/ . Biochem., 80, 1401-1409 (1976)
Peptidoglycan Sacculus of Escherichia coli K-12 1 Yoshinori HASEGAWA, Hisami YAMADA, and Shoji MIZUSHIMA 1 Department of Agricultural Chemistry, Faculty of-Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464 Received for publication, May 17, 1976
The outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution. Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions. When the outer membrane was preheated in SDS solution, the binding did not take place. Optimum binding was observed at pH 8 in the presence of 5 ITIM Mg t + . A high concentration of sodium chloride strongly inhibited the binding. The effects of these factors on the bindings of O-8 and O-9 were very similar. The binding of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other. Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60°. A dilution experiment showed that the complex was not an artifact. The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8 • O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature. The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.
The outer membrane and the peptidoglycan layer form the outermost layer of gram-negative bacteria. Recently, evidence has been accumulated which supports the view that they are interrelated in bacterial cells: (a) Hydrolysis of the peptidoglycan layer by enzymes such as lysozyme is indispensable for the isolation of the outer membrane (1-3). (b) A lipoprotein which binds covalently to the peptido1 This study was supported in part by a grant (No. 047060) from the Ministry of Education, Science and Culture of Japan. "* To whom correspondence should be addressed. Abbreviation: SDS, sodium dodecyl sulfate.
Vol. 80, No. 6, 1976
glycan layer (4) has also been found in outer membrane preparation which was prepared after hydrolysis of the peptidoglycan layer by lysozyme (5, 6). The results strongly indicate that the outer membrane and the peptidoglycan layer are linked by lipoprotein molecules, (c) Rosenbusch has reported that an envelope protein, matrix protein, of Escherichia coli B was specifically attached to the piptidoglycan layer even in sodium dodecyl sulfate (SDS) solution at 60° (7). Based on the migration velocity on polyacrylamide gel and the amino acid composition, the protein was believed to be one of the outer membrane proteins (8). (d) An asymmetric distribution of lipopolysaccharide in the
1401
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Interactions of Outer Membrane Proteins 0-8 and 0-9 with
1402
Y. HASEGAWA, H. YAMADA, and S. MIZUSHIMA.
Preparation of Peptidoglycan Sacculus—The peptidoglycan sacculus bearing lipoprotein was prepared based on the method of Yanai (personal communication). Details of the procedure are a& follows: E. coli YA21 grown in glycerol-casamino acids medium was cooled rapidly in ice-water at the late exponential phase of growth (about 10" cells per ml) and harvested by centrifugation. Bacterial cells (about 15 g wet weight from 4 liters of culture) were added to 200 ml of boiling 4% SDS. The mixture was boiled for one hour with stirring and then stirred for several hours until the solution had cooled to room temperature. The cell lysate thus obtained was centrifuged at 77,000 x g for 80 min at 20° and the white precipitate was carefully resuspended in warm water and centrifuged again under the same conditions. The pellet was resuspended in 20 ml of hot water and poured into 20O ml of boiling 4% SDS and the procedure described MATERIALS AND METHODS above was repeated. The resulting pellet was suspended in warm water and centrifuged at 1,000 x g~ Chemicals—SDS was obtained from Yonefor 10 min to remove large particles. Peptidoyama Chemical Industries Ltd. (Osaka). Lysozyme glycan sacculus was recovered form the supernatant [EC 3. 2. 1. 17] was a gift from Eizai Co. (Tokyo). solution by centrifugation at 77,000 x g for 80 min L-[4, 5-*H2]leucine (spec. act. 32 /iCi/mmole) was at 20°, washed by centrifugation with warm water purchased from Daiichi Pure Chemicals Co. under the same conditions and finally suspended ia (Tokyo). a small volume of water. The preparation conBacterium and Media—Escherichia coli YA21 tained a lipoprotein which covalently binds to a used was a leucine auxotrophic mutant derived peptidoglycan network. Unless otherwise noted, from E. coli (K-12; met~F-,T). Unless otherwise "peptidoglycan sacculus" refers to the lipoprostated, cells were grown with reciprocating shaking tein-bearing material in this paper. at 37° in a glycerol-casamino acids medium which contained, per liter; Na,HPO4-12Hi.O 26.5 g; Removal of Lipoprotein from Peptidoglycan KH,PO4 4.5g; NH,C1 1 g; casamino acids (Difco, Sacculus—The method of Yanai (personal comcertified) 5 g; glycerol 10 g; MgSO4-7H,O 0.3 g; munication) was used. The peptidoglycan sacCaCl,-2H,O 44 mg; FeSO4-7H2O I mg; pH 7.0. culus (about 20 mg dry weight) was treated with 1 For the preparation of cells lacking O-9 in the outer mg of pronase (Sigma Chem. Co) in 10 ml of 10 mM membrane, the medium was supplemented with Tris-HCl (pH 7.4) at 37° for 4 hr. One-tenth 3% (w/v) NaCl and glycerol was replaced by volume of 10% SDS was added and the solution glucose. For the preparation of cells lacking O-8 was boiled for 30 min to denature pronase. A in the outer membrane, Bacto-nutrient broth lipoprotein-free peptidoglycan sacculus was re(Difco) was used. covered by centrifugation and the treatment with Membrane Preparations—The outer membrane boiling SDS was repeated. Finally, the preparawas prepared as described previously (2). Outer tion was washed twice with water and suspended in membrane free from lysozyme was used in the pres- water. The preparation was free from lipoproent study. Lysozyme was removed from the outer tein, since its hydrolyzed product with either lysomembrane as described previously (11). For the zyme or trypsin did not give any protein band oa labelling of proteins, cells were grown in the SDS-polyacrylamide gel. glycerol-casamino acids medium supplemented Preparation of Peptidoglycan Sacculus Bearing with 200 fid L-[4, 5-'HJleucine (spec. act. 32 //Ci/ Outer Membrane Proteins OS and/or O-9—The mmole) per liter. method described by Rosenbusch (7) was applied /. Biochem.
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outer membrane was destroyed when the cell envelope was treated with lysozyme (9, 10). (e) The reassembly of outer membrane from constituents dissociated in SDS solution took place preferentially on the peptidoglycan layer (Yamada, H. & Mizushima, S., /. Biochem. in press). During the course of studies on the reassembly of outer membrane on the peptidoglycan sacculus, we have found that the outer membrane proteins O-8 and 0-9 were specifically bound to the peptidoglycan even in SDS solution. The proteins were the same as those found on the peptidoglycan sacculus when the sacculus was prepared from E. coli K-12 in SDS solution at 60° according to the method of Rosenbusch (7). In the present paper, the nature of the interaction between proteins O-8 and O-9 and the peptidoglycan sacculus is discussed.
OUTER MEMBRANE PROTEINS 0-8 AND 0-9 AND PEPTIDOGLYCAN OF £. coli
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using cells grown in three different media. In Osborn et al. (3). Purified lipopolysaccharide brief, cells were broken with glass beads and the prepared from the same strain was used as a standenvelope fraction was treated with SDS solution at ard. Organic phosphorus in phospholipid was 60°. In this paper, the envelope preparation after assayed by the method of Bartlett (14) and the SDS treatment at 60° is referred to as " Rosenbusch amount of phospholipid was calculated by assuming envelope." 25 fig of phospholipid per fig of lipid-phosphorus. Assay of Binding of Outer Membrane Proteins Glucosamine and muramic acid in peptidoglycan to Peptidoglycan Sacculus and Preparation of preparations were assayed by the Elson-Morgan Reassembled OS • O-9-Peptidoglycan Complex—Thereaction (75) and the amount of peptidoglycan was outer membrane (230 fig of protein) was dissolved calculated by assuming 5 fig of peptidoglycan per in 150 /il of 1 % SDS-1 % 2-mercaptoethanol at 37° fig of glucosamine. for 30min and centrifuged at 110,000 xg for 30 min. The supernatant solution thus obtained was RESULTS mixed well with peptidoglycan sacculus (80 /ig of peptidoglycan) suspended in 150 /il of 1 % SDS-1 % Binding of Proteins OS and O-9 to Peptido2-mercaptoethanol-20 mM Tris-HCl (pH 8.0), and glycan Sacculus—When the outer membrane was then 15 ^1 of 0.1 M MgCl, was added. The mix- dissolved in SDS solution and mixed with the ture was incubated at 30° for 60 min and centri- peptidoglycan sacculus, O-8 and O-9 were found in fuged at UO.OOOxg for 30 min to recover the the psptidoglycan fraction, which was recovered by peptidoglycan sacculus. The pellet was resus- centrifugation (Fig. 1). Sines the amount of 0-8 pended in 300 fi\ of 1 % SDS-1 % 2-mercaptoetha- and O-9 in the fraction was negligible when the nol and heated at 100° for 5 min to extract proteins. peptidoglycan sacculus was omitted from the reacThe heated sample was centrifuged at 110,000xg tion mixture, the results strongly indicate that these for 30 min to remove peptidoglycan sacculus and proteins were able to interact with the peptidogly100 fi\ of the supernatant solution was used for the can sacculus in SDS solution. The binding was analysis of proteins by polyacrylamide gel electro- highly specific, and none of the proteins in other phoresis. For the quantitative determination of preparations (cytoplasmic membrane, cytoplasm, O-8 and O-9, stained gels were scanned on paper and ribosomes) was able to bind to peptidoglycan with a Chromoscan (Joyce Loebl, filter 5-042) and sacculus under the same conditions. peaks of individual proteins were cut out and Identity of Proteins OS and O-9 and Proteins weighed. Proteins O-8 and O-9 purified according in Rosenbusch Envelope—An envelope protein of E. to the method of Nakamura and Mizushima (12) coli B has been found on the peptidoglycan sacculus were used as standards. in Rosenbusch envelope (7). Since the protein is The pellet before the extraction of O-8 and O9 in 1% SDS-1 % 2-mercaptoethanol at 100° was used as a reassembled 0-8-0-9-peptidoglycan complex. Polyacrylamide Gel Electrophoresis—Polyacryl0-80-9amide gel electrophoresis in the presence of SDS and urea was performed as described previously (2, 11). Nomenclature of Outer Membrane Proteins— The nomenclature of Uemura and Mizushima is used (11). B Analytical Methods—Protein content was determined by the method of Lowry et al. (13) with Fig. 1. Binding of O-8 and O-9 to peptidoglycan bovine serum albumin as a standard. The amount sacculus. The binding experiment was carried out of lipopolysaccharide was estimated from the 2- with (B) and without (Q peptidoglycan sacculus. A; keto-3-deoxyoctonic acid content, which was outer membrane (50 fi% of protein) was used as a assayed with thiobarbituric acid as described by reference. Vol. 80, No. 6, 1976
Y. HASEGAWA, H. YAMADA, and S. MIZUSHIMA
1#4
r
n
40 60 80 100° Temperature
Fig. 2. Proteins in outer membranes and Rosenbusch envelopes. Outer membranes (A, C, and E) and Rosenbusch envelopes (B, D, and F) were prepared from cells grown on glycerol-casamino acids medium (A and B), on glucose-casamino acids medium containing 3% sodium chloride (C and D) and on nutrient broth (E and F). Before electrophoresis, Rosenbusch envelopes were heated in 1% SDS-1% 2-mercaptoethanol at 100° for 5 min and centrifuged at 110,000 xg for 30 min to remove peptidoglycan sacculus. Outer membranes were also heated under the same conditions. The amounts of proteins applied to gels were 70 fig (A, C, and E) and 30 fig (B, D, and F).
Fig. 3. Effect of preheating of outer membranes in SDS solution on the binding of O-8 and O-9 to peptidoglycan sacculus. Outer membrane (300 fig of protein) was dissolved in ISO //I of 1% SDS-1% 2-mercaptoethanol in a centrifuge tube and heated at the indicated temperature for 30 min. After centrifugation at 1LO,OOOX0 for 30 min, the entire supernatant solution was used for binding experiments with peptidoglycan sacculus. O, O-8 bound to peptidoglycan; • , O-9 bound to peptidoglycan. The amount of protein bound after preheating at 37° was taken as 100%. Control experiments without peptidoglycan sacculus gave no detectable protein bands on the gels.
believed to be one from the outer membrane, proteins in Rosenbusch envelope were compared with those in the outer membrane in E. coli K-12. First we confirmed the result obtained by Rosenbusch, that is, Rosenbusch envelope from E. coli B gave a single band on polyacrylamide gel electrophoresis. It migrated to the position of O-9 (data not shown). However, Rosenbusch envelope from E. coli K-12 grown in glycerol-casamino acids medium gave two protein bands, the positions of which were the same as those of O-8 and O-9 on polyacrylamide gel electrophoresis (Fig. 2, B). When the concentration of sodium chloride in the culture medium was increased and glycerol was replaced by glucose, the lower band almost disappeared (Fig. 2, D). On the other hand, the upper band almost disappeared when the cells were grown in nutrient broth where the concentration of sodium chloride was very low (Fig. 2, F). Figure 2 also shows the protein compositions of outer membranes prepared from different cultures. Differences in the protien composition were exactly the same as those in Rosenbusch envelopes. The outer membrane prepared from E. coli B lacked O-8, consistent with the result for Rosenbusch envelope (Ichihara, S., &
Mizushima, S., /. Biochem. in press). These results strongly indicate that the proteins in Rosenbusch envelopes of E. coli K-12 are outer membrane proteins O-8 and O-9. The cytoplasmic membrane did not gave any significant protein band around this area on gel electrophoresis. Factors Influencing the Interaction of Proteins 0-8 and O-9 with Peptidoglycan Sacculus—Figure 3 shows that when the outer membrane was preheated in SDS solution, the binding of O-8 and O-9 to the peptidoglycan sacculus was strongly suppressed under the standard conditions for binding assay. The heat inactivation curves for O-8 and O-9 were very similar. Figure 4 shows the effect of temperature on the extraction of O-8 and O-9 from peptidoglycan. Similar curves were obtained with both Rosenbusch envelope and the reassembled O-8-O9-peptidoglycan complex. Curves for 0-8 and O-9 were also similar. Furthermore, these curves were similar to the heat inactivation curves for the binding of O-8 and O-9 to the peptidoglycan sacculus, shown in Fig. 3. Figure 5 shows the effect of pH on the binding of O-8 and O-9 to the peptidoglycan sacculus. The binding was most significant at pH 8 and the pH/. Biochem.
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0-80-9~
OUTER MEMBRANE PROTEINS O-8 AND O-9 AND PEPTIDOGLYCAN OF E. coli
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Fig. 4. Effect of temperature on the extraction of O-8 and O-9 from peptidoglycan. Rosenbusch envelopes were prepared in SDS solution at 37° instead of 60°. The preparation (70 fig of protein) was suspended in 500 ftl of 1% SDS-1% 2-mercaptoethanol and heated -at the indicated temperature for 30min. After centrifugation at 110,000 xg for 30 min, precipitates were •suspended in 500 i*\ of the same solution, heated at 100° for 5 min and centrifuged at 110,000X5 for 30 min. 200 fi\ of supernatant solution was used for polyacrylamide gel electrophoresis. O, O-8 and • , O-9. Reassembled 0-8-0-9-peptidoglycan complex was prepared as described in "MATERIALS AND METHODS." The complex was washed once with 1% SDS-1% 2-mercaptoethanol and suspended in'the same solution. The preparation (150//I; 30 /*g of protein) was heated at the indicated temperature for 30 min and centrifuged at 110,000xff for 30 min. Precipitates were suspended in 150 ft\ of the same buffer, heated at 100° for 5 min and centrifuged again to remove peptidoglycan sacculus. The whole supernatant solution was subjected to polyacrylamide gel electrophoresis. • , O-8 and • , O-9.
•dependency curves were essentially the same with both proteins. The effect of magnesium ions on the binding was also studied. Magnesium ions up t o 5 mM stimulated the binding of both O-8 and O-9 t o the peptidoglycan sacculus. However, higher concentrations of magnesium ions caused the precipitation of proteins without the peptidoglycan sacculus. Figure 6 shows the effect of sodium chloride o n the binding of O-8 and O-9 to the peptidoglycan sacculus. The binding was inhibited almost completely by 0.5 M sodium chloride, indicating the importance of electrostatic forces in the binding. The inhibition curves for O-8 and O-9 were very similar and also resembled those of the effect of •sodium chloride on the extraction of these proteins
Vol. 80, No. 6, 1976
Fig. 5. Effect of pH on the binding of O-8 and O-9 to peptidoglycan sacculus. Binding experiments were carried out under standard conditions at the indicated pH with 10 mM sodium succinate buffer (O, • ) and 10 mM Tris-HCl buffer ( • , • ) . O, D, O-8 and • , • , O-9. Control experiments without peptidoglycan sacculus gave no detectable protein bands on the gels.
0.2
0.4
NaCI (M)
Fig. 6. Effect of sodium chloride on the binding of O-8 and O-9 to peptidoglycan sacculus. Binding experiments were carried out under standard conditions with the indicated concentration of sodium chloride. O, O-8; • , O-9. Control experiments without peptidoglycan sacculus gave no detectable protein bands on the gels. from Rosenbusch envelope and from the reassembled O-8 • O-9-peptidoglycan complex, shown in Fig. 7. Due to the high concentration of salt in the reaction mixture, fractional determination of O-8 and O-9 on the gels was not carried out in Fig. 7.
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40 60 80 100° Temperature
Y. HASEGAWA, H. YAMADA, and S. MIZUSHIMA
1406
Fig. 7. Effect of sodium chloride on the extraction of O-8 and O-9 from peptidoglycan. Rosenbusch envelope (40 fig of protein) and reassembled O-8 • O-9peptidoglycan complex (42 fig of protein) were incubated in 200 fi\ of 1% SDS-10mM Tris-HCl buffer (pH 7.4) containing the indicated concentration of sodium chloride at 37° for 30 min. The reaction mixtures were centrifuged at 110,000 x g for 30 min and protein in the supernatant solution was determined. • , Rosenbusch envelope; O, reassembled O-8• O-9-peptidoglycan complex. The time required for the binding of these proteins on the peptidoglycan sacculus was examined and one hour was found to be sufficient to obtain the maximal binding. Quantitative Studies on the Binding of Proteins OS and O-9 to Peptidoglycan Sacculus—Under the standard conditions of the binding experiment, the amounts of O-8 and O-9 bound per mg of peptidoglycan were 0.2-0.4 mg and 0.4-0.8 mg, respectively, while those in Rosenbusch envelope were estimated to be 1.2-2.5 mg and 2.5-5 mg, respectively. When the amount of peptidoglycan sacculus was increased with a fixed amount of outer membrane proteins, the amounts of O-8 and O-9 bound to the peptidoglycan sacculus increased to a certain
100 200 Peptidoglycan (ug)
400
Fig. 8. Relationship between the amount of peptidoglycan and the amount of O-8 and O-9 bound. Binding experiments were carried out under standard conditions, with various amounts of peptidoglycan sacculus. Or O-8 and • , O-9. extent (Fig. 8). However, the curve levelled off, leaving more than half of each protein in solution. The results indicate that more than half of O-8 and O-9 was incapable of interacting with the peptidoglycan sacculus. Direct Binding of Proteins OS and O-9 ta Peptidoglycan—So far, we have used peptidoglycan sacculus,to which lipoprotein was bound covalently. The role of the bound form of lipoprotein in the interaction was examined by using peptidoglycan sacculus from which the lipoprotein had been removed by pronase treatment. Table I shows that the amounts of O-8 and O-9 bound per unit amount of peptidoglycan were the same irrespective of pronase treatment. The results indicate that the lipoprotein is not involved in the interaction. Although Rosenbusch envelope contained a certain amount of free lipoprotein, the following evidence rules out its involvement in the binding of O-8 and O-9; 1) the reassembled O 8 • O-9-peptidoglycan
TABLE I. Binding of O-8 and O-9 to lipoprotein-free peptidoglycan sacculus. Binding experiments were carried out under standard conditions. Control experiments without peptidoglycan sacculus gave no detectable bands on gels. Amount of protein bound (mg) Peptidoglycan sacculus O-8 Lipoprotein-bearing (0.3 mg as peptidoglycan) Pronase-treated (0.24 mg as peptidoglyc an) PG:
O-8/mg PG»
O-9
O-9/mg PG*
0.075
0.25
0.15
0.50
0.06
0.25
0.12
0.50
Peptidoglycan / . Biochem.
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02 0.4 NaCI (M)
OUTER MEMBRANE PROTEINS O-8 AND O-9 AND PEPTCDOGLYCAN OF E. colt
1407
Experiment No. I II
O-8 and O-9 in outer membrane addedb
Radioactivity in O-8 and O-9 (calculated)"
O-8 and O-9 in cell envelope0
0.60 mg 0.60
7630 cpm 8790
0.41 mg 0.41
Radioactivity found in Rosenbusch envelope 86 cpm 112
a
Cell envelope was prepared as described by Rosenbusch (7). b The fraction of O-8 plus O-9 in the outer membrane protein was estimated to be 33% from densitometric tracing of stained gel electrophoretograms of outer membrane. c The fraction of O-8 plus O-9 in the cell envelope protein was estimated to be 22% from densitometric tracing of stained gel electrophoretograms of cell envelope. complex was essentially free from these molecules and 2) the free form of lipoprotein in Rosenbusch envelope could be extracted almost completely at 80°, while significant amounts of O-8 and O-9 remained on the peptidoglycan sacculus under the same conditions (data not shown). The role of outer membrane components other than protein was also examined. The amounts of phospholipid and lipopolysaccharide in the reassembled complex were determined to be 0.21 mg and 0.12 mg per mg of peptidoglycan, respectively. Since the sum of O-8 and O-9 contents per mg of peptidoglycan has been estimated to be 0.6-1.2 mg, phospholipid and lipopolysaccharide in these amount may play some role in the binding of O-8 and O-9 to peptidoglycan. In all the experiments so far presented, the binding of O-8 and that of O-9 took place simultaneously and to the same extent. However, this did not mean that the binding of one of them required the simultaneous binding of the other, since the binding of O-8 or O-9 alone took place when outer membrane lacking either one of them was used (data not shown). Interaction between Proteins 0-8 and O-9 and Peptidoglycan in Native Cell Envelope—So far, we have shown that O-8 and O-9 are specifically bound to the peptidolgycan in SDS solution, and that the mode of binding is similar to that in Rosenbusch envelope. The specificity of the protein-peptidoglycan interaction strongly suggests, but does not prove, that the complex corresponds to that exist-
Vol. 80, No. 6, 1976
ing in the native cell envelope. We performed the following experiment to confirm this. Outer membrane labelled with L-[4, 5-*HJleucine was dissolved in SDS solution and the solution was used for dissolving a cell envelope fraction at 60° to prepare Rosenbusch envelope. If the O-8 • O-9peptidoglycan complex in Rosenbusch envelope is not the physiological one, but an artifact formed as a result of the SDS treatment, we would expect a significant incorporation of radioactive O-8 and O-9 into Rosenbusch envelope, as a result of dissociation of O-8 and O-9 from both the outer membrane and the csll envelope, mixing of the nonradioactive with radioactive protein and its subsequent binding to the peptidoglycan. As shown in Table II, the radioactivity incorporated in Rosenbusch envelope was only 86 cpm. Assuming that complete mixing of labelled O-8 and O-9 with non-labelled material preceded their binding to the peptidoglycan layer, one would expect about 3,100 cpm to be incorporated from the data shown in Table II. In experiment II in Table II, radioactive proteins dissolved in SDS solution were added after the formation of Rosenbusch envelope. The radioactivity incorporated was comparable to that in experiment I, indicating that this amount of radioactivity can be accounted for simply by the additional binding of O-8 and O-9 to Rosenbusch envelope. From this result we concluded that the 0-8-0-9-peptidoglycan complex is physiologically specific.
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TABLE II. Interaction between O-8 and O-9 and peptidoglycan in the native cell envelope.1 Experiment I: L-[4,5-'HJIeucine-labelled outer membrane (1.8 mg of protein, radioactivity 22,880 cpm) was dissolved in 2.6 ml^of extraction buffer (7) containing 2% SDS, 10 mM Tris-HCl (pH 7.3), 0.01% 2-mercaptoethanol and 10% glycerol. Cell envelope was suspended in the solution (1.8 mg of protein) and the suspension was incubated at 60° for 30 min. Rosenbusch envelope fraction was then recovered by centrifugation at 110,000 xg for 30 min, washed once with water and the radioactivity counted. Experiment II: Outer membrane and cell envelope was dissolved separately in 1.3 ml each of extraction buffer, incubated separately at 60° for 30 min, then combined. Rosenbusch envelope fraction was recovered as described in Experiment I.
1408
Y. HASEGAWA, H. YAMADA, and S. MIZUSfflMA
We have shown that outer membrane proteins O-8 and O-9 are specifically bound to the peptidoglycan sacculus in SDS solution. We have also shown that the same proteins are found on the peptidoglycan sacculus prepared in SDS solution according to the method of Rosenbusch ( 7 ) . Contrary to the present results, Rosenbusch has reported that only one protein was found in the peptidoglycan sacculus from E. colt B. We repeated his experiment and found that B strain lacks O-8, and accordingly Rosenbusch envelope contains only O-9. From these results, we conclude that strain K-12 possesses two proteins, O-8 and O-9, which interact with peptidoglycan, while strain B possesses only O-9. O-8 and O-9 are not separable on polyacrylamide gel in the absence of In the present paper, we have studied the nature of interaction of O-8 and 0-9 with the peptidoglycan sacculus using the native complex (Rosenbusch envelope) and the reassembled O-8 • O-9-peptidoglycan complex. Individual factors so far examined all influenced the interaction in both preparations to the same extent. This strongly indicates that the mode of interaction in the reassembled complex was the same as that in the native cell envelope.
Binding experiments under various conditions showed that O-8 and 0-9 are very similar. This is compatible with our previous finding that O-8 and O-9 were difficult to separate during purification (77). Furthermore, circular dichroism spectra of 0-8 and O-9 are essentially the same, as shown in the accompanying paper (72). Chemical analyses of 0-8 and O-9 are now under way in our laboratory. Because of the similarity between O-8 and O-9, the binding site on the peptidoglycan sacculus for these proteins is thought to be the same. However, data confirming this view have not yet been obtained.
The binding of 0-8 and O-9 to the peptidoglycan sacculus was strongly suppressed by preheating of the proteins in SDS solution. It is well known that many outer membrane proteins are heat-modifiable, that is, the migration velocity in SDS-poIyacrylamide gel changes when the sample is preheated in SDS solution. We have previously shown that heat modifiability also occurs in the conformation of outer membrane proteins, that is, /3-structured conformation in outer membrane proteins is stable in SDS solution and disappears on heating in SDS solution (76). Recently we succeeded in purifying 0-8 and O-9 without heating in SDS solution and found that both O-8 and O-9 are very rich in /5-stmctured polypeptide and that this conformation disappears only when they are heated in SDS solution (72). These results suggest that ^-structured polypeptide, which is stable in SDS solution at low temperature, is important for the binding of proteins to the peptido-
The amount of O-8 and O-9 per mg of peptidoglycan in the reassembled complex was about onefifth to one-seventh of that in Rosenbusch envelope. Even in the presence of a large excess of peptidoglycan sacculus, about a half of O-8 and O-9 remained unbound (Fig. 8), suggesting that these proteins were incapable of interacting with peptidoglycan. In the accompanying paper (72) O-8 and O-9 showed heterogeneous profiles on polyacrylamide gel electrophoresis in SDS solution unless they were heated. A certain conformation may be active in the interaction with the peptidoglycan sacculus. Another possibility is that a portion of O-8 and O-9 was masked by peptidoglycan fragments in the outer membrane preparation from the first. Finally, we would like to discuss the asymmetric assembly of the outer membrane. Miihlradt and Golecki (9) reported that an asymmetric distribution of lipopoiysaccharide in the outer
urea {11).
/ . Biochem.
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DISCUSSION
glycan sacculus. Both O-8 and O-9 seem to interact directly with the peptidoglycan network. Complete removal of the bound form of lipoprotein from the peptidoglycan sacculus by pronase did not cause any difference in its ability to bind O-8 and O-9. The free form of the lipoprotein was not involved in the binding. On the other hand, phospholipid and lipopolysaccharide may play some role in the binding, since the reassembled O-8-O-9-peptidoglycan complex contained considerable amounts of these materials. In addition, Rosenbusch envelope, which possesses larger amounts of O-8 and O-9, contained more phospholipid and lipopolysaccharide. Finally, the binding of either O-8 or O-9 did not require the simultaneous binding of the other.
OUTER MEMBRANE PROTEINS 0-8 AND O-9 AND PEPTIDOGLYCAN OF E. coli
REFERENCES 1. Miura, T. & Mizushima, S. (1968) Biochim. Biophys. Acta 150, 159-161 2. Mizushima, S. & Yamada, H. (1975) Biochim. Biophys. Acta 375, 44-53 3. Osborn, M.J., Gander, J.E., Parisi, E., & Carson, J. (1972) J. Biol. Chem. 247, 3962-3972 4. Braun, V. (1975) Biochim. Biophys. Acta 415, 335377
Vol. 80, No. 6, 1976
5. Bosch, V. & Braun, V. (1973) FEBS Lett. 34,307-310 6. Lee, N. & Inouye, M. (1974) FEBS Lett. 39,167-170 7. Rosenbusch, J.P. (1974) J. Biol. Chem. 249, 80198029 8. Garten, W., Hindennach, I., & Henning, U. (1975) Eur. J. Biochem. 59, 215-221 9. MUhlradt, P.E. & Golecki, J.R. (1975) Eur. J. Biochem. 51, 343-352 10. Nakamura, K. & Mizushima, S. (1975) Biochim. Biophys. Acta 413, 371-393 11. Uemura, J. & Mizushima, S. (1975) Biochim. Biophys. Acta 413, 163-176 12. Nakamura, K. & Mizushima, S. (1976) / . Biochem. 80, 1411-1422 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 14. Bartlett, G.R. (1959) / . Biol. Chem. 234, 466-468 15. Herbert, D., Phipps, P.J.P., & Strange, R.E. (1971) Methods in Microbiology Vol. 5B, pp. 284-285 Academic Press, London & New York 16. Mizushima, S. (1974) Biochem. Biophys. Res. Commim. 61,1221-1226 17. Smit, J., Kamio, Y., & Nikaido, H. (1975) J. Bacteriol. 124, 942-958
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membrane bilayer was maintained by the peptidoglycan layer, and that the asymmetry disappeared when the peptidoglycan layer was digested with lysozyme. We have also reported a structural asymmetry of the outer membrane on the surface of intact cells, while no asymmetry was observed with the isolated outer membrane or reassembled outer membrane {10). An asymmetric distribution of particles in the outer membrane was also observed in freeze-fractured electron micrographs of cells (77). The specific interaction of O-8 and O-9 with the peptidoglycan layer may provide an basis for asymmetric assembly of the outer membrane on the peptidoglycan surface.
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Peptidoglycan Sacculus of Escherichia coli K-12 1 Yoshinori HASEGAWA, Hisami YAMADA, and Shoji MIZUSHIMA 1 Department of Agricultural Chemistry, Faculty of-Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464 Received for publication, May 17, 1976
The outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution. Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions. When the outer membrane was preheated in SDS solution, the binding did not take place. Optimum binding was observed at pH 8 in the presence of 5 ITIM Mg t + . A high concentration of sodium chloride strongly inhibited the binding. The effects of these factors on the bindings of O-8 and O-9 were very similar. The binding of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other. Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60°. A dilution experiment showed that the complex was not an artifact. The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8 • O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature. The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.
The outer membrane and the peptidoglycan layer form the outermost layer of gram-negative bacteria. Recently, evidence has been accumulated which supports the view that they are interrelated in bacterial cells: (a) Hydrolysis of the peptidoglycan layer by enzymes such as lysozyme is indispensable for the isolation of the outer membrane (1-3). (b) A lipoprotein which binds covalently to the peptido1 This study was supported in part by a grant (No. 047060) from the Ministry of Education, Science and Culture of Japan. "* To whom correspondence should be addressed. Abbreviation: SDS, sodium dodecyl sulfate.
Vol. 80, No. 6, 1976
glycan layer (4) has also been found in outer membrane preparation which was prepared after hydrolysis of the peptidoglycan layer by lysozyme (5, 6). The results strongly indicate that the outer membrane and the peptidoglycan layer are linked by lipoprotein molecules, (c) Rosenbusch has reported that an envelope protein, matrix protein, of Escherichia coli B was specifically attached to the piptidoglycan layer even in sodium dodecyl sulfate (SDS) solution at 60° (7). Based on the migration velocity on polyacrylamide gel and the amino acid composition, the protein was believed to be one of the outer membrane proteins (8). (d) An asymmetric distribution of lipopolysaccharide in the
1401
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Interactions of Outer Membrane Proteins 0-8 and 0-9 with
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Y. HASEGAWA, H. YAMADA, and S. MIZUSHIMA.
Preparation of Peptidoglycan Sacculus—The peptidoglycan sacculus bearing lipoprotein was prepared based on the method of Yanai (personal communication). Details of the procedure are a& follows: E. coli YA21 grown in glycerol-casamino acids medium was cooled rapidly in ice-water at the late exponential phase of growth (about 10" cells per ml) and harvested by centrifugation. Bacterial cells (about 15 g wet weight from 4 liters of culture) were added to 200 ml of boiling 4% SDS. The mixture was boiled for one hour with stirring and then stirred for several hours until the solution had cooled to room temperature. The cell lysate thus obtained was centrifuged at 77,000 x g for 80 min at 20° and the white precipitate was carefully resuspended in warm water and centrifuged again under the same conditions. The pellet was resuspended in 20 ml of hot water and poured into 20O ml of boiling 4% SDS and the procedure described MATERIALS AND METHODS above was repeated. The resulting pellet was suspended in warm water and centrifuged at 1,000 x g~ Chemicals—SDS was obtained from Yonefor 10 min to remove large particles. Peptidoyama Chemical Industries Ltd. (Osaka). Lysozyme glycan sacculus was recovered form the supernatant [EC 3. 2. 1. 17] was a gift from Eizai Co. (Tokyo). solution by centrifugation at 77,000 x g for 80 min L-[4, 5-*H2]leucine (spec. act. 32 /iCi/mmole) was at 20°, washed by centrifugation with warm water purchased from Daiichi Pure Chemicals Co. under the same conditions and finally suspended ia (Tokyo). a small volume of water. The preparation conBacterium and Media—Escherichia coli YA21 tained a lipoprotein which covalently binds to a used was a leucine auxotrophic mutant derived peptidoglycan network. Unless otherwise noted, from E. coli (K-12; met~F-,T). Unless otherwise "peptidoglycan sacculus" refers to the lipoprostated, cells were grown with reciprocating shaking tein-bearing material in this paper. at 37° in a glycerol-casamino acids medium which contained, per liter; Na,HPO4-12Hi.O 26.5 g; Removal of Lipoprotein from Peptidoglycan KH,PO4 4.5g; NH,C1 1 g; casamino acids (Difco, Sacculus—The method of Yanai (personal comcertified) 5 g; glycerol 10 g; MgSO4-7H,O 0.3 g; munication) was used. The peptidoglycan sacCaCl,-2H,O 44 mg; FeSO4-7H2O I mg; pH 7.0. culus (about 20 mg dry weight) was treated with 1 For the preparation of cells lacking O-9 in the outer mg of pronase (Sigma Chem. Co) in 10 ml of 10 mM membrane, the medium was supplemented with Tris-HCl (pH 7.4) at 37° for 4 hr. One-tenth 3% (w/v) NaCl and glycerol was replaced by volume of 10% SDS was added and the solution glucose. For the preparation of cells lacking O-8 was boiled for 30 min to denature pronase. A in the outer membrane, Bacto-nutrient broth lipoprotein-free peptidoglycan sacculus was re(Difco) was used. covered by centrifugation and the treatment with Membrane Preparations—The outer membrane boiling SDS was repeated. Finally, the preparawas prepared as described previously (2). Outer tion was washed twice with water and suspended in membrane free from lysozyme was used in the pres- water. The preparation was free from lipoproent study. Lysozyme was removed from the outer tein, since its hydrolyzed product with either lysomembrane as described previously (11). For the zyme or trypsin did not give any protein band oa labelling of proteins, cells were grown in the SDS-polyacrylamide gel. glycerol-casamino acids medium supplemented Preparation of Peptidoglycan Sacculus Bearing with 200 fid L-[4, 5-'HJleucine (spec. act. 32 //Ci/ Outer Membrane Proteins OS and/or O-9—The mmole) per liter. method described by Rosenbusch (7) was applied /. Biochem.
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outer membrane was destroyed when the cell envelope was treated with lysozyme (9, 10). (e) The reassembly of outer membrane from constituents dissociated in SDS solution took place preferentially on the peptidoglycan layer (Yamada, H. & Mizushima, S., /. Biochem. in press). During the course of studies on the reassembly of outer membrane on the peptidoglycan sacculus, we have found that the outer membrane proteins O-8 and 0-9 were specifically bound to the peptidoglycan even in SDS solution. The proteins were the same as those found on the peptidoglycan sacculus when the sacculus was prepared from E. coli K-12 in SDS solution at 60° according to the method of Rosenbusch (7). In the present paper, the nature of the interaction between proteins O-8 and O-9 and the peptidoglycan sacculus is discussed.
OUTER MEMBRANE PROTEINS 0-8 AND 0-9 AND PEPTIDOGLYCAN OF £. coli
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using cells grown in three different media. In Osborn et al. (3). Purified lipopolysaccharide brief, cells were broken with glass beads and the prepared from the same strain was used as a standenvelope fraction was treated with SDS solution at ard. Organic phosphorus in phospholipid was 60°. In this paper, the envelope preparation after assayed by the method of Bartlett (14) and the SDS treatment at 60° is referred to as " Rosenbusch amount of phospholipid was calculated by assuming envelope." 25 fig of phospholipid per fig of lipid-phosphorus. Assay of Binding of Outer Membrane Proteins Glucosamine and muramic acid in peptidoglycan to Peptidoglycan Sacculus and Preparation of preparations were assayed by the Elson-Morgan Reassembled OS • O-9-Peptidoglycan Complex—Thereaction (75) and the amount of peptidoglycan was outer membrane (230 fig of protein) was dissolved calculated by assuming 5 fig of peptidoglycan per in 150 /il of 1 % SDS-1 % 2-mercaptoethanol at 37° fig of glucosamine. for 30min and centrifuged at 110,000 xg for 30 min. The supernatant solution thus obtained was RESULTS mixed well with peptidoglycan sacculus (80 /ig of peptidoglycan) suspended in 150 /il of 1 % SDS-1 % Binding of Proteins OS and O-9 to Peptido2-mercaptoethanol-20 mM Tris-HCl (pH 8.0), and glycan Sacculus—When the outer membrane was then 15 ^1 of 0.1 M MgCl, was added. The mix- dissolved in SDS solution and mixed with the ture was incubated at 30° for 60 min and centri- peptidoglycan sacculus, O-8 and O-9 were found in fuged at UO.OOOxg for 30 min to recover the the psptidoglycan fraction, which was recovered by peptidoglycan sacculus. The pellet was resus- centrifugation (Fig. 1). Sines the amount of 0-8 pended in 300 fi\ of 1 % SDS-1 % 2-mercaptoetha- and O-9 in the fraction was negligible when the nol and heated at 100° for 5 min to extract proteins. peptidoglycan sacculus was omitted from the reacThe heated sample was centrifuged at 110,000xg tion mixture, the results strongly indicate that these for 30 min to remove peptidoglycan sacculus and proteins were able to interact with the peptidogly100 fi\ of the supernatant solution was used for the can sacculus in SDS solution. The binding was analysis of proteins by polyacrylamide gel electro- highly specific, and none of the proteins in other phoresis. For the quantitative determination of preparations (cytoplasmic membrane, cytoplasm, O-8 and O-9, stained gels were scanned on paper and ribosomes) was able to bind to peptidoglycan with a Chromoscan (Joyce Loebl, filter 5-042) and sacculus under the same conditions. peaks of individual proteins were cut out and Identity of Proteins OS and O-9 and Proteins weighed. Proteins O-8 and O-9 purified according in Rosenbusch Envelope—An envelope protein of E. to the method of Nakamura and Mizushima (12) coli B has been found on the peptidoglycan sacculus were used as standards. in Rosenbusch envelope (7). Since the protein is The pellet before the extraction of O-8 and O9 in 1% SDS-1 % 2-mercaptoethanol at 100° was used as a reassembled 0-8-0-9-peptidoglycan complex. Polyacrylamide Gel Electrophoresis—Polyacryl0-80-9amide gel electrophoresis in the presence of SDS and urea was performed as described previously (2, 11). Nomenclature of Outer Membrane Proteins— The nomenclature of Uemura and Mizushima is used (11). B Analytical Methods—Protein content was determined by the method of Lowry et al. (13) with Fig. 1. Binding of O-8 and O-9 to peptidoglycan bovine serum albumin as a standard. The amount sacculus. The binding experiment was carried out of lipopolysaccharide was estimated from the 2- with (B) and without (Q peptidoglycan sacculus. A; keto-3-deoxyoctonic acid content, which was outer membrane (50 fi% of protein) was used as a assayed with thiobarbituric acid as described by reference. Vol. 80, No. 6, 1976
Y. HASEGAWA, H. YAMADA, and S. MIZUSHIMA
1#4
r
n
40 60 80 100° Temperature
Fig. 2. Proteins in outer membranes and Rosenbusch envelopes. Outer membranes (A, C, and E) and Rosenbusch envelopes (B, D, and F) were prepared from cells grown on glycerol-casamino acids medium (A and B), on glucose-casamino acids medium containing 3% sodium chloride (C and D) and on nutrient broth (E and F). Before electrophoresis, Rosenbusch envelopes were heated in 1% SDS-1% 2-mercaptoethanol at 100° for 5 min and centrifuged at 110,000 xg for 30 min to remove peptidoglycan sacculus. Outer membranes were also heated under the same conditions. The amounts of proteins applied to gels were 70 fig (A, C, and E) and 30 fig (B, D, and F).
Fig. 3. Effect of preheating of outer membranes in SDS solution on the binding of O-8 and O-9 to peptidoglycan sacculus. Outer membrane (300 fig of protein) was dissolved in ISO //I of 1% SDS-1% 2-mercaptoethanol in a centrifuge tube and heated at the indicated temperature for 30 min. After centrifugation at 1LO,OOOX0 for 30 min, the entire supernatant solution was used for binding experiments with peptidoglycan sacculus. O, O-8 bound to peptidoglycan; • , O-9 bound to peptidoglycan. The amount of protein bound after preheating at 37° was taken as 100%. Control experiments without peptidoglycan sacculus gave no detectable protein bands on the gels.
believed to be one from the outer membrane, proteins in Rosenbusch envelope were compared with those in the outer membrane in E. coli K-12. First we confirmed the result obtained by Rosenbusch, that is, Rosenbusch envelope from E. coli B gave a single band on polyacrylamide gel electrophoresis. It migrated to the position of O-9 (data not shown). However, Rosenbusch envelope from E. coli K-12 grown in glycerol-casamino acids medium gave two protein bands, the positions of which were the same as those of O-8 and O-9 on polyacrylamide gel electrophoresis (Fig. 2, B). When the concentration of sodium chloride in the culture medium was increased and glycerol was replaced by glucose, the lower band almost disappeared (Fig. 2, D). On the other hand, the upper band almost disappeared when the cells were grown in nutrient broth where the concentration of sodium chloride was very low (Fig. 2, F). Figure 2 also shows the protein compositions of outer membranes prepared from different cultures. Differences in the protien composition were exactly the same as those in Rosenbusch envelopes. The outer membrane prepared from E. coli B lacked O-8, consistent with the result for Rosenbusch envelope (Ichihara, S., &
Mizushima, S., /. Biochem. in press). These results strongly indicate that the proteins in Rosenbusch envelopes of E. coli K-12 are outer membrane proteins O-8 and O-9. The cytoplasmic membrane did not gave any significant protein band around this area on gel electrophoresis. Factors Influencing the Interaction of Proteins 0-8 and O-9 with Peptidoglycan Sacculus—Figure 3 shows that when the outer membrane was preheated in SDS solution, the binding of O-8 and O-9 to the peptidoglycan sacculus was strongly suppressed under the standard conditions for binding assay. The heat inactivation curves for O-8 and O-9 were very similar. Figure 4 shows the effect of temperature on the extraction of O-8 and O-9 from peptidoglycan. Similar curves were obtained with both Rosenbusch envelope and the reassembled O-8-O9-peptidoglycan complex. Curves for 0-8 and O-9 were also similar. Furthermore, these curves were similar to the heat inactivation curves for the binding of O-8 and O-9 to the peptidoglycan sacculus, shown in Fig. 3. Figure 5 shows the effect of pH on the binding of O-8 and O-9 to the peptidoglycan sacculus. The binding was most significant at pH 8 and the pH/. Biochem.
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0-80-9~
OUTER MEMBRANE PROTEINS O-8 AND O-9 AND PEPTIDOGLYCAN OF E. coli
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Fig. 4. Effect of temperature on the extraction of O-8 and O-9 from peptidoglycan. Rosenbusch envelopes were prepared in SDS solution at 37° instead of 60°. The preparation (70 fig of protein) was suspended in 500 ftl of 1% SDS-1% 2-mercaptoethanol and heated -at the indicated temperature for 30min. After centrifugation at 110,000 xg for 30 min, precipitates were •suspended in 500 i*\ of the same solution, heated at 100° for 5 min and centrifuged at 110,000X5 for 30 min. 200 fi\ of supernatant solution was used for polyacrylamide gel electrophoresis. O, O-8 and • , O-9. Reassembled 0-8-0-9-peptidoglycan complex was prepared as described in "MATERIALS AND METHODS." The complex was washed once with 1% SDS-1% 2-mercaptoethanol and suspended in'the same solution. The preparation (150//I; 30 /*g of protein) was heated at the indicated temperature for 30 min and centrifuged at 110,000xff for 30 min. Precipitates were suspended in 150 ft\ of the same buffer, heated at 100° for 5 min and centrifuged again to remove peptidoglycan sacculus. The whole supernatant solution was subjected to polyacrylamide gel electrophoresis. • , O-8 and • , O-9.
•dependency curves were essentially the same with both proteins. The effect of magnesium ions on the binding was also studied. Magnesium ions up t o 5 mM stimulated the binding of both O-8 and O-9 t o the peptidoglycan sacculus. However, higher concentrations of magnesium ions caused the precipitation of proteins without the peptidoglycan sacculus. Figure 6 shows the effect of sodium chloride o n the binding of O-8 and O-9 to the peptidoglycan sacculus. The binding was inhibited almost completely by 0.5 M sodium chloride, indicating the importance of electrostatic forces in the binding. The inhibition curves for O-8 and O-9 were very similar and also resembled those of the effect of •sodium chloride on the extraction of these proteins
Vol. 80, No. 6, 1976
Fig. 5. Effect of pH on the binding of O-8 and O-9 to peptidoglycan sacculus. Binding experiments were carried out under standard conditions at the indicated pH with 10 mM sodium succinate buffer (O, • ) and 10 mM Tris-HCl buffer ( • , • ) . O, D, O-8 and • , • , O-9. Control experiments without peptidoglycan sacculus gave no detectable protein bands on the gels.
0.2
0.4
NaCI (M)
Fig. 6. Effect of sodium chloride on the binding of O-8 and O-9 to peptidoglycan sacculus. Binding experiments were carried out under standard conditions with the indicated concentration of sodium chloride. O, O-8; • , O-9. Control experiments without peptidoglycan sacculus gave no detectable protein bands on the gels. from Rosenbusch envelope and from the reassembled O-8 • O-9-peptidoglycan complex, shown in Fig. 7. Due to the high concentration of salt in the reaction mixture, fractional determination of O-8 and O-9 on the gels was not carried out in Fig. 7.
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40 60 80 100° Temperature
Y. HASEGAWA, H. YAMADA, and S. MIZUSHIMA
1406
Fig. 7. Effect of sodium chloride on the extraction of O-8 and O-9 from peptidoglycan. Rosenbusch envelope (40 fig of protein) and reassembled O-8 • O-9peptidoglycan complex (42 fig of protein) were incubated in 200 fi\ of 1% SDS-10mM Tris-HCl buffer (pH 7.4) containing the indicated concentration of sodium chloride at 37° for 30 min. The reaction mixtures were centrifuged at 110,000 x g for 30 min and protein in the supernatant solution was determined. • , Rosenbusch envelope; O, reassembled O-8• O-9-peptidoglycan complex. The time required for the binding of these proteins on the peptidoglycan sacculus was examined and one hour was found to be sufficient to obtain the maximal binding. Quantitative Studies on the Binding of Proteins OS and O-9 to Peptidoglycan Sacculus—Under the standard conditions of the binding experiment, the amounts of O-8 and O-9 bound per mg of peptidoglycan were 0.2-0.4 mg and 0.4-0.8 mg, respectively, while those in Rosenbusch envelope were estimated to be 1.2-2.5 mg and 2.5-5 mg, respectively. When the amount of peptidoglycan sacculus was increased with a fixed amount of outer membrane proteins, the amounts of O-8 and O-9 bound to the peptidoglycan sacculus increased to a certain
100 200 Peptidoglycan (ug)
400
Fig. 8. Relationship between the amount of peptidoglycan and the amount of O-8 and O-9 bound. Binding experiments were carried out under standard conditions, with various amounts of peptidoglycan sacculus. Or O-8 and • , O-9. extent (Fig. 8). However, the curve levelled off, leaving more than half of each protein in solution. The results indicate that more than half of O-8 and O-9 was incapable of interacting with the peptidoglycan sacculus. Direct Binding of Proteins OS and O-9 ta Peptidoglycan—So far, we have used peptidoglycan sacculus,to which lipoprotein was bound covalently. The role of the bound form of lipoprotein in the interaction was examined by using peptidoglycan sacculus from which the lipoprotein had been removed by pronase treatment. Table I shows that the amounts of O-8 and O-9 bound per unit amount of peptidoglycan were the same irrespective of pronase treatment. The results indicate that the lipoprotein is not involved in the interaction. Although Rosenbusch envelope contained a certain amount of free lipoprotein, the following evidence rules out its involvement in the binding of O-8 and O-9; 1) the reassembled O 8 • O-9-peptidoglycan
TABLE I. Binding of O-8 and O-9 to lipoprotein-free peptidoglycan sacculus. Binding experiments were carried out under standard conditions. Control experiments without peptidoglycan sacculus gave no detectable bands on gels. Amount of protein bound (mg) Peptidoglycan sacculus O-8 Lipoprotein-bearing (0.3 mg as peptidoglycan) Pronase-treated (0.24 mg as peptidoglyc an) PG:
O-8/mg PG»
O-9
O-9/mg PG*
0.075
0.25
0.15
0.50
0.06
0.25
0.12
0.50
Peptidoglycan / . Biochem.
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02 0.4 NaCI (M)
OUTER MEMBRANE PROTEINS O-8 AND O-9 AND PEPTCDOGLYCAN OF E. colt
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Experiment No. I II
O-8 and O-9 in outer membrane addedb
Radioactivity in O-8 and O-9 (calculated)"
O-8 and O-9 in cell envelope0
0.60 mg 0.60
7630 cpm 8790
0.41 mg 0.41
Radioactivity found in Rosenbusch envelope 86 cpm 112
a
Cell envelope was prepared as described by Rosenbusch (7). b The fraction of O-8 plus O-9 in the outer membrane protein was estimated to be 33% from densitometric tracing of stained gel electrophoretograms of outer membrane. c The fraction of O-8 plus O-9 in the cell envelope protein was estimated to be 22% from densitometric tracing of stained gel electrophoretograms of cell envelope. complex was essentially free from these molecules and 2) the free form of lipoprotein in Rosenbusch envelope could be extracted almost completely at 80°, while significant amounts of O-8 and O-9 remained on the peptidoglycan sacculus under the same conditions (data not shown). The role of outer membrane components other than protein was also examined. The amounts of phospholipid and lipopolysaccharide in the reassembled complex were determined to be 0.21 mg and 0.12 mg per mg of peptidoglycan, respectively. Since the sum of O-8 and O-9 contents per mg of peptidoglycan has been estimated to be 0.6-1.2 mg, phospholipid and lipopolysaccharide in these amount may play some role in the binding of O-8 and O-9 to peptidoglycan. In all the experiments so far presented, the binding of O-8 and that of O-9 took place simultaneously and to the same extent. However, this did not mean that the binding of one of them required the simultaneous binding of the other, since the binding of O-8 or O-9 alone took place when outer membrane lacking either one of them was used (data not shown). Interaction between Proteins 0-8 and O-9 and Peptidoglycan in Native Cell Envelope—So far, we have shown that O-8 and O-9 are specifically bound to the peptidolgycan in SDS solution, and that the mode of binding is similar to that in Rosenbusch envelope. The specificity of the protein-peptidoglycan interaction strongly suggests, but does not prove, that the complex corresponds to that exist-
Vol. 80, No. 6, 1976
ing in the native cell envelope. We performed the following experiment to confirm this. Outer membrane labelled with L-[4, 5-*HJleucine was dissolved in SDS solution and the solution was used for dissolving a cell envelope fraction at 60° to prepare Rosenbusch envelope. If the O-8 • O-9peptidoglycan complex in Rosenbusch envelope is not the physiological one, but an artifact formed as a result of the SDS treatment, we would expect a significant incorporation of radioactive O-8 and O-9 into Rosenbusch envelope, as a result of dissociation of O-8 and O-9 from both the outer membrane and the csll envelope, mixing of the nonradioactive with radioactive protein and its subsequent binding to the peptidoglycan. As shown in Table II, the radioactivity incorporated in Rosenbusch envelope was only 86 cpm. Assuming that complete mixing of labelled O-8 and O-9 with non-labelled material preceded their binding to the peptidoglycan layer, one would expect about 3,100 cpm to be incorporated from the data shown in Table II. In experiment II in Table II, radioactive proteins dissolved in SDS solution were added after the formation of Rosenbusch envelope. The radioactivity incorporated was comparable to that in experiment I, indicating that this amount of radioactivity can be accounted for simply by the additional binding of O-8 and O-9 to Rosenbusch envelope. From this result we concluded that the 0-8-0-9-peptidoglycan complex is physiologically specific.
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TABLE II. Interaction between O-8 and O-9 and peptidoglycan in the native cell envelope.1 Experiment I: L-[4,5-'HJIeucine-labelled outer membrane (1.8 mg of protein, radioactivity 22,880 cpm) was dissolved in 2.6 ml^of extraction buffer (7) containing 2% SDS, 10 mM Tris-HCl (pH 7.3), 0.01% 2-mercaptoethanol and 10% glycerol. Cell envelope was suspended in the solution (1.8 mg of protein) and the suspension was incubated at 60° for 30 min. Rosenbusch envelope fraction was then recovered by centrifugation at 110,000 xg for 30 min, washed once with water and the radioactivity counted. Experiment II: Outer membrane and cell envelope was dissolved separately in 1.3 ml each of extraction buffer, incubated separately at 60° for 30 min, then combined. Rosenbusch envelope fraction was recovered as described in Experiment I.
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Y. HASEGAWA, H. YAMADA, and S. MIZUSfflMA
We have shown that outer membrane proteins O-8 and O-9 are specifically bound to the peptidoglycan sacculus in SDS solution. We have also shown that the same proteins are found on the peptidoglycan sacculus prepared in SDS solution according to the method of Rosenbusch ( 7 ) . Contrary to the present results, Rosenbusch has reported that only one protein was found in the peptidoglycan sacculus from E. colt B. We repeated his experiment and found that B strain lacks O-8, and accordingly Rosenbusch envelope contains only O-9. From these results, we conclude that strain K-12 possesses two proteins, O-8 and O-9, which interact with peptidoglycan, while strain B possesses only O-9. O-8 and O-9 are not separable on polyacrylamide gel in the absence of In the present paper, we have studied the nature of interaction of O-8 and 0-9 with the peptidoglycan sacculus using the native complex (Rosenbusch envelope) and the reassembled O-8 • O-9-peptidoglycan complex. Individual factors so far examined all influenced the interaction in both preparations to the same extent. This strongly indicates that the mode of interaction in the reassembled complex was the same as that in the native cell envelope.
Binding experiments under various conditions showed that O-8 and 0-9 are very similar. This is compatible with our previous finding that O-8 and O-9 were difficult to separate during purification (77). Furthermore, circular dichroism spectra of 0-8 and O-9 are essentially the same, as shown in the accompanying paper (72). Chemical analyses of 0-8 and O-9 are now under way in our laboratory. Because of the similarity between O-8 and O-9, the binding site on the peptidoglycan sacculus for these proteins is thought to be the same. However, data confirming this view have not yet been obtained.
The binding of 0-8 and O-9 to the peptidoglycan sacculus was strongly suppressed by preheating of the proteins in SDS solution. It is well known that many outer membrane proteins are heat-modifiable, that is, the migration velocity in SDS-poIyacrylamide gel changes when the sample is preheated in SDS solution. We have previously shown that heat modifiability also occurs in the conformation of outer membrane proteins, that is, /3-structured conformation in outer membrane proteins is stable in SDS solution and disappears on heating in SDS solution (76). Recently we succeeded in purifying 0-8 and O-9 without heating in SDS solution and found that both O-8 and O-9 are very rich in /5-stmctured polypeptide and that this conformation disappears only when they are heated in SDS solution (72). These results suggest that ^-structured polypeptide, which is stable in SDS solution at low temperature, is important for the binding of proteins to the peptido-
The amount of O-8 and O-9 per mg of peptidoglycan in the reassembled complex was about onefifth to one-seventh of that in Rosenbusch envelope. Even in the presence of a large excess of peptidoglycan sacculus, about a half of O-8 and O-9 remained unbound (Fig. 8), suggesting that these proteins were incapable of interacting with peptidoglycan. In the accompanying paper (72) O-8 and O-9 showed heterogeneous profiles on polyacrylamide gel electrophoresis in SDS solution unless they were heated. A certain conformation may be active in the interaction with the peptidoglycan sacculus. Another possibility is that a portion of O-8 and O-9 was masked by peptidoglycan fragments in the outer membrane preparation from the first. Finally, we would like to discuss the asymmetric assembly of the outer membrane. Miihlradt and Golecki (9) reported that an asymmetric distribution of lipopoiysaccharide in the outer
urea {11).
/ . Biochem.
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DISCUSSION
glycan sacculus. Both O-8 and O-9 seem to interact directly with the peptidoglycan network. Complete removal of the bound form of lipoprotein from the peptidoglycan sacculus by pronase did not cause any difference in its ability to bind O-8 and O-9. The free form of the lipoprotein was not involved in the binding. On the other hand, phospholipid and lipopolysaccharide may play some role in the binding, since the reassembled O-8-O-9-peptidoglycan complex contained considerable amounts of these materials. In addition, Rosenbusch envelope, which possesses larger amounts of O-8 and O-9, contained more phospholipid and lipopolysaccharide. Finally, the binding of either O-8 or O-9 did not require the simultaneous binding of the other.
OUTER MEMBRANE PROTEINS 0-8 AND O-9 AND PEPTIDOGLYCAN OF E. coli
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5. Bosch, V. & Braun, V. (1973) FEBS Lett. 34,307-310 6. Lee, N. & Inouye, M. (1974) FEBS Lett. 39,167-170 7. Rosenbusch, J.P. (1974) J. Biol. Chem. 249, 80198029 8. Garten, W., Hindennach, I., & Henning, U. (1975) Eur. J. Biochem. 59, 215-221 9. MUhlradt, P.E. & Golecki, J.R. (1975) Eur. J. Biochem. 51, 343-352 10. Nakamura, K. & Mizushima, S. (1975) Biochim. Biophys. Acta 413, 371-393 11. Uemura, J. & Mizushima, S. (1975) Biochim. Biophys. Acta 413, 163-176 12. Nakamura, K. & Mizushima, S. (1976) / . Biochem. 80, 1411-1422 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 14. Bartlett, G.R. (1959) / . Biol. Chem. 234, 466-468 15. Herbert, D., Phipps, P.J.P., & Strange, R.E. (1971) Methods in Microbiology Vol. 5B, pp. 284-285 Academic Press, London & New York 16. Mizushima, S. (1974) Biochem. Biophys. Res. Commim. 61,1221-1226 17. Smit, J., Kamio, Y., & Nikaido, H. (1975) J. Bacteriol. 124, 942-958
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membrane bilayer was maintained by the peptidoglycan layer, and that the asymmetry disappeared when the peptidoglycan layer was digested with lysozyme. We have also reported a structural asymmetry of the outer membrane on the surface of intact cells, while no asymmetry was observed with the isolated outer membrane or reassembled outer membrane {10). An asymmetric distribution of particles in the outer membrane was also observed in freeze-fractured electron micrographs of cells (77). The specific interaction of O-8 and O-9 with the peptidoglycan layer may provide an basis for asymmetric assembly of the outer membrane on the peptidoglycan surface.
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