/ . Biochem. 84, 179-191 (1978)

Separation and Characterization of the Outer Membrane of Pseudomonas aeruginosa

Takeshi MIZUNO and Makoto KAGEYAMA Mitsubishi-Kasei Institute of Life Sciences, Machida, Tokyo 194 Received for publication, January 14, 1978

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (l'POpolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa {P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)} were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.

The cell envelope of gram-negative bacteria, including P. aeruginosa, consists of two typical membranous structures; outer membrane and cytoplasmic membrane (see reviews: 1). The separation of outer and cytoplasmic membranes was first achieved in E. coli by Miura and Mizushima by a technique involving isopycnic sucrose density Abbreviations: SDS, sodium dodecyl sulfate; KDO, l-ketoO-deoxyoctonate; EDTA, ethylcnediaminetetraacetate; TCA, trichloroacetic acid; TEMED, N.N, N'.N'-tetramethylethylenediamine. Vol. 84, No. 1, 1978

179

gradient centrifugation of membranes obtained from a spheroplast lysate (2), and a similar method modified for S. typhimurium was developed by Osborn et al. (3). These techniques have led to a greater understanding of the structure and function of the outer membrane of gram-negative bacteria (see reviews: 4-6). However, most of the information has come from the analysis of the outer membranes of Enterobacteriaceae. Reports of individual components of the cell envelope of P. aeruginosa have appeared: protein (7, 8), peptidoglycan (°), lipopolysaccharide {10, 11), and

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lipid (12). However, only a few authors have reported on the separation of outer and cytoplasmic membranes of P. aerugionsa, and on the characterization of outer membrane proteins (13, 14), moreover, only a little is known at present of the role of individual components in the structure and function of the outer membrane of P. aeruginosa. Bacteriocins (pyocins) of P. aeruginosa have been investigated by us from various standpoints; genetics of pyocinogenic factors (75,16), mechanism of killing action (17-19), interaction of pyocins and their receptors (20, 21), and assembly of pyocin particles (22). In the course of these studies, our attention was focused on the cell envelope of P. aeruginosa, since the cell envelope should play an important role in the action of pyocins. As the first step, we decided to examine more closely the outer membrane of P. aeruginosa. In the present paper, we describe the separation and characterization of outer and cytoplasmic membranes of P. aeruginosa, and also the protein composition and some characteristic features of major outer membrane proteins. We will also make a comparison with the outer membrane of E. coli. MATERIALS AND METHODS Chemicals—SDS and Coomassie brilliant blue R250 were obtained from Merck., KDO and egg white lysozyme [EC 3.3.1.17] and trypsin [EC 3.4.21.4] from Sigma Chem. Co., acrylamide from Eastman Kodak Co., and deoxyribonuclease from Miles Laboratories Ltd. Bacterial Strains and Growth Conditions— Pseudomonas aeruginosa P14 (23), PI 5 (16), M92 (PAO1) (24), M2008 (PAT) (25), and PAC1 (26), were used for the preparation of membranes. P. aeruginosa PI 5 was also used for the preparation of pyocin Rl. P. aeruginosa SL44 and SL109 were used for the preparation of phage 44 and phage 109, respectively. These are Sjoberg and Lindberg's typing phages (27). Cells were grown in nutrient broth with vigorous shaking at 37°C as described previously (28). Separation of Outer and Cytoplasmic Membranes—The separation of outer and cytoplasmic membranes was carried out by the method of Mizushima and Yamada (29) with some modifica-

tions, since P. aeruginosa is appreciably more sensitive than E. coli to treatment with EDTA and cold-shock. Cells were harvested at the late log phase by centrifugation at 8,000 rprn for 8 min at room temperature, and washed with 20% (w/v) sucrose. Cells (ca. 1.5 g wet weight) were suspended in 18 ml of ice-cold 20% (w/v) sucrose and ice-cold reagents were slowly added to the suspension in an ice-bath in the following order; 9 ml of 2 M sucrose, 10 ml of 0.1 M Tris-HCl (pH 7.8 at 25°C), 0.8 ml of 1 % Na-EDTA (pH 7.0), and 1.8 ml of 0.5% lysozyme. Then the mixture was warmed to 30°C within a few minutes and kept at that temperature for 60 min. During the incubation, the mixture became viscous due to breakage of a part of the cells, and so, after 30 min incubation deoxyribonuclease was added (ca. 3 /ig/ml). Then the suspension was centrifuged to remove the the spheroplasts at 13,000 rpm for 15 min at 30°C. Crude outer membranes were recovered from the supernatant by centrifugation at 30,000 rpm for 60 min. The spheroplasts were burst in 40 ml of 5 mM MgCli and the spheroplast membranes were recovered by centrifugation at 15,000 rpm for 20 min, and washed with the same solution. The crude outer and spheroplast membranes were suspended in distilled water and dialyzed overnight at 4°C against 1 mM Na-EDTA (pH 7.0) and 27 mM Na-EDTA (pH 7.0), respectively. Purification of outer and cytoplasmic membranes was carried out by isopycnic sucrose density gradient centrifugation. The membrane fractions suspended in a small volume of distilled water were layered onto discontinuous sucrose gradients, and centrifuged at 38,000 rpm for 15 h in a Hitachi rotor, RPS-40-T. Step gradients were prepared by layering 55% (1 ml), 50% (2 ml), 45% (3 ml), 40% (3 ml), and 37% (0.5 ml) sucrose solutions (w/w) over a cushion (1.5 ml) of 60% sucrose. For small scale preparation (ca. 5 mg protein), step gradients were prepared by layering 50% (0.5ml), 45% (1.5ml), 40% (1.0ml), and 37% (0.5 ml) sucrose solutions (w/w) over a cushion (1.0 ml) of 55% sucrose and centrifuged at 38,000 rpm for 4 h in a Hitachi rotor, RPS-40-T2. All sucrose solutions were made up in distilled water. Gradients were fractionated by an auto DensiFlow IIC (Buchler Instruments). Membrane fractions were collected and washed with distilled water by centrifugation at 30,000 rpm for 60 min. J. Biochem.

OUTER MEMBRANE OF P. atruginosa

181

Preparation of Peptidoglycan and Digestion P. aeruginosa P15-16d was used. After incubation with Trypsin or Lysozyme—Peptidoglycan was for 15 min or 30 min at 37°C, lipid was extracted prepared from P. aeruginosa PI 4 by the method of by the method of Bligh and Dyer (32). The Braun {30). Trypsin digestion of peptidoglycan chloroform phase was evaporated and the residue was carried out in 10 mM Tris-HCl (pH 8.0) at suspended in a small volume of chloroform: 37°C for 4 h with an enzyme substrate ratio of methanol (2:1). This solution was applied to a 1 : 60. The lysozyme digestion was carried out silica gel thin-layer plate (Merck code no. 5721) and overnight in 10 mM Tris-HCl (pH 8.0) at 37°C developed with petroleum ether : diethylether : acetic acid (70 : 30 : 2, by volume). The spots of with an enzyme substrate ratio of 1 : 40. reaction products corresponding to free fatty acid Analytical Procedures Protein—The method of Lowry et al. (31) was were counted by a gas-flow GM-counter (Aloka). used with bovine serum albumin as a standard. Assay of Activity Inactivating Pyocin Rl and 2-Keto-3-Deoxyoctonate (KDO)—The KDO Bactenophages—Pyocin Rl was purified from a content was determined by the thiobarbituric acid mitomycin C-lysate of P. aeruginosa PI5 by the method according to the method of Osborn (3) method described previously (34). The activity of the membrane fraction to neutralize pyocin Rl with KDO as a standard. Phosphohpid—The extraction of phospholipid was estimated by the methpd of Ikeda (35). The from the membrane fraction was carried out neutralization activity of the membrane fraction according to the method of Bligh and Dyer (32). towards phages was estimated by the inhibition of The amount of organic phosphorus in phospholipid plaque formation. A mixture of phage and memwas determined by the method of Bartlett (55). brane fraction (0.2 to 30 fig of protein) was incuThe relative amount .of phospholipid in the mem- bated, in 100 ft] of DB solution containing 5 mM brane fraction was calculated by assuming 25 fig CaCl2) and the remaining phage was counted by the agar overlay technique. Phages 109, 44, and of phospholipid per fig of lipid-phosphorus. and E79 were prepared on P. aeruginosa SL109, Enzyme Assays Succinate Dehydrogenase, D-Lactate Dehydro- SL44, and PI 4, respectively, which were also used genase, Malate Dehydrogenase, Gluconate Dehydro-as indicator strains for phage infectivity, respecgenase, Glucose Dehydrogenase—Incubation mix- tively. SDS Polyacylamide Gel Electrophoresis tures contained 60 mM phosphate buffer (pH 7.2), i) Urea-SDS Gel System— Urea-SDS gel 10 mM KCN, 10 fig of phenazine methosulfate, 20 fig of dichlorophenol indophenol, 25 mM suc- electrophoresis was carried out according to the cinate (2.5 mM D-lactate, 20 mM malate, 30 mM method of Uemura and Mizushima (36). The gluconate, or 30 mM glucose), and the membrane concentrations in the gel were as follows; 8% fraction (20 to 100 fig of protein) in a volume of acrylamide, 0.1% N,N'-methylene-bisacrylamide 1.0 ml. Absorbance at 600 nm was measured for (bis-acrylamide), 0.5% SDS, 0.1 M sodium phosmore than lOmin at 25°C by a Shimadzu recording phate (pH 7.2), and 0.06% N,N,N',N'-tetramethylethylenediamine (TEMED). Except where otherspectrophotometer UV200. Nicotinamide Adenine Dinucleotide (NADH) wise noted, samples were solubilized in 1 % SDS Oxidase—Incubation mixtures contained 50 mM and 2% j9-mercaptoethanol at 100°C for 5 min. Tris-HCl (pH 8.0 at 25°C), 0.12 mM NADH, 0.2 mM Gels were fixed with 20% (w/w) sulfosalicylic acid dithiothreitol, and the membrane fraction (20 to and stained with Coomassie brilliant blue R250 100 fig of protein) in a volume of 1.0 ml. The rate according to the method of Maizel (37). ii) SDS Slab-Gel System—Slab-gel electroof decrease in absorbance at 340 nm was measured phoresis using a discontinuous buffer system was at 25°C. Phospholipase Activity—Incubation mixtures carried out according to the method of Laemmli contained in a final volume of 1.0 ml : 20 nmol of (38), with slight modification. The concentrations ["qphospholipid, 0.05% Triton X-100, 30 mM in the separation gel were as follows; 12% acrylTris-HCl (pH8.0 at 25°Q, 5 mM CaCl, and amide, 0.33% bis-acrylamide, 0.375 M Tris-HCl membrane fraction (200 fig of protein). Crude (pH 8.8), 0.1 % SDS, and 0.025% TEMED. The total phospholipid labeled with [14C]acetate from concentrations in the stacking gel were as follows; Vol. 84, No. 1, 1978

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T. MIZUNO and M. KAGEYAMA

3% acrylamide, 0.75% bis-acrylamide, 0.1% SDS, 0.625 M Tris-HCl (pH 6.8), and 0.025% TEMED. The electrode buffer (pH 8.3) contained 0.025 M Tris, 0.192 M glycine, and 0.1% SDS. Samples were solubilized in 0.0625 M Tris-HCl (pH 6.8), 2% SDS, 10% sucrose, and 2% 0-mercaptoethanol at 100°C for 5 min. Electrophoresis was carried out first with a current of 10 mA per ge! until the bromophenol blue marker reached the top of the separation gel, then with a current of 20 mA per gel until the marker reached the bottom of the gel. Slab-gels were fixed and stained with 10% acetic acid, 25% isopropanol, and 0.1% Coomassie brilliant blue R250 at 37°C for 1 h.

genase activity and the amount of KDO between these two kinds of membrane fractions are also presented in Fig. 1. Succinate dehydrogenase activity was specifically located in the L band fraction, while KDO was predominant in the H band fraction. The protein composition of these two fractions in the sucrose density gradient was determined by SDS polyacrylamide gel electrophoresis. As shown in Fig. 1C, the protein composition of the H band fractions was markedly different from that of the L band fractions e.g. the protein composition of H band fractions showed a simpler profile containing several major bands, while L band fractions consisted of many protein The following standard proteins were used bands with heterogeneous molecular weights. The for molecular weight calibration, 1) RNA poly- nature of the low density fractions in Fig. IB (a merase (£ : 165,000, a : 39,000), 2) bovine serum and b) is unclear. The protein composition of albumin (68,000), 3) trypsin inhibitor (21,000), and these fractions was similar to that of H band fractions but the KDO content was low. They 4) egg white Iysozyme (14,300). Electron Microscopy—Negative staining was may be defective H band materials formed artifiperformed with 1 % sodium phosphotungstate (pH cially during preparation. 6.2) or 2% uranyl acetate. For thin sectioning, Indentification and Characterization of Memsamples were fixed overnight with 1 % OsO4 in brane Fractions—H band and L band fractions were acetate-veronal buffer, dehydrated with ethanol isolated from P. aeruginosa P14 grown in nutrient and embedded in Epon. Sections were stained broth, the chemical compositions of which are with uranyl acetate and lead citrate. Observation presented in Table I. Both the band fractions was carried out with a Nihon-Denshi JEM-100B. consisted mainly of protein, lipid, and lipopolysaccharide. However, asymmetric distribution of these constituents was observed. KDO was rich RESULTS in the H band fraction, while only a small amount of Separation of Outer and Cytoplasmic Mem- it was observed in the L band fraction. KDO is branes—For spheroplast formation, the method of a component of lipopolysaccharide which is located Mizushima and Yamada (29) was modified, since on the outer cell surface of E. coli and S. typhiP, aeruginosa was more sensitive than E. coli to the murium (1). The relative amount of phosphotreatment with lysozyme-EDTA. Thus, the con- lipid in the L band fraction was higher than centration of EDTA for spheroplast formation and that in the H band fraction. for dialysis of membrane fractions was reduced to The localization of several enzyme activities the minimum (see " MATERIALS AND METH- in the two membrane fractions was determined. ODS ")• Profiles of two membrane fractions in Membrane fractions were prepared from two the sucrose density gradient are presented in Figs. different strains {P14 and M92 (PAO1)}. The 1A and B. The membrane fraction prepared enzyme activities of the electron transfer systems from spheroplasts was rich in low density materials were predominantly localized in the L band fraction (L band, p=ca. 1.16 g/cc). On the other hand, (Table II): for example, the specific activity of the membrane fraction prepared from the super- succinate dehydrogenase was 200 to 300 fold higher natant of the spheroplast formation medium was than that of H band fraction. Phospholipase A rich in high density materials (H band, p=ca. 1.22 activity was rich in the H band fraction. It has g/cc). In some experiments, middle density been reported for E. coli that phospholipase Ax materials (M band) were also obtained in the is localized in the outer membrane (39). The data membrane fraction prepared from spheroplasts. presented above indicate that the H band fraction Asymmetrical distributions of succinate dehydro- was derived from the outer membrane and the L / . Biochem.

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OUTER MEMBRANE OF P. aerugmosa

a

W 15 20 Fraction No

b c d e

h

i

j

k

I

30

Fig. 1. Profiles of sucrose density gradient centrifugation of membrane fractions of P. aerugmosa P14. A Membrane fraction prepared from spheroplasts B: Membrane fraction prepared from supernatant of spheroplast formation medium (see " MATERIALS AND METHODS"), O, Protein; • , KDO, • , succmate dehydrogenase activity. C SDS slab-gel electrophoretic pattern of fractions by sucrose density gradient centrifugation (see " MATERIALS AND METHODS "), a-1; protein composition of the fractions indicated by arrows in A and B. Each two adjacent fractions were combined and used for KDO determination, succinate dehydrogenase assay, and analysis of protein composition.

TABLE I. Composition of membrane fractions. Membrane fractions were prepared from strain PI4 grown in nutrient broth Analytical methods are described under "MATERIALS AND METHODS." Membrane fraction H band Buoyant density Protein Phospholipid

Lband

1.16g/cc 1 22g/cc (pg/mg dry weight) 540

480

190

340

KDO

32

3

Phosphorus

33

14

Vol. 84, No. 1, 1978

band fraction from the cytoplasmic membrane of the bacteria. Electron Microscopy of Outer and Cytoplasmic Membranes—Morphologies of isolated outer and cytoplasmic membranes were clearly distinct from each other (Fig. 2). The outer membrane consisted of vesicles of homogeneous size (approximately 0.15 [im in diameter) bound by a single unit membrane. Some, however, appeared as open C-shaped structures in the thin-section picture. These characteristics of the morphology of isolated outer membranes are similar to these observed in other gram-negative bacteria (J). The cytoplasmic membrane appeared as amorphous fragments of various sizes.

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TABLE II. Localization of enzyme activities. Membrane fractions were prepared from strain P14 and strain M92 (PAO1) grown in nutrient broth. Enzyme assays were carried out as described under "MATERIALS AND METHODS" Specific activities are expressed as nanomoles per min per mg of protein. Specific activity Strain P14

Enzyme L band Succinate dehydrogenase D-Lactate dehydrogenase Malate dehydrogenase Glucose dehydrogenase Gluconate dehydrogenase NADH oxidase Phospholipase A

Strain M92(PAO1)

H band

207 38 57 19 nd» 54