Permeability of Pseudomonas aeruginosa Outer Membrane to Hydrophilic Solutes

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 1982, P /82/ $02.00/0 Copyright 1982, American Society for Microbiology Vol. 152, No. 2 Permeability of Pseudomonas aeruginosa Outer Membrane to Hydrophilic Solutes FUMINOBU YOSHIMURAt AND HIROSHI NIKAIDO* Department of Microbiology and Immunology, University of California, Berkeley, California Received 21 June 1982/Accepted 5 August 1982 Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20,uM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5,M. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate-limiting step in active transport processes with high Vmax values. The outer membrane of gram-negative bacteria plays an important role as a solute sizedependent permeability barrier. A group of major proteins, porins, located in the outer membrane, form water-filled pores which allow the trans-membrane diffusion of hydrophilic substances below a certain molecular size (25). Porin protein has been isolated from Pseudomonas aeruginosa (12, 23). Reconstitution studies with purified porins revealed that the P. aeruginosa porin produced a significantly larger pore than those of enteric bacteria such as Escherichia coli and Salmonella typhimurium (12, 13). On the other hand, P. aeruginosa shows a strong intrinsic resistance to a wide range of antibiotics, and this resistance has often been attributed to the poor permeability of its outer membrane (4, 5, 35). The resistance alone, however, does not constitute proof that the outer membrane or the cell wall of this species is unusually low in permeability, because the resistance could easily be due to the efficient inactivation of these agents. More pertinent in this respect are studies in which increased sensitivity to deleterious agents was found after damage or removal of the outer membrane (35). However, spheroplasts and inner membranes of E. coli are often more sensitive to antibiotics than are intact E. coli cells (for example, see reference 19); therefore, this finding does not prove that the P. aeruginosa outer membrane is t Present address: Department of Microbiology, School of Dentistry, Hokkaido University, Sapporo 060, Japan. in any sense unusual in its barrier properties. In fact, the crypticity ratios (i.e., ratio of,-lactam hydrolysis rate with sonicated extract to that with intact cells) for many,b-lactam compounds were similar in magnitude for P. aeruginosa and E. coli, although they were quite different with a few compounds, such as cephaloridine (see Table 19 of reference 31). Similar comments apply to the finding of elevated sensitivity in mutants with altered outer membranes (30, 38). The ratio of minimal inhibitory concentration in wild type to that in such a supersensitive mutant may sometimes be higher with P. aeruginosa than with E. coli, but this could be a reflection of differences in the drug-inactivating activity rather than a consequence of different barrier properties of the outer membrane. While this work was in progress, a result of the direct measurement of P. aeruginosa outer membrane permeability appeared (1). These workers compared the nitrocefin (28) permeability in P. aeruginosa with the permeability of E. coli outer membranes toward P-lactam compounds of very different structure and concluded that P. aeruginosa outer membrane had a much lower intrinsic permeability than did that of E. coli. This conclusion is unconvincing, however, as the structure of P-lactam compounds has a strong influence on their permeability through the outer membrane (H. Nikaido, E. Y. Rosenberg, and J. Foulds, submitted for publication). Thus, to our knowledge the low permeability of P. aeruginosa outer membrane has never been proven by a direct assay and instead has 636

2 VOL. 152, 1982 remained an inference. We show that the cell wall, and probably the outer membrane, of P. aeruginosa indeed has very low permeability to a wide range of solutes and that this low permeability appears to affect the kinetics of active transport of various nutrients by intact cells of this organism. MATERIALS AND METHODS Bacterial strais and growth conditions. P. aeruginosa K799 (38), and PAO1 (17) were used. E. coli K-12 JC6724 (enda thi gal) was also used for comparison. These organisms were always grown at 37 C with aeration by shaking (batch cultures) or bubbling (chemostat cultures). PERMEABILITY OF P. AERUGINOSA OUTER MEMBRANE For measurement of growth rates and growth Ki,, i.e., the substrate concentration giving half-maximal growth rate, M63 medium (8) supplemented with a carbon source was used in both batch and chemostat cultures. Harvesting and washing of cells were done by centrifuging usually at 1,000 x g for 15 min and always at room temperature to avoid autolysis. Dry weight of the cells was obtained from optical density of the suspension at 600 nm. When the cells were sonicated, this was done at 0 C by using the medium-sized probe of a Braunsonic 1510 sonicator with four 30-s bursts at an output level of 100 W; the concentration of the cell suspension was usually 2 to 5 mg/ml. L broth (1% yeast extract [Difco Laboratories], 1% tryptone [Difco], 0.5% NaCI) containing 6-aminopenicillanic acid was used for induction of the chromosomally determined P-lactamase. At 50,ug of 6-aminopenicillanic acid per ml, 3-lactamase levels in induced cultures were at least 10- to 20-fold higher than those in noninduced cultures. These cells were washed and suspended in 10 mm potassium phosphate buffer (ph 6.0)-S mm MgC92. Low-phosphate medium (0.02 M NH4CI, 0.02 M KCI, 0.12 M Tris-chloride, 0.5% glucose, 0.5% proteose peptone [Difco], M MgSO4, ph 7.4) was used for inducing periplasmic alkaline phosphatase (7). Cells were harvested at mid-exponential phase, washed once with 10 mm Tris-chloride (ph 7.6)-i mm MgCl2-1 mm KCI, and suspended in the same medium for phosphatase assay (see below). Production of spheroplasts. P. aeruginosa PAO1 grown in the low-phosphate medium (see above) was washed once in 20%o (wtlvol) sucrose-20 mm Trischloride buffer, ph 8.0, and suspended in this mixture at a concentration of 5 mg (dry weight)/ml, and EDTA (final concentration, 10 mm) was added. Finally, lysozyme was added to 1 mg/ml, and after 30 min of incubation at 25 C, MgCI2 was added to a final concentration of 15 mm, followed by about 0.1 mg each of pancreatic DNase and RNase per ml; spheroplasts were recovered by centrifugation at 3,000 x g for 15 min. Assay of glucose 6-phosphate dehydrogenase showed that only about 15% of this cytoplasmic enzyme leaked out into the "spheroplast supernatant" or "periplasmic" fraction. Direct measurement of outer membrane permeability. The permeability coefficient, P, of outer membrane was determined by measuring the rate of hydrolysis of,b-lactam compounds by intact cells (27, 39). By using sonicated extracts of cells grown in the presence of 50,ug of 6-aminopenicillanic acid per ml (see above), we 637 first determined the K,,, values of P. aeruginosa periplasmic P-lactamase for cephacetrile and cephaloridine to be 100 and 130,uM, respectively; there were only insignificant differences between the K799 enzyme and the PAO1 enzyme. Washed, induced cells of P. aeruginosa (see above) were divided into two portions. One portion was sonicated to release all 1Blactamase activity into solution, and the other portion did not receive any treatment. The rates of hydrolysis of cephalosporins by each of these preparations were determined spectrophotometrically (Nikaido et al., submitted), and the permeability coefficient was calculated by combining Fick's first law of diffusion with the Michaelis-Menten behavior of the 13-lactamase (27, 39). Because we could not find data on the surface area of P. aeruginosa cells, we used the surface area/weight ratio obtained with Salmonella typhimurium, i.e., 131 cm2/mg (dry weight) (34). Since a fraction of,b-lactamase leaked out of the cell during the centrifugationresuspension process, the supernatant obtained by the centrifugation of the "intact cell" preparation was also assayed for 1-lactamase activity, and the rate of hydrolysis by this preparation was subtracted from the intact cell rates to correct for the hydrolysis by the leaked-out enzyme. In different preparations, the rate of hydrolysis by this supernatant was between 1 and 18% of the rate obtained by using sonic extracts of the same amount of cells. Permeability of the outer membrane was measured also by using the rates of hydrolysis, by intact cells and sonic extracts, of several phosphate ester compounds. Cultures with derepressed alkaline phosphatase (see above) were used. The phosphatase activity was proven to be located in the periplasm because (i) spheroplasting as described above released 95% of the activity into the medium, a result suggesting that the enzyme is located either in the periplasmic space or in the outer membrane, whereas (ii) the outer and inner membrane fractions isolated according to reference 10 contained <5% of the activity originally present. The hydrolysis of sugar phosphates was determined by assaying for Pi by using a scaled-down version of the method of Chen et al. (6), both before and after incubation for 20 min at 37 C in 50 mm Tris-chloride buffer, ph 8.0, containing 10 mm substrate. The Km values for glucose 6-phosphate and fructose 6-phosphate were 830 and 670,uM, respectively, and there were no significant differences between K799 and PAO1. Two controls were required for the determination of intact cell activity. (i) The rate of hydrolysis by leaked-out enzyme molecules was determined by using, as the enzyme, the supernatant obtained by centrifuging the washed cell suspension. This activity corresponded to 7 to 20%o of the activity of sonicated extracts. (ii) The "spontaneous" release of Pi from cells was determined by incubating the cells alone without the substrate. The activities in these control tubes were subtracted from the rate observed in the tube containing the complete reaction mixture. A control similar to the second one above was also run with the assay with sonic extracts to correct for the rate of generation of Pi by the hydrolysis of cellular constituents. The hydrolysis of p-nitrophenylphosphate was determined in a reaction mixture containing 50 mm Trischloride buffer, ph 8.0, and 1 mm substrate and by stopping the reaction after 10 min at 37 C by adding

3 638 YOSHIMURA AND NIKAIDO NaOH to a final concentration of 0.4 M. The released p-nitrophenol was determined by its absorbance at 400 nm (24). The Km values for this substrate were 190 and 70,uM with the P. aeruginosa and E. coli enzymes, respectively. In this case, the only control needed was the rate of hydrolysis by the leaked-out enzyme [see (i) above]. Measurement of "growth K,." by batch culture method (26). The cells were first grown in M63 medium usually containing 0.5 to 2 mm concentrations of the carbon source. In mid-exponential phase, portions of this culture were diluted at various ratios (usually 3- to 20-fold dilution) into flasks containing prewarmed M63 medium without the carbon source. (Alternatively, in some experiments the cells at the end of the exponential phase were diluted into flasks containing M63 medium with various concentrations of the carbon source compound.) These flasks were shaken at 37 C, and the growth was followed by determining optical density at 310 nm. The concentrations of the carbon source at the time of dilution were calculated from the dilution ratio, the initial concentration of carbon source in the original culture, growth yield, and the optical density of the culture at the time of dilution, assuming that a fixed amount of carbon source was consumed by a unit amount of cellular growth. The reciprocals of initial rates of growth after dilution were plotted against the reciprocals of the carbon source concentrations at the time of dilution calculated as described, and the growth Km was obtained from these plots. Measurement of growth K,. in chemostat culture. Measurement of growth Km in chemostat culture was done by using a New Brunswick Bioflo model C-30 chemostat, by estimating either steady-state concentrations of the substrate in the working vessel at various dilution rates or the dilution rate that gave the maximum output of organisms in unit time (16, 36). Chemostat cultures were started by using medium 63 containing a 1 to 2 mm concentration of carbon source compound in the reservoir. At steady state, a small portion (4 to 5 ml) of the culture was taken out from the working vessel (volume, 250 ml) and was immediately centrifuged, using a Brinkmann centrifuge 3200 to remove bacterial cells, followed by passage through a membrane filter (type GS, 0.22,um; Millipore Corp.). The concentrations of glucose, fructose, and gluconate in the supernatant were determined spectrophotometrically by coupled enzymatic assays that produced NADPH from NADP by consuming these substrates (3). To minimize error, measurements were made at several dilution rates that would "bracket" the dilution rate corresponding to the half-maximal rate of growth. It was difficult to quantitate fumarate enzymatically with sufficient sensitivity and accuracy. Therefore, the growth Km was determined, from the maximum growth rate of batch culture and the dilution rate that gave the maximum output, by using equation 14 of Herbert et al. (16). RESULTS Measurement of outer membrane permeability. Although P. aeruginosa outer membrane has been suspected to exhibit low permeability to various solutes, we have not been able to find in J. BACTERIOL. the literature any attempt to measure directly the permeability. We therefore tried the approach of Zimmermann and Rosselet (39), in which the permeability coefficient of the outer membrane is calculated from the rate of hydrolysis of substrates by enzymes located in the periplasmic space. Table 1 shows the results with two cephalosporin substrates. The hydrolytic enzyme P- lactamase is chromosomally determined and inducible (32). Since induction with antibiotics with efficient bactericidal activity will damage the cell wall, we used 6-aminopenicillanic acid, which has very little antibiotic activity (29). Since the hydrolysis rates with suspensions of intact cells were so low, the correction for the hydrolysis by leaked-out, extracellular enzyme was sometimes as large as 50% of the rates measured with intact cells, and the precision of the permeability coefficients obtained might not have been very high. Nevertheless, the permeability coefficients for cephacetrile and cephaloridine were close to 10-6 cm/s. When these values are compared with the permeability coefficients of E. coli K-12 outer membrane toward the same compounds (0.8 x 10-4 and 5 x 10-4 cm/s; Nikaido et al., submitted), it is clear that the P. aeruginosa outer membrane shows permeability 100- to 500-fold lower than E. coli K- 12 outer membrane. To make certain that this apparent low permeability is not the result of an unusual location of the,-lactamase or is we also measured the hydrolysis rates of phosphorylated compounds by intact cells. As reported by Cheng et al. (7), P. aeruginosa produces a periplasmic alkaline phosphatase. The not limited to P-lactams, TABLE 1. Outer membrane permeability of P. aeruginosa PAO1 estimated from,b-lactam hydrolysis rates Rate of 1-lactam hydrolysis (nmol/ min per mg, dry Permeability Expt Substrate wt)a coefficient (nm/s) Intact Sonicated cellsb extract c Cephacetrile Cephaloridine d Cephacetrile Cephaloridine Substrate concentration was 1 mm. bactivity in intact cells was corrected for hydrolysis due to enzyme leaking out into the suspension buffer. c Cells were induced by growth in the presence of 50 g of 6-aminopenicillanic acid per ml. d Cells were induced by growth in the presence of 75 pg of 6-aminopenicillanic acid per ml.

4 VOL. 152, 1982 PERMEABILITY OF P. AERUGINOSA OUTER MEMBRANE 639 periplasmic location of this enzyme was confirmed by the release, upon spheroplast formation, of 95% of the enzyme into the medium. Regardless of the nature of the substrate, the P. aeruginosa wild-type strains had permeability coefficients in the neighborhood of 10-7 cm/s (Table 2). These values were about a few hundredfold lower than the permeability coefficients of the E. coli K-12 outer membrane (Table 2). Again due to the low permeability of the P. aeruginosa outer membrane, even extremely small amounts of enzyme that had leaked out into the medium contributed quite significantly to the rates of hydrolysis by intact cell suspensions (see Materials and Methods). To minimize the leakage of the enzyme, in some experiments the P. aeruginosa cells were grown at ph 6.3 (see reference 6). These cells gave similar permeability coefficients (Table 2), although the leakage was not significantly decreased. Growth K. for carbon sources. The determination of outer membrane permeability in the experiments described above relied on the assumption that the periplasmic space is a homogeneous compartment, in which substrates are fully exposed to the action of degradative enzymes. Although this is very likely, there is no guarantee that this assumption is correct. We have therefore studied the kinetics of transport of various compounds that acted as carbon and energy sources for P. aeruginosa. In gramnegative bacteria, various substrates first diffuse through the outer membrane, usually through the porin channels, and then are taken up by the active transport systems located in the cytoplasmic membrane. The diffusion through the outer membrane is a simple diffusion process and follows Fick's first law of diffusion. Thus, the penetration rate per unit area of the membrane is proportional to P x (CO - Cp), where P, CO, and Cp represent the permeability coefficient, concentration of the solute in the outside medium, and its concentration in the periplasmic space, respectively. Since the solute is efficiently removed from the periplasmic space by the active transport system, Cp is usually much smaller than C0, and we can assume the rate to be roughly proportional to P x Co. Thus, if the outer membrane has high intrinsic permeability (i.e., a high permeability coefficient), the diffusion rates through the outer membrane would remain reasonably high even at low values of CO, and the rate-limiting process in the overall transport process will be the active transport step through the inner membrane. However, when the permeability coefficient of the outer membrane is low, the diffusion through the outer membrane becomes the rate-limiting step, especially at low values of C,. Since this is a process with an infinitely large value of K,,, the Km of the overall transport process becomes elevated, regardless of the Km value of the active transport step. Thus, the Km value of the overall transport processes in whole cells can give us valuable clues to the magnitude of permeability coefficients of the outer membrane. If this Km is low (i.e., <10 FxM for high throughput systems such as the transport systems for carbon sources), then the outer membrane must have rather high permeability. If the Km is high, then the result is at least consistent with the low permeability of the outer membrane (see Discussion). We measured the substrate concentration that gave the half-maximal growth rates (growth Kin) rather than the actual transport rates, because at low concentrations of carbon and energy source, growth rate is obviously being limited by the rates of transport of this compound and because growth Km could be measured more easily and more precisely than the transport Km. Two methods were used. In the batch culture method, an exponentially growing culture was diluted, at various ratios, into prewarmed media without the carbon source, to decrease the carbon source concentration, and initial rates of growth were measured. This method could not be used for the PA01 strain, because simple dilution into prewarmed media tended to produce significant autolysis. Strain K799 showed much less autolysis, but even with this strain high ratios of dilution appeared to produce a lag phase lasting for several minutes (Fig. 1). Initial growth rates at various concentrations of the carbon source were obtained by disregarding TABLE 2. Outer membrane permeability estimated by phosphate hydrolysis rates Rate of hydrolysis of substrate (nmol/min per Permeability Strain Expt Substratea mg, dry wt) coefficient (nm/s) Sictd Intact esxoctd P. aeruginosa K799 1 F-6-P G-6-P PA01 1 G-6-P b G-6-P P-NPP P-NPP E. coli K-12 1 G-6-P P-NPP a F-6-P, Fructose 6-phosphate; G-6-P, glucose 6- phosphate; P-NPP, p-nitrophenyl-phosphate. b Cells were grown in medium with an initial ph of 6.3.

5 640 YOSHIMURA AND NIKAIDO o 0.20L (3 X ) (5X)* 0.10/ 0.06 (lox) (20X) Culture time (hr) FIG. 1. Batch culture dilution experiment with P. aeruginosa K799. The cells were grown, with shaking, in an Erlenmeyer flask containing medium 63 with 0.83 mm 2-ketogluconate. At the time indicated by the arrow, portions of the culture were diluted at indicated ratios into prewarmed medium 63 without the carbon source, and growth was followed. For details, see text. A310, Absorbance (optical density) at 310 nm. this short lag (Fig. 1), and the concentration giving the half-maximal growth rate was found from the double-reciprocal plot of (1/growth rate) versus (1/concentration). The growth Km values were all higher than 20,uM (with the exception of glucose) and were frequently higher than 100 gxm (Table 3). These values were far higher than the values observed with E. coli, where growth Km for most compounds were below 5 to 10 pum (15, 26, 37). The batch culture dilution method, however, was plagued by the lag and autolysis produced at the time of dilution. Therefore, we tried to confirm these results by using a second method, i.e., chemostat culture. Because here we are dealing with a population in continuous, exponential growth, more reliable values of growth Km can be obtained. The results confirmed the conclusions from batch culture studies (Table 3), although the Km for gluconate was substantially lower. DISCUSSION In view of the inferential nature of the evidence on the P. aeruginosa outer membrane permeability so far published, we felt that it was necessary to carry out a critical study of this problem. By using the rate of hydrolysis of 1- lactams by intact cells of P. aeruginosa, we found that the permeability coefficients of its outer membrane toward cephacetrile or cepholoridine were about 10-6 cm/s (Table 1), about 100- to several hundredfold lower than in E. coli (Nikaido et al., submitted). (A similarly low value for the penetration of nitrocefin, a chromogenic substrate for 1-lactamase [28], was obtained by Angus and co-workers [1].) It is possible, however, that,b-lactamases are sequestered in a special place within the periplasmic space. To rule out this possibility of an "additional barrier" between the bulk periplasmic space and,b-lactamases, we repeated the permeability coefficient determination by measuring phosphate ester hydrolysis by periplasmic alkaline phosphatase (Table 2). The precision of the results was low owing to the low activity of the enzyme, apparently low outer membrane permeability, and the often significant leakage of the enzyme out of the cell. However, there is no question that the permeability of P. aeruginosa outer membrane to the phosphorylated compounds tested was indeed very low, probably two orders of magnitude lower than in E. coli outer membrane. The two assays described above depended on the assumption that periplasmic degradative enzymes were uniformly distributed within the periplasmic space. The growth Km experiments circumvent this restriction. According to Fick's first law of diffusion, the rate of influx (V) of nutrients across the outer membrane is V = P * A * (Co - Cp) (1) where A denotes the area of the membrane and the other symbols have the meanings defined TABLE 3. J. BACTERIOL. Growth Km in P. aeruginosa Growth Km (ILM) Chemostat cul- Substrate Batch culture ture (K799)a K799 PAO1 Glucose <10 <1 <1 Fructose Gluconate Ketogluconate 50 Mannitol 20 Glycerol 20 Acetate 100 Fumarate Citrate 150 a The maximal growth rate constants (in hour-') for these substrates were: glucose, 0.56; fructose, 0.59; gluconate, 0.63; 2-ketogluconate, 0.55; mannitol, 0.54; glycerol, 0.35; acetate, 0.48; fumarate, 0.91; citrate, 0.78.

6 VOL. 152, 1982 PERMEABILITY OF P. AERUGINOSA OUTER MEMBRANE 641 earlier. The rate of active transport is governed by the Michaelis-Menten equation V = Vmax Cpl(Km + Cp) (2) We can combine equations 1 and 2 to eliminate Cp and define the growth Km or overall transport Km (Kin') as the external concentration at which V = 1/2 * Vmax. Thus, P = Vmax/[2 * A * (Km' - Km)] (3) If we assume the Vma value of 4 nmol/mg (dry weight) per s (26), A of 131 cm2/mg (dry weight) (34), and Km of 1 F.M, then equation 3 shows that P is 1 x 10-4 to 3 x 10-4 cm/s for the range (50 to 150,uM) of values of Km' observed for most solutes in P. aeruginosa (Table 3). (The values of P are affected very little by the assumed Km value for the active transport system, up to a Km of about 10,uM.) As the outer membrane of E. coli is calculated to have permeability coefficients in the vicinity of 4 x 10-3 cm/s for glucose (26), these permeability coefficients calculated from growth Km of P. aeruginosa suggest that the outer membrane of this organism has permeability 10- to 50-fold lower than that of E. coli for small nutrient molecules. (However, it is still possible that the P. aeruginosa outer membrane has much higher permeability, in the unlikely event that all of the cytoplasmic membrane-associated transport systems have low affinity and high Km values.) Glucose constitutes a clear exception, as its growth Km was <1 p,m, in contrast to the value of approximately 60 F.M for its structural isomer fructose (Table 3). This apparent exception, however, actually strengthens our tentative conclusion, because Hancock and Carey (11) found that P. aeruginosa grown in the presence of glucose produced a special "glucose channel" protein in the outer membrane. It appears likely that the large difference between the transport behavior of glucose and fructose is largely due to the difference in their diffusion rates through the outer membrane. Hancock and associates (14) have recently found that phosphate starvation of P. aeruginosa results in the production of another channel, protein P. Nevertheless, our results showed that cells grown under phosphate starvation conditions still produced outer membrane that had low permeability toward sugar phosphates and p-nitrophenylphosphate (Table 2). This observation, however, does not contradict Hancock's results (14), because the P protein is reported to produce an extremely narrow channel that barely accommodates Pi (14) and is not expected to allow the passage of these large phosphate esters. We found several reports on the kinetic parameters of the active transport systems in P. aeruginosa. Midgley and Dawes (22) as well as Eagon and Phibbs (9) reported a Km of 7 to 9,uM for glucose. Although these values are somewhat higher than ours (Table 3), it is difficult to obtain accurate results with transport assays with an incubation period of <1 min, and we believe that the growth Km determined by the chemostat method is a more reliable indicator of the magnitude of Km for glucose transport. For other carbohydrates, transport Km values have been reported at 16 to 77,uM for D-fructose (9), 40,uM for D-gluconate (10), 14,uM for D- mannitol (9), and 13,uM for glycerol (33); these values are similar to the growth Km values obtained in this study. In contrast, transport Km values for various amino acids have been found to be in the range of 0.4 to 1.4,uM (18, 20, 21). Although at first these findings appear to suggest the presence of outer membrane of high permeability, this impression is not necessarily correct. The Vmax values of these amino acid transport systems are only around 0.1 nmollmg per s (21), with one exceptionally high value of 0.4 nmol/mg per s (18) reported for a branched amino acid transport system. With the former value, equation 3 shows that these transport parameters can be produced with an outer membrane with a vermeability coefficient of 4 x 10-4 to 9 x 10- cm/s. The permeability coefficients of E. coli outer membrane toward amino acids have not been measured, but would probably be close to, or higher than, that estimated for glucose, 4 x 10-3 cm/s (26). Thus, even these low Km values for amino acids are not incompatible with the assumption that P. aeruginosa outer membrane is far less permeable than that of E. coli. Our growth Km data are therefore consistent with, and strengthen, the finding of low permeability coefficients toward P-lactam compounds and phosphate esters; we feel it unlikely that the latter observation was the result of sequestration of the periplasmic enzymes behind the second barrier. Our results in this study, however, do not shed any light on the nature of the permeability barrier in P. aeruginosa, although we assumed it to be the outer membrane. Reconstitution of P. aeruginosa porins into black lipid films (2) as well as liposomes (F. Yoshimura and H. Nikaido, manuscript in preparation) showed that this assumption was correct and that the low permeability of the outer membrane was *caused by the intrinsic properties of the porin molecule. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases and grant BC-20 from the American Cancer Society.

7 642 YOSHIMURA AND NIKAIDO We thank one of the anonymous reviewers of the first version of this paper for pointing out some of the references on the active transport processes in P. aeruginosa. LITERATURE CITED 1. Angus, B. L., A. M. Carey, D. A. Caron, A. M. B. Kropinski, and R. E. W. Hancock Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptibile mutant. Antimicrob. Agents Chemother. 21: Benz, R., and R. E. W. Hancock Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes. Biochim. Biophys. Acta 646: Bergmeyer, H. U. (ed.) Methods of enzymatic analysis. Academic Press, Inc., New York. 4. Brown, M. R. W The role of cell envelope in resistance, p In M. R. W. Brown (ed.), Resistance of Pseudomonas aeruginosa. John Wiley & Sons Ltd., London. 5. Bryan, L. E Resistance to antimicrobial agents: the general nature of the problem and the basis of resistance, p In R. G. Dogget (ed.), Pseudomonas aeruginosa: clinical manifestations of infection and current therapy. Academic Press, Inc., New York. 6. Chen, P. S., T. Y. Toribara, and H. Warner Microdetermination of phosphorus. Anal. Chem. 28: Cheng, K.-J., J. M. Ingram, and J. W. Costerton Release of alkaline phosphatase from cells of Pseudomonas aeruginosa by manipulation of cation concentration and of ph. J. Bacteriol. 104: Cohen, G. N., and H. V. Rickenberg Concentration specifique reversible des amino acides chez Escherichia coli. Ann. Inst. Pasteur Paris 91: Eagon, R. G., and P. V. Phibbs, Jr Kinetics of transport of glucose, fructose, and mannitol by Pseudomonas aeruginosa. Can. J. Biochem. 49: Guynon, L. F., and R. G. Eagon Transport of glucose, gluconate, and methyl a-d-glucoside by Pseudomonas aeruginosa. J. Bacteriol. 117: Hancock, R. E. W., and A. M. Carey Protein D1-A glucose-inducible, pore-forming protein from the outer membrane of Pseudomonas aeruginosa. FEMS Microbiol. Lett. 8: Hancock, R. E. W., G. M. Decad, and H. Nikaido Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01. Biochim. Biophys. Acta 554: Hancock, R. E. W., and H. Nlkaido Outer membrane of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PA01 and use in reconstitution and definition of the permeability barrier. J. Bacteriol. 136: Hancock, R. E. W., K. Poole, and R. Benz Outer membrane protein P of Pseudomonas aeruginosa: regulation by phosphate deficiency and function of small anionspecific channels in lipid bilayer membranes. J. Bacteriol. 150: Hayash, S., and E. C. C. Lin Capture of glycerol by cells of Escherichia coli. Biochim. Biophys. Acta 94: Herbert, D., R. Elsworth, and R. C. Telling The continuous culture of bacteria; a theoretical and experimental study. J. Gen. Microbiol. 14: Holloway, B. W., V. Krishnapillai, and A. F. Morgan Chromosomal genetics of Pseudomonas. Microbiol. Rev. 43: Hoshino, T Transport systems for branched-chain amino acids in Pseudomonas aeruginosa. J. Bacteriol. 139: J. BACTERIOL. 19. Izakl, K., M. Matsuhashi, and J. L. Stremlnger Glycopeptide transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reactions. Proc. Natl. Acad. Sci. U.S.A. 55: Kay, W. W., and A. F. Gronlund Amino acid transport in Pseudomonas aeruginosa. J. Bacteriol. 97: Kay, W. W., and A. F. Gronlund Transport of aromatic amino acids by Pseudomonas aeruginosa. J. Bacteriol. 105: Mldgley, M., and E. A. Dawes The regulation of transport of glucose and methyl a-glucoside in Pseudomonas aeruginosa. Biochem. J. 132: Mizuno, T., and M. Kageyama Separation and characterization of the outer membrane of Pseudomonas aeruginosa. J. Biochem. (Tokyo) 84: Neu, H. C., and L. A. Heppel The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J. Biol. Chem. 240: Nlkaldo, H., and T. Nakse The outer membrane of Gram-negative bacteria. Adv. Microb. Physiol. 20: Nikaldo, H., and E. Y. Rosenberg Effect of solute size on diffusion rates through the transmembrane pore of the outer membrane of Escherichia coli. J. Gen. Physiol. 77: Nikaldo, H., S. A. Song, L. Shaltiel, and M. Nurminen Outer membrane of Salmonella. XIV. Reduced transmembrane diffusion rates in porin-deficient mutants. Biochem. Biophys. Res. Commun. 76: O'Callaghan, C. H., A. Morris, S. Kirby, and A. H. Shingler A novel method for detection of,-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1: Price, K. E Structure-activity relationships of semisynthetic penicillins. Adv. Appl. Microbiol. 11: Richmond, M. H., D. C. Clark, and S. Wotton Indirect method for assaying the penetration of betalactamase-nonsusceptible penicillins and cephalosporins in Escherichia coli strains. Antimicrob. Agents Chemother. 10: Richmond, M. H., and R. B. Sykes The P-lactamases of Gram-negative bacteria and their possible physiological role. Adv. Microb. Physiol. 9: Sabath, L. D., M. Jago, and E. P. Abraham Cephalosporinase and penicillinase activities of a P-lactamase from Pseudomonas pyocyanea. Biochem. J. 96: Siegel, L. S., and P. V. Phibbs, Jr Glycerol and L-aglycerol-3-phosphate uptake by Pseudomonas aeruginosa. Curr. Microbiol. 2: Smit, J., Y. Kamlo, and H. Nikaido Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J. Bacteriol. 124: Sykes, R. B., and A. Morrb Resistance of Pseudomonas aeruginosa to antimicrobiol drugs. Prog. Med. Chem. 12: Tempest, D. W The continuous cultivation of microorganisms. I. Theory of the chemostat, p In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 2. Academic Press, Inc., New York. 37. Von Meyenburg, K Transport-limited growth rates in a mutant of Escherichia coli. J. Bacteriol. 107: Zlmmermann, W Penetration of P-lactam antibiotics into their target enzymes in Pseudomonas aeruginosa: comparison of a highly sensitive mutant with its parent strain. Antimicrob. Agents Chemother. 18: Z nmmean, W., and A. Rosselet The function of the outer membrane of Escherichia coli as a permeability barrier to,b-lactam antibiotics. Antimicrob. Agents Chemother. 12: