surface of the molecule. Interest- tion (7 x 11 A in the wild type) to be subdivided into two ingly, Gly-119 belongs to the sequence motif PEFG119G that intercommunicating subcompartments of 3-4 A in diameter. is found in several enterobacterial porins (12). It is evident The functional consequences of this structural modification from the x-ray structure of the wild-type protein that in this consist of a reduction of the channel conductance by about position, no side chain can be accommodated without per- one-third, of altered ion selectivity and voltage gating, and of turbing the backbone structure. Here, we describe the struc- a decrease of permeation rates of various sugars by factors of tural and functional properties ofthis mutated porin.** While 2-12. The structural modification of the mutant protein the results cannot explain unambiguously the effect on colicin affects neither the «-barrel structure nor those regions of the N entry, the distinct structural alterations do explain the molecule that are exposed at the cell surface. Considering the pronounced functional changes observed in the mutant at the colicin resistance of the mutant, it is inferred that in vivo, atomic level. colicin N traverses the outer membrane through the porin channel or that the dynamics ofthe exposed loops are affected MATERIALS AND in the mutant such that these may impede the binding of the METHODS toxin. Bacterial Strains, , Media, and Mutagenesis of ompF . A "porin-deficient" strain, BZB 1107 (E. coli The unspecific porin, encoded by the ompF gene, is one of BE, ompF::TnS), was derived from the wild-type E. coli BE the major outer membrane proteins expressed in wild-type (BZB 3000BE) from the Biozentrum collection. Plasmids Escherichia coli K-12 cells growing under standard labora- pLG361 and pFD119 have been described (7, 13) and encode tory conditions (1). The protein forms three large water-filled wild-type or mutant (G119D) OmpF porin, respectively. Cells channels per trimer, allowing the diffusion of small hydro- were routinely grown in Luria-Bertani (LB) broth, at 37C philic molecules across the outer bacterial membrane (2-4). with gentle shaking. Kanamycin and tetracyclin were added It also serves as a cell-surface-exposed receptor for many as required. Mutagenesis and isolation of the G119D OmpF phages and colicins (1, 5). The role ofthe OmpF porin during have been described (7). Briefly, pLG361 encoding entry of colicin N (and A) in the binding step to the bacterial OmpF (13) was incubated overnight with 1 M hydroxylamine surface and in translocation across the outer membrane has in 0.5 M sodium phosphate (pH 6) at 37TC. DNA was purified recently been investigated (6, 7). Several antibiotics, includ- and used to transform BZB1107 cells. The cells were plated ing (3-lactams, use the porin pathway to cross the outer and incubated with colicin N. From the resistant clones on membrane and to find their targets (8). Deletion and substi- antibiotic plates, cells expressing OmpF were selected. Four tution mutations in the lumen of the porin channel dramati- types of substitutions were identified on the ompF gene by cally modify cell growth conditions and outer membrane sequencing 34 selected clones (7). In addition to resistance to permeability to hydrophobic antibiotics (9). colicin N, the mutant G119D also exhibited resistance to The three-dimensional structure of the OmpF porin has colicin A. been solved at 2.4-A resolution (10). Each monomer consists Purification and Characterization of the Mutated OmpF of a (3-barrel (16 antiparallel (-strands) that contains the Porin. The level of mutated OmpF synthesized was deter- channel. Six loops (each 11-17 residues long) are exposed to mined by immunoblot analysis and was similar to the wild- the surface ofthe cell, and one is involved in subunit contact. type OmpF (data not shown). Extraction and purification The longest loop (L3 with 34 residues) is bent into the channel were performed as described (14). The cells were broken at a height corresponding to the center of the membrane, forming the constriction site or selectivity gate (10). In this Present address: Ecole Superieure de Biotechnologie de Stras- narrow region, a positively charged cluster, formed by basic bourg, Pole Universitaire Illkirch, rue Sebastien Brant, 67400 Illkirch, France. ItTo whom reprint requests should be addressed. The publication costs ofthis article were defrayed in part by page charge **The atomic coordinates and structure factors have been deposited payment. This article must therefore be hereby marked "advertisement" in the Protein Data Bank, Chemistry Department, Brookhaven in accordance with 18 U.S.C. §1734 solely to indicate this fact. National Laboratory, Upton, NY 11973 (I.D. code 1MPF). 10675 Downloaded by guest on September 24, 2021 10676 Biophysics: Jeanteur et al. Proc. Natl. Acad. Sci. USA 91 (1994)

using a French press, and envelopes were recovered by FAST area detector and processed by the program MADNES centrifugation. Porins were then treated by preextractig (24). The crystals of space group P321 have cell constants a eight times with a buffer containing 0.5% octyl-polyoxyeth- = b = 117.9 A, c = 52.8 A, a = 13 = 90°, and fy = 1200. The ylene, which eliminated the majority of contaminants. Five agreement between symmetry related reflections was R8y. = extraction steps with a buffer containing 3% octyl- 9.4% (34% in the highest resolution shell). Data reduction and polyoxyethylene allowed solubilization ofintegral membrane map calculations were performed by programs from the proteins. The porin obtained after this last step was purified CCP4 package (25). The wild-type OmpF model including by ion-exchange chromatography (DEAE-cellulose, Merck) ordered water molecules and detergent fragments (10) served followed by chromatofocusing (PBE94, Pharmacia) and fi- as the starting model. The crystallographic R factor was nally by gel filtration on Sephadex G-150 (Pharmacia). Purity 23.9% (20-3.0 A). After remodeling of the site of mutation was checked by SDS/PAGE and isoelectrofocusing. and conventional positional refinement using the program Bacteriodn Sensitiity. The colicin survival tests and the XPLOR (26), the final model had an R factor of16.6% and good various (6, 15) were tested with cells grown in stereochemistry (rms deviations of bond lengths and angles LB medium (0.1 ml of suspension at OD600 = 0.5 unit). from ideal values are 0.014 A and 1.8°, respectively). Tem- Various dilutions (101 to 106) of colicins A or N were added perature factors were taken from the wild-type structure to the cells in 0.15 M NaCl/3 mM KCl/1 mM potassium without further refinement. The negative difference electron phosphate/10 mM sodium phosphate, pH 7, and incubated density at residue Asp-119 disappeared after assigning a for 20 min at 37"C. The cell suspension was then diluted with temperature factor of 45 A2 to its atoms. The final difference 15 vol of fresh LB medium. In the direct test, the percentage electron density has a rms deviation of0.034 electron perA3 of surviving cells with or without treatment was and extreme values of ±0.16 electron per Ak. monitored after 2 hr at 37"C by determining the ratio of the optical densities at 600 nm. When the normal pathway was RESULTS bypassed (6, 16) by treatment at low ionic strength ("bypass" experiments), cells were washed twice in 10 mM sodium High-Resolution Structure of G119D OmpF Porn. Trigonal phosphate (pH 6.8) and resuspended in the same buffer at the crystals (space group P321) of the mutant porin were readily initial density (ODWO = 0.5 unit). Portions (0.1 ml) of the cell obtained using the established conditions for the wild-type suspension were incubated with various dilutions of bacte- protein (23). The structure was solved by the difference riocins (101 to 106) in this buffer and treated as in the direct Fourier method at a 3.0-A resolution. The low R value assay. The extent of cell survival was monitored by the obtained with the wild-type model indicated that wild-type procedure described for the direct test. and mutant crystals were virtually isomorphous. The differ- Measurement of K+ efflux resulting from the insertion of ence electron density map showed a distinctly altered course colicin in the cytoplasmic membrane of sensitive bacteria of the protein backbone for segment at positions 119 and 120 allows a quantitative determination of the colicin action. and a positive density for the aspartyl side chain at position Variation of K+ concentrations was followed using a K+- 119 (Fig. 1A). In the wild type, the segment at positions 119 selective electrode (17, 18). The buffer was 0.11 M sodium and 120 fit snugly to the inner barrel wall, whereas in the phosphate/0.5 mM KCl, pH 7.2, and 109 cells per ml were mutant, it swung out toward the pore axis, with the carboxyl energized by addition of 0.2% glucose. Initial rates were group of Asp-119 near the cluster of basic residues at the calculated from the linear part of the kinetic of potassium opposite side of the pore (Fig. 1B). van der Waals distances efflux after addition of colicin at various multiplicities (18). to residues Arg42, Arg-82, and Arg-132 were on the order of Planr Lipid Bilayers and Veside Swelling Assays. Double 3 A. This effectively subdivides the channel into two sub- quartz-distilled water and reagent grade chemicals were compartments, each with diameters of 3-4 A. Comparison of used. Bilayers were formed across a 0.15-mm hole in Teflon the mutant structure with that ofthe wild type shows no effect septa, pretreated with a solution of 2% n-hexadecane in on the a-barrel. After superposition of all Ca-positions, the n-hexane. Conductance measurements and the criteria for rms deviation was 0.26 A, i.e., within the range of the bilayer formation were as -described (19, 20). The trans estimated coordinate error. compartment was held to virtual earth. The sign of the Alterations of Channel Properties. In native porin, the membrane potential referred to that on the cis side of the conductance of OmpF porin channels is large (0.85 nS). The membrane, and the values quoted, therefore, refer to Vci, - channels are slightly cation-selective and voltage-gated (21, Vtra8. Porins were always added to the subphase on the cis 27, 28). Channel activation usually occurs with high cooper- side of preformed bilayers, with the aqueous solution stirred ativity of three channels, while inactivation and fluctuations by a magnetic bar. The membrane current was amplified with occur independently as single steps. In vitro channel closings a current-voltage converter with an operational amplifier are induced by characteristic threshold potentials (Vj), irre- (Burr Brown, model 3528) and feedback resistors ranging spective of the polarity of the potential. The mutant porn from 106 to 109 fl. Recordings were filtered at 1 kHz with a showed significant changes in its electrical properties com- low-pass filter [EG & G (Salem, MA) model 113] and re- pared to the wild type (Table 1). Conductance values were corded on an FM tape recorder (Racal FM, Southampton, decreased, the pores were more cation-selective, and the U.K.). All experiments were performed at room tempera- threshold potential for channel closing was increased (Fig. 2). ture. Measurements of channel conductivities and voltage The permeation rates of sugars across the pores were altered dependence were performed in 1 M NaCl/10 mM Hepes/5 drastically. Also shown in Table 1 are the reductions of the mM CaCl2, with a final pH 7.0. For the evaluation of ion flux rates of glucose and mannose by factors of 5- to 12-fold selectivities, the reverse potential was generated by applying and that of arabinose by a factor of -2. 0.1 M NaCl in the trans side and 1 M NaCl in the cis side. Sensitivity to Colicin N. Resistance ofthe mutant G119D to Selectivities are expressed as the ratio of the permeability of colicin N, which requires the OmpF porin as the sole protein Na+ and Cl- ions, PNa/Pci (21). Swelling assays were per- to bind and to enter the bacterial cell, has been reported (7). formed as described (22). In view of the structural results, it was of interest to deter- X-Ray Crystallography. Crystallization of the mutant mine quantitatively the level of the sensitivity. Fig. 3 (A and (G119D) was performed as described for wild-type protein B) shows that the sensitivity to colicin N ofthe G119D OmpF (23). The crystals diffracted to 2.7 A but suffered from porin is decreased by a factor of =1000 relative to the wild radiation damage. A data set was, therefore, collected to type. The effect on K+ efflux during colicin N action on E. 3.0-A resolution (85% complete) from a single crystal on a coli cells is fully compatible with this result (Fig. 3C). Colicin Downloaded by guest on September 24, 2021 Biophysics: Jeanteur et al. Proc. Nadl. Acad. Sci. USA 91 (1994) 10677

E u 1 7

FiG. 1. (A) Stereoscopic view ofsegment containing positions 117-121 ofthe mutant G119D (the peptide backbone is indicated by solid bonds; the wild-type model is indicated by open bonds). Also shown is the electron density ofthe mutant porin, calculated with (2Fa,8-Fgjl) coefficients and model phases (residues 118-121 were not used in the structure factor calculation). The contour level ofthe map is la,. (B) Model ofthe mutant OmpF porin structure in the channel constriction. The view is approximately along the pore axis. The slab shown is -25 A thick. Strands in the a-barrel (periphery) are represented by broad arrows; the short helical segment is represented by a ribbon. Residues lining the pore constriction are shown in full, and other loop segments are indicated by double lines. (C) For comparison, the model ofthe wild-type porin (10) is shown in the same view as in B.

binding, and its translocation across the outer membrane, is these conditions, the G119D mutation did not confer signif- contingent upon OmpF (6, 7) unless, at low ionic strength, icant resistance to colicin N (Fig. 3B). colicin uses an alternative "bypass" pathway (6, 16). Under DISCUSSION Table 1. Comparison of the functional properties of the wild-type porin and G119D In the structure of wild-type OmpF porin, there is no space for a side chain in position 119. A change in the backbone Wild type Mutant Ratio conformation of the G119D mutation was, therefore, to be OmpF porin parameter (K-12) (G119D) mutant/wt anticipated. X-ray structure analysis showed that the local Electrical properties structural perturbation in the mutant protein is substantial Conductance, nS 0.85 0.55 0.65 and well defined (Fig. 1). Probably facilitated by the inher- Selectivity, PNa/Pca 1.91 4.45 2.33 ent conformational flexibility of the neighboring residue Threshold voltage (Vj), mV 150 190 1.27 Gly-120, the dipeptide (Asp119-Gly'20) adopts a new con- Sugar permeability* formation by protruding into the channel lumen. The con- Glucose 0.354 0.060 0.17 formation of this segment may be largely stabilized by Mannose 0.388 0.030 0.08 electrostatic interactions between the carboxylate group of Arabinose 0.478 0.198 0.41 Asp-119 and the cluster of basic residues on the opposite *Data are swelling rates (22) expressed as initial rates ofdecrease of side of the channel. The center-to-center distance to the OD4. unit(s)/mm. All experiments were performed six times. SD nearest arginine residue (Arg-42) is 6.0 A and is, therefore, values were 1-10% for OD values >0.3 unit and 5-20%6 for OD too large for a salt bridge, but the strong transversal values <0.2 unit. electrostatic field that exists across the pore (11) is further Downloaded by guest on September 24, 2021 10678 Biophysics: Jeanteur et al. Proc. Natl. Acad. Sci. USA 91 (1994)

1L 1 min

-0.1

FIG. 2. Channel properties of the G119D OmpF in asolectin bilayers. Purified trimers from the G119 OmpF mutant were incorporated into planar bilayers by injecting detergent-solubilized protein into the bathing solution. The scanned trace shows the stepwise increases of the membrane current that followed protein injections. Each upward step corresponds to the conductance ofatrimer(1.6 nS). The closing downward step at the end ofthe recording corresponds to the closing ofa single channel. (Inset) Current-voltage curve for G119D. The ionic current starts to decrease at applied potentials >200 mV. This negative resistance is due to channel closings. The critical threshold voltage (VY) ofthe wild-type OmpF occurs >160 mV. The buffer used was 10 mM Hepes/1 M NaCl/5 mM CaCl2 at pH 7.0. enhanced by the additional negative charge in the anionic with respect to channel closing is significant, as these values cluster. Due to the changed backbone at positions 119 and are highly reproducible. It is conceivable thatthe energy level 120 and the protruding side chain of Asp-119, the cross- of the closed state, the kinetic barrier for the transition, or section ofthe mutant pore is drastically reduced. In the wild both are increased due to steric hindrance by the additional type, the elliptical cross-section of the pore at the constric- side chain. In this context, it is interesting to compare a tion site is 7 x 11 A (using van der Waals radii ofthe atoms), mutation in OmpC porin (29) that conveys to cells the ability whereas in the mutant, the carboxylate group of Asp-119 to grow on substrates larger than the exclusion size of600 Da approaches Arg-42, Arg-82, and Arg-132 as close as 3 A, (30). Thus, maltodextrin is unable to diffuse across channels resulting in an effective division of the pore constriction of native OmpC porin but is able to diffuse into strains with into two subcompartments having diameters of 3 and 4 A, a constriction-forming L3 loop that either carry mutations and a concomitant reduction ofthe overall cross-section by encoding smaller side-chain residues or short deletions in this approximately one-third. loop. These changes increase the conductance of the porin The reduced conductance of the G119D mutant and the and its sensitivity to voltage. These opposite effects in the drastically reduced permeation rates of uncharged sugars OmpC mutants and that presented here indicate that the L3 (Table 1) may thus be attributed to the steric alterations and loop is indeed critical in the pore closing mechanism and to the altered charge distribution. With ions, dehydration suggest that the voltage gating might correlate with the may play a vital role in the diffusion rates across the con- inherent flexibility of the L3 loop. striction sites, since the hydrodynamic diameter of a hy- A challenging problem persists: although the structural drated sodium ion is -5.5 A. The observed increase in cation alteration observed in the mutant porin explains the func- selectivity is likely to be explained qualitatively by the tional modifications rather well, its resistance to colicin N, on presence of the additional negatively charged carboxylate the basis ofwhich it was selected, remains puzzling. The gene group at the pore constriction, analogous to the anion selec- sequence (7) and our present results show that the G119D tivity that in PhoE porn is due to a single lysine residue mutation has a strictly local effect in the constriction loop. (Lys-131). To assess this phenomenon quantitatively, mo- The x-ray data reveal that the conformation ofthe barrel and lecular dynamics calculations based on the x-ray structures of the six surface-exposed loops are unchanged. Since these are required. The decreased sensitivity of the mutant porn loops are involved in only a few weak crystal lattice inter- Dilution Dilution Multiplicity 10 20 30 40 50 WT

FIG. 3. Sensitivity to colicin, as revealed by bacterial survival or by K+ efflux. (A and B) The mutant strain (G119D) is significantly less sensitive to colicin in the presence ofporin, exceptunder "bypass" conditions (low ionic strength), butis considerably more sensitive than strains lacking porin. (C) Colicin N causes rapid K+ efflux from the wild type (WT) but very little from the G119D mutant. Survival was evaluated from culture turbidity (OD600) after the addition ofcolicin N (circles) to the final dilution indicated. Colicin A (squares) is shown for comparison. Open symbols and dotted lines show the porin-dependent pathway, and "bypass" conditions are indicated by solid symbols. The strains used were the "porin-deficient" strain, the wild-type strain (WT), and the G119D mutant. For efflux measurements, 2 x 109 cells per ml, corresponding to 1 mg (dry weight), were incubated for 15 min at 37TC in 10 mM Hepes/0.15 M NaCl, pH 7.2, supplemented with 0.2%6 glucose and 0.6 mM KCl. Initial rates were calculated from the linear part of the kinetics of K+ efflux after colicin addition and expressed in nmol per mg per min for colicin N at the multiplicities indicated in the top of the panel. Downloaded by guest on September 24, 2021 Biophysics: Jeanteur et al. Proc. Natl. Acad. Sci. USA 91 (1994) 10679 actions, their structures are unlikely affected by forces sta- 8. Nikaido, H. (1989) Antimicrob. Agents Chemother. 33, 1831- bilizing crystal contacts. Moreover, the same loop structures 1836. are also seen in a different (tetragonal) crystal form of 9. Benson, S. A., Occi, J. L. L. & Sampson, B. A. (1988) J. Mol. wild-type OmpF porin (S. W. Cowan, R. M. Garavito, J. N. Biol. 203, 961-970. 10. Cowan, S. W., Schirmer, T., Rummel, G., Steiert, M., Ghosh, Jansonius, J. Jenkins, R. M. Karlsson, N. Konig, E. Pai, R., Pauptit, R. A., Jansonius, J. N. & Rosenbusch, J. P. (1992) R. A. Pauptit, P. J. Rizkallah, J.P.R., G.R., and T.S., un- Nature (London) 358, 727-733. published data). Finally, the antigenic profile of the mutant 11. Karshikoff, A., Cowan, S. W., Spassov, V., Ladenstein, R. & porin is unchanged relative to the wild-type protein (7). The Schirmer, T. (1994) J. Mol. Biol. 240, 372-384. external surface thus appears very similar, suggesting the 12. Jeanteur, D., Lakey, J. H. & Pattus, F. (1991) Mol. Microbiol. following explanations. (i) The receptor function of the 5, 2153-2164. mutant porin is impeded. The constriction site may be 13. Jackson, M. E., Pratt, J. M., Stoker, N. G. & Holland, I. B. directly involved in the binding ofcolicin N or, alternatively, (1985) EMBO J. 4, 2377-2383. 14. Garavito, R. M. & Rosenbusch, J. P. (1986) Methods Enzymol. the dynamics of the external loops may be more severely 125, 309-328. affected by long-range conformational changes than appears 15. El Kouhen, R., Fierobe, H.-P., Scianimanico, S., Steiert, M., from the data. (ii) Translocation of colicin N occurs through Pattus, F. & Pages, J.-M. (1993)Eur. J. Biochem. 214, 635-639. the porin channel. In the mutant, this process could be 16. Cavard, D. & Lazdunski, C. (1981) FEMS Microbiol. Lett. 12, prevented due to steric hindrance at the pore constriction. 311-316. This latter possibility is intriguing, as unfolding has been 17. Boulanger, P. & Letellier, L. (1988) J. Biol. Chem. 263, suggested for the passage of colicin A (31, 32). The differ- 9767-9775. 18. Bourdineaud, J.-P., Boulanger, P., Lazdunski, C. & Letellier, ences observed between the two colicins appear to support L. (1990) Proc. Natl. Acad. Sci. USA 87, 1037-1041. this hypothesis, as the size of the receptor-translocator 19. Schindler, H. (1980) FEBS Lett. 122, 77-79. domain of colicin A is nearly twice as large as that of colicin 20. Wilmsen, H. U., Pugsley, A. P. & Pattus, F. (1990) Eur. N (15, 33). This difference could indeed affect the unfolding Biophys. J. 18, 149-158. process significantly. Approaching this question by studying 21. Benz, R., Janko, K., Boos, W. & Lauger, P. (1978) Biochim. the molecular dynamics of porin appears interesting also Biophys. Acta 511, 305-319. because it may give, at the same time, clues to the equilibrium 22. Nikaido, H. & Rosenberg, E. Y. (1983) J. Bacteriol. 153, of and the native in vivo. 241-252. open closed states of porin 23. Pauptit, R. A., Zhang, H., Rummel, G., Schirmer, T., Janso- nius, J. N. & Rosenbusch, J. P. (1991) J. Mol. Biol. 218, We thank Drs. D. Cavard and B. I. Holland for the generous gifts 505-507. of colicins and plasmids and Dr. M. Luckey for expert advice with 24. Messerschmidt, A. & Pflugrath, J. W. (1987) J. Appl. Crystal- the vesicle swelling assays. We gratefully acknowledge J.-M. Bolla, logr. 20, 306-315. D. Cavard, R. El Kouhen, J. H. Lakey, and M. Mallea for helpful 25. CCP4 (1979) Science and Engineering Research Council Col- discussions. We thank S. Scianimanico and N. Bleimling for excel- laborative Computing Project No. 4 (Daresbury Laboratory, lent technical assistance. This work was supported by the Centre Warrington, U.K.). National de la Recherche Scientifique and the Institut National de la 26. Brunger, A. T. (1990) X-PLOR Manual (Yale Univ., New Haven, Sante et de la Recherche Mddicale (CRE 930610) to J.-M.P. and by CT). grants of the Swiss National Science Foundation to T.S. and J.P.R. 27. Schindler, H. & Rosenbusch, J. P. (1978) Proc. Natl. Acad. Sci. USA 75, 3751-3755. 1. Nikaido, H. & Vaara, M. (1985) Microbiol. Rev. 49, 1-32. 28. Schindler, H. & Rosenbusch, J. P. (1981) Proc. Natl. Acad. 2. Benz, R. & Bauer, K. (1988) Eur. J. Biochem. 176, 1-19. Sci. USA 78, 2302-2306. 3. Buehler, L. K., Kusumoto, S., Zhang, H. & Rosenbusch, J. P. 29. Lakey, J. H., Lea, E. J. A. & Pattus, F. (1991) FEBS Lett. 278, (1991) J. Biol. Chem. 266, 24446-24450. 31-34. 4. Nikaido, H. (1994) J. Biol. Chem. 269, 3905-3908. 30. Misra, R. & Benson, S. A. (1988) J. Bacteriol. 170, 3611-3617. 5. Pugsley, A. P. (1984) Microbiol. Sci. 1, 168-176. 31. Bdn6detti, H., Lloubes, R., Lazdunski, C. & Letellier, L. 6. Fourel, D., Hikita, S., Bola, J.-M., Mizushima, S. & Pages, (1992) EMBO J. 11, 441-447. J.-M. (1990) J. Bacteriol. 172, 3675-3680. 32. Webster, R. E. (1991) Mol. Microbiol. 5, 1005-1011. 7. Fourel, D., Mizushima, S., Bernadac, A. & Pages, J.-M. (1993) 33. Baty, D., Frenette, M., Lloubes, R., Geli, V., Howard, S. P., J. Bacteriol. 175, 2754-2757. Pattus, F. & Lazdunski, C. (1988) Mol. Microbiol. 2, 807-811. Downloaded by guest on September 24, 2021