Vol. 140, No. 3
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 843-847 0021-9193/79/12-0843/05$02.00/0
Influence of Molecular Size and Osmolarity of Sugars and Dextrans on the Synthesis of Outer Membrane Proteins 0-8 and 0-9 of Escherichia coli K-12 HIROSHI KAWAJI, TAKESHI MIZUNO,t AND SHOJI MIZUSHIMA* Laboratory of Microbiology, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464, Japan Received for publication 3 September 1979
Supplementation of the growth medium with high concentrations of sugars or low-molecular-weight dextrans results in a drastic change in the ratio of outer membrane proteins 0-8 and 0-9, due to induction of 0-8 synthesis and suppression of 0-9 synthesis. Sugars and dextrans of molecular weights greater than 600 to 700 switched the synthesis of 0-9 to that of 0-8 more effectively than those of lower molecular weight, although the effect was almost the same within each of the two groups irrespective of the differences in molecular weight within the group. Proteins 0-8 or 0-9, or both, are responsible for the formation of pores that allow the passive diffusion of hydrophilic molecules whose molecular weights are smaller than about 600 (T. Nakae, Biochem. Biophys. Res. Commun. 71:877884, 1976). The results indicate that substances that cannot pass through the outer membrane switch the synthesis of 0-9 to that of 0-8 more effectively than those that can penetrate this membrane with the aid of 0-8, 0-9, or both. It is suggested that the osmotic pressure exerted on the outer membrane plays an imnportant role in the regulation of synthesis of the two proteins. Two major outer membrane proteins of Escherichia coli K-12, 0-8, and 0-9, resemble each other in many biochemical and physicochemical respects. They are so-called peptidoglycan-associated proteins (3, 14) and exist as trimers (12, 23). The circular dichroism spectra (13), amino acid compositions, and N-terminal sequences (5) are also very similar. Furthermnore, both proteins form a hexagonally latticed structure with lipopolysaccharide (22). Although it is almost certain that the two proteins are products of different structural genes (5), their similarity can be explained by the hypothesis that these genes developed from a single ancestral gene through the duplication of the ancestral genome of E. coli (5). On the other hand, the biosynthesis of the two proteins is affected differentially by high concentrations of substances like sucrose and NaCl in the culture media (3, 19). The influence of these substances on the biosynthesis of 0-8 and 0-9 was found to be opposite: inductive for 0-8 and suppressive for 0-9. This fact indicates the possible existence of a regulatory mechanism which controls the biosynthesis of both proteins. Proteins 0-8 and 0-9 have also been called Ib and Ia, c and b, and lb and la, respectively (1, 4, 7, 15). In the present work, we examined the influt Present address: Mitsubishi-Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan.
ence of carbohydrates of different molecular sizes on the biosynthesis of 0-8 and 0-9. Carbohydrates whose molecular weights were larger than 600 to 700 switched the synthesis of 0-9 to that of 0-8 more effectively than those of smaller molecular weight. However, the effect on the switching was almost the same within each of the two groups of carbohydrates irrespective of the differences in molecular weight. Protein 0-8 or 0-9 or both are responsible for the formation of pores that allow the passive diffusion of hydrophilic molecules whose molecular weights are smaller than 600 (11). The results strongly suggest that 0-8 and 0-9 are involved in the regulation of their own synthesis through their pore
functions. MATERLA1S AND METHODS Bacterial strain and growth conditions. E. coli W4626Phe- [K-12, F- purE trp phe lac-85 gal-2 xyl-2 mal mtl ara rpsL (A)] (9) was grown in medium A supplemented with different concentrations of sugars or dextrans. Medium A contained, per liter, 7 g of nutrient broth, 1 g of yeast extract, 2 g of glycerol, 3.7 g of K2HPO4, and 1.3 g of KH2PO4. Cell envelopes. Cells from a 10-ml culture were washed with 15 mM sodium phosphate buffer (pH 7.2) and broken with a sonic disintegrator in water. Cell envelopes were recovered by differential centrifugation, suspended in 2 ml of water, and extracted with an equal volume of 4% Triton X-100-20 mM Tris-
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KAWAJI, MIZUNO, AND MIZUSHIMA
hydrochloride (pH 8.0)-20 mM MgCl2 at 37°C for 30 min, based on the method of Schnaitman (16). Insoluble residues were recovered by centrifugation, and protein profiles were analyzed by urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis as described previously (10, 18). For quantitative determination of individual proteins, stained gels were scanned with a Joyce-Loebl densitometer, and the peaks of major outer membrane proteins 0-8, 0-9, and 0-10 plus 011 were cut out and weighed (for the nomenclature of 0-10 and 0-11, see reference 4). The amount of protein in envelope preparations was determined by the method of Lowry et al. (6). Sugars and dextrans of small molecular weights. Sucrose and lactose were obtained from Katayama Chemical Ind.; raffinose, maltose and galactose were obtained from Yoneyama Chemical Co.; mannitol came from Wako Chemicals; and stachyose came from Sigma Chemical Co. a-1,6-Glucans with number average molecular weights of 2,100, 1,300, 1,100, and 700 were gifts from Meito Sangyo Co. In this paper they are referred to as dextran-2100, dextran-1300, dextran-1100 and dextran-700, respectively. The contents as molar percentages of the sum of mono-, di-, and trisaccharides in these preparations were about 2, 10, 20, and 40%, respectively. The mixture of isomaltose and isomaltotriose (1:1 in weight ratio) was prepared from an enzymatic hydrolysate of dextran (DXase-D), a gift from Meito Sangyo Co., as follows. DXase-D (20 g) was applied to a column of Norit A-celite (1:1 in weight ratio; 1 liter) and washed with 1.5 liters of water. The mixture of isomaltose and isomaltotriose was eluted from the column with 5% ethanol and concentrated under reduced pressure. All preparations derived from dextran were successively treated with Amberlite IR120 (H+) and Dowex 1-X8 (OH-) to remove possible contamination of ionic substances. Spheroplast-stabilizing activity of sugars and dextrans. E. coli W4626Phe- was grown in medium A, and spheroplasts were prepared from the cells as described previously (10). When the spheroplast stabilizing activity of lactose, maltose, and galactose was studied, cells were grown in medium A supplemented with 0.05% of the individual sugars. Spheroplast suspensions were diluted with a 10-fold volume of 25 mM Tris-hydrochloride (pH 7.8) containing different concentrations of sugars or dextrans and incubated at 37°C. The lysis of spheroplasts was followed by measuring the turbidity decrease at 660 nm.
RESULTS The ratio of 0-8 to 0-9 in the outer membrane of E. coli W4626Phe- grown in medium A
strictly depended on the sucrose concentration of the medium, whereas the relative amounts of 0-10 and 0-11, two other major outer membrane proteins, were almost constant irrespective of the sucrose concentration (Fig. 1). When cells grown in medium A were transferred to the same medium supplemented with 0.6 M sucrose, the synthesis of 0-9 was immediately replaced by that of 0-8. Conversely, transfer from the me-
J. BACTERIOL. 0, c
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FIG. 1. Influence of sucrose in the growth medium on relative amounts of major outer membrane proteins 0-8, 0-9, 0-10, and 0-11 as percentages of the sum of the four proteins. Cells were grown in medium A supplemented with the indicated concentrations of sucrose, and outer membrane proteins were analyzed on urea-sodium dodecyl sulfate polyacrylamide gel. Symbols: E, 0-8; *, 0-9; A, the sum of 0-10 and 011.
dium with 0.6 M sucrose to that without sucrose resulted in the immediate cessation of 0-8 synthesis with a concomitant initiation of 0-9 synthesis (data not shown). An inhibition experiment with chloramphenicol and a pulse-chase experiment with ["4C]leucine revealed that this phenomenon is due to the switching of the net synthesis of these proteins. These results, being consistent with our previous observations (3), confirmed the results of van Alphen and Lugtenberg (19). Since sucrose is not taken up by E. coli cells, these results strongly suggest that the phenomenon was caused by the increased osmotic strength of the medium rather than a specific action of sucrose. Pursuing this, we examined the effect of sugars of different molecular weights on the switching phenomenon. None of the sugars examined supported the growth of this strain. They were all able to stabilize spheroplasts of W4626Phe-. More than 80% of the spheroplasts were protected against osmotic lysis during the 30-min incubation with 0.6 M sugars, indicating that the rate of uptake of these sugars by cells, if any, is slow. However, the degree of stabilization was slightly different among the sugars, galactose, mannitol, and maltose being less potent than the others. In any case, the results indicate that these sugars exert osmotic pressure on the outer surface of the cytoplasmic membrane. Without exception, the effect on the switching of the mono-, di-, and trisaccharides examined was the same as that of sucrose regardless of molecular weight (Fig. 2), indicating that the phenomenon was caused by the osmotic strength of the medium. On the other hand, stachyose, a tetrasaccharide, was
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FIG. 2. Influence of different sugars in the growth medium on relative amounts ofouter membrane proteins 0-8 and 0-9 as percentages of the sum of 0-8, 0-9, 0-10, and 0-11. Cells were grown in medium A supplemented with the indicated concentrations of sugars, and outer membrane proteins were analyzed on urea-sodium dodecyl sulfate polyacrylamide gel. Symbols: Open, 0-8; closed, 0-9. (A) A A, Lactose (molecular weight [MW] 342); 0 , raffinose (MW 504); 0 *, stachyose (MW 666). (B). V , Galactose (MW 180); A A, mannitol (MW 182); 0 I, maltose (MW 342). The broken lines in (B) are a reproduction of the curves for stachyose in (A).
more effective than the other sugars, suggesting that the mode of action of this sugar may be somewhat different from that of smaller sugars. To study this possibility further, a-1,6-glucans of different molecular sizes were prepared from dextran, and their effect on the switching was examined (Fig. 3). None of the glucans used was utilized by this strain, and all were active in stabilizing the spheroplasts of this strain. The mixture of di- and trisaccharides, isomaltose and isomaltotriose, respectively, was as effective on the switching as the other mono-, di- and trisaccharides examined (see Fig. 1 and 2). On the other hand, the effect of dextran-2100, dextran1300, and dextran-1100 was the same irrespective of the differences in molecular weight, but this effect was appreciably greater than that of the smaller sugars. The effect of dextran-700 was slightly weaker than that of higher-molecularweight dextrans but still appreciably greater than that of the mixture of isomaltose and isomaltotriose. In Fig. 4 we summarize the effects of sugars and dextrans on the switching phenomenon. The molarities of individual sugars that caused 50% reduction of the 0-9 content were plotted against their molecular weights. It is clear that the sugars and dextrans examined can be divided into two groups. All substances whose molecular weights were larger than 700 showed the same effect regardless of molecular weight. The effect of mono-, di-, and trisaccharides was also the same for all irrespective of molecular weight. However, the amount of these sugars required, for the 50% reduction of 0-9 was about three times as much as that of the dextrans of higher molecular weights. Oligosaccharides of molecu-
lar weights 600 to 700 showed an intermediate effect.
DISCUSSION In the present work, we examined the influence of sugars and dextrans that function as osmotic stabilizers for E. coli spheroplasts on the synthesis of major outer membrane proteins 60 0 10
0
30
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E
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0.4 0.2 d-1.6- Glucans in Medium (M)
FIG. 3. Influence of a-1,6-glucans of different molecular weights in the growth medium on relative amounts of outer membrane proteins 0-8 and 0-9 as percentages of the sum of 0-8, 0-9, 0-10, and 0-11. Cells were grown in medium A supplemented with the indicated concentrations of a-1,6-glucans, and the outer membrane proteins were analyzed on urea-sodium dodecyl sulfate polyacrylamide gel. Symbols: Open, 0-8; closed, 0-9. V V, Mixture of isomaltose and isomaltotriose (1:1 in weight ratio, average molecular weight was about 420); A A, O *, ], and 0 0 were dextran-700, dextran-1100, dextran-1300, and dextran-2100, respectively.
846
KAWAJI, MIZUNO, AND MIZUSHIMA Lac
Rot
Gal Mal
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A-XJ
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.2 0220
EaSta
O01 _
~Dex7 t ex.. Dexi Dexl 3
C
2 5 10 Molecular Weights ( 10
20
2)
FIG. 4. Relationships between molecular weight and effect of sugars and dextrans on the switching phenomenon. All data were taken from Fig. 1, 2, and 3. Molar concentrations of individual carbohydrates that caused 50% reduction of the 0-9 content were plotted against molecular weights. Gal, galactose; Mtl, mannitol; Mal, maltose; Lac, lactose; Suc, sucrose; Raf, raffinose; Sta, stachyose; Dex2.3, mixture of isomaltose and isomaltotriose; Dex7, dextran- 700; Dexll, dextran-1100; Dexl3, dextran-1300; Dex2l, dextran-2100.
J. BACTERIOL.
permeable to water and unable to support a pressure gradient, the periplasm and the cytoplasm are most probably isoosmotic, although substances responsible for the periplasmic osmotic strength have not been determined yet (17). On the other hand, the outer membranepeptidoglycan layer is believed to be resistant to the osmotic pressure exerted by the periplasm. Stock et al. reported that the osmotic pressure across the outer membrane is around 0.15 osM (17). The presence of an impermeable solute at 0.1 M will reduce it to 0.05 osM, i.e., to one-third of the original value. To achieve the same degree of reduction with a carbohydrate that can freely pass through the outer membrane, the volume of the periplasmic space has to be expanded by a factor of 3, which seems to require a carbohydrate of around 0.5 M (17). Contrary to the case of the outer membrane, the difference of osmotic pressure between both sides of the cytoplasmic membrane should always be negligible, provided that the periplasm and the cytoplasm are isoosmotic. With these considerations, it is probable that the outer membrane-peptidoglycan layer recognizes the difference of osmotic pressure exerted on both its sides to regulate the biosynthesis of 0-8 and 0-9. It is unlikely that the increase in osmotic strength itself influences the synthesis of 0-8 and 0-9, since the intracellular osmotic strength raised by carbohydrates that can penetrate the outer membrane should be larger than that raised by carbohydrates that cannot penetrate this membrane. Finally, it should also be asked why E. coli cells switch the synthesis of 0-9 to that of 0-8 in a medium of high osmotic strength. Analyses of mutants which lack one or both of 0-8 and 0-9 suggest that the two proteins are involved in the entry of different molecules (8, 21). Although the specificity of the individual proteins in the entry of sugars has not been determined yet, the switching might be related to a specificity difference. Alternatively, the rigidity of the outer membrane-peptidoglycan layer might be different depending on whether this layer contains 08 or 0-9, and the switching might be related to this possible difference.
0-8 and 0-9. Supplementation of the growth medium with high concentrations of sugars or dextrans resulted in a decrease in the 0-9 content, accompanied by a roughly equal increase in the 0-8 content. Irrespective of molecular weight and species, all mono-, di-, and trisaccharides examined caused the switching of synthesis of 0-9 to that of 0-8 to almost the same extent, indicating that the switching was due to the osmotic strength of the medium caused by them. Carbohydrates whose molecular weights were larger than 600 to 700 also caused the switching, the extent of which was also almost the same for all irrespective of molecular weight. However, they were appreciably more effective than sugars of smaller molecular weight. The outer membranes of E. coli and Salmonella typhimurium possess aqueous pores by which hydrophilic molecules of molecular weights smaller than 600 can pass through the outer ACKNOWLEDGMENTS membrane (2). In E. coli K-12, 0-8 and 0-9 have We thank Meito Sangyo Co. for providing us with dextrana. been demonstrated to be mainly responsible for This work was supported by grants from the Ministry of this pore function (8, 11, 20, 21). Taking this fact into consideration, we can probably conclude Education, Science and Culture of Japan. that carbohydrates that cannot pass through the LITERATURE CITED outer membrane switch the synthesis of 0-9 to 1. Bassford, P. J., Jr., D. L. Diedrich, C. A. Schnaitman, that of 0-8 more effectively than those that can and P. Reeves. 1977. Outer membrane proteins of Escherichia coli. VI. Protein alteration in bacteriopenetrate the outer membrane. A possible explaphage-resistant mutants. J. Bacteriol. 131:608-622. nation for these differential effects of carbohy- 2. Decad, G. M., and H. Nikaido. 1976. Outer membrane drates is as follows. of gram-negative bacteria. XII. Molecular sieving funcis membrane Provided that the cytoplasmic tion of cell wall. J. Bacteriol. 128:325-336.
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3. Hasegawa, Y., H. Yamada, and S. Mizushima. 1976. Interactions of outer membrane proteins 0-8 and 0-9 with peptidoglycan sacculus of Escherichia coli K-12. J. Biochem. (Tokyo) 80:1401-1409. 4. Ichihara, S., and S. Mizushima. 1977. Strain specificity of outer membrane proteins in Escherichia coli. J. Biochem. (Tokyo) 81:1525-1530. 5. Ichihara, S., and S. Mizushima. 1978. Characterization of major outer membrane proteins 0-8 and 0-9 of Escherichia coli K-12. Evidence that structural genes for the two proteins are different. J. Biochem. (Tokyo) 83:1095-1100. 6. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 7. Lugtenberg, B., J. MeiJers, R. Peters, P. van der Hoek, and L. van Alphen. 1975. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 58:254258. 8. Lutkenhaus, J. F. 1977. Role of a major outer membrane protein in Escherichia coli. J. Bacteriol. 131:631-637. 9. Miyoshi, Y., and H. Yamagata. 1976. Sucrose-dependent, spectinomycin-resistant mutants of Escherichia coli. J. Bacteriol. 125:142-148. 10. Mizushima, S., and H. Yamada. 1975. Isolation and characterization of two outer membrane preparations from Escherichia coli. Biochim. Biophys. Acta 375:4453. 11. Nakae, T. 1976. Identification of the outer membrane protein of Escherichia coli that produces transmembrane channels in reconstituted vesicle membrane. Biochem. Biophys. Res. Commun. 71:877-884. 12. Nakae, T., J. Ishii, and M. Tokunaga. 1979. Subunit structure of functional porin oligomers that form permeability channels in the outer membrane of Escherichia coli. J. Biol. Chem. 254:1457-1461. 13. Nakamura, K., and S. Mizushima. 1976. Effects of heating in dodecyl sulfate solution on the conformation and electrophoretic mobility of isolated major outer membrane proteins from Escherichia coli K-12. J. Bio-
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chem. (Tokyo) 80:1411-1422. 14. Rosenbusch, J. P. 1974. Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidogylcan and unusual dodecyl sulfate binding. J. Biol. Chem. 249:8019-8029. 15. Schmitges, C. J., and U. Henning. 1976. The major protein of the Escherichia coli outer cell envelope membrane. Heterogeneity of protein I. Eur. J. Biochem. 63:47-52. 16. Schnaitman, C. A. 1971. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J. Bacteriol. 108:545-552. 17. Stock, J. B., B. Rauch, and S. Roseman. 1977. Periplasmic space in Salmonella typhimurium and Escherichia coli. J. Biol. Chem. 252:7850-7861. 18. Uemura, J., and S. Mizushima. 1975. Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel. Biochimn. Biophys. Acta 413:163-176. 19. van Alphen, W., and B. Lugtenberg. 1977. Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli. J. Bacteriol. 131:623-630. 20. van Alphen, W., R. van Boxtel, N. van Selm, and B. Lugtenberg. 1978. Pores in the outer membrane of E. coli K-12. Involvement of proteins b and c in the permeation of cephaloridine and ampicillin. FEMS Microbiol. Lett. 3:103-106. 21. van Alphen, W., N. van Selm, and B. Lugtenberg. 1978. Pores in the outer membrane of Escherichia coli K12. Involvement of proteins b and e in the functioning of pores for nucleotides. Mol. Gen. Genet. 159:7543. 22. Yamada, H., and S. Mizushima. 1978. Reconstitution of an ordered structure from major outer membrane constitutents and the lipoprotein-bearing peptidoglycan sacculus of Escherichia coli. J. Bacteriol. 135:10241031. 23. Yu, F., S. Ichihara, and S. Mizushima. 1979. A major outer membrane protein, 0-8, of Escherichia coli K-12 exists as a trimer in sodium dodecyl sulfate solution. FEBS Lett. 100:71-74.
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 843-847 0021-9193/79/12-0843/05$02.00/0
Influence of Molecular Size and Osmolarity of Sugars and Dextrans on the Synthesis of Outer Membrane Proteins 0-8 and 0-9 of Escherichia coli K-12 HIROSHI KAWAJI, TAKESHI MIZUNO,t AND SHOJI MIZUSHIMA* Laboratory of Microbiology, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464, Japan Received for publication 3 September 1979
Supplementation of the growth medium with high concentrations of sugars or low-molecular-weight dextrans results in a drastic change in the ratio of outer membrane proteins 0-8 and 0-9, due to induction of 0-8 synthesis and suppression of 0-9 synthesis. Sugars and dextrans of molecular weights greater than 600 to 700 switched the synthesis of 0-9 to that of 0-8 more effectively than those of lower molecular weight, although the effect was almost the same within each of the two groups irrespective of the differences in molecular weight within the group. Proteins 0-8 or 0-9, or both, are responsible for the formation of pores that allow the passive diffusion of hydrophilic molecules whose molecular weights are smaller than about 600 (T. Nakae, Biochem. Biophys. Res. Commun. 71:877884, 1976). The results indicate that substances that cannot pass through the outer membrane switch the synthesis of 0-9 to that of 0-8 more effectively than those that can penetrate this membrane with the aid of 0-8, 0-9, or both. It is suggested that the osmotic pressure exerted on the outer membrane plays an imnportant role in the regulation of synthesis of the two proteins. Two major outer membrane proteins of Escherichia coli K-12, 0-8, and 0-9, resemble each other in many biochemical and physicochemical respects. They are so-called peptidoglycan-associated proteins (3, 14) and exist as trimers (12, 23). The circular dichroism spectra (13), amino acid compositions, and N-terminal sequences (5) are also very similar. Furthermnore, both proteins form a hexagonally latticed structure with lipopolysaccharide (22). Although it is almost certain that the two proteins are products of different structural genes (5), their similarity can be explained by the hypothesis that these genes developed from a single ancestral gene through the duplication of the ancestral genome of E. coli (5). On the other hand, the biosynthesis of the two proteins is affected differentially by high concentrations of substances like sucrose and NaCl in the culture media (3, 19). The influence of these substances on the biosynthesis of 0-8 and 0-9 was found to be opposite: inductive for 0-8 and suppressive for 0-9. This fact indicates the possible existence of a regulatory mechanism which controls the biosynthesis of both proteins. Proteins 0-8 and 0-9 have also been called Ib and Ia, c and b, and lb and la, respectively (1, 4, 7, 15). In the present work, we examined the influt Present address: Mitsubishi-Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan.
ence of carbohydrates of different molecular sizes on the biosynthesis of 0-8 and 0-9. Carbohydrates whose molecular weights were larger than 600 to 700 switched the synthesis of 0-9 to that of 0-8 more effectively than those of smaller molecular weight. However, the effect on the switching was almost the same within each of the two groups of carbohydrates irrespective of the differences in molecular weight. Protein 0-8 or 0-9 or both are responsible for the formation of pores that allow the passive diffusion of hydrophilic molecules whose molecular weights are smaller than 600 (11). The results strongly suggest that 0-8 and 0-9 are involved in the regulation of their own synthesis through their pore
functions. MATERLA1S AND METHODS Bacterial strain and growth conditions. E. coli W4626Phe- [K-12, F- purE trp phe lac-85 gal-2 xyl-2 mal mtl ara rpsL (A)] (9) was grown in medium A supplemented with different concentrations of sugars or dextrans. Medium A contained, per liter, 7 g of nutrient broth, 1 g of yeast extract, 2 g of glycerol, 3.7 g of K2HPO4, and 1.3 g of KH2PO4. Cell envelopes. Cells from a 10-ml culture were washed with 15 mM sodium phosphate buffer (pH 7.2) and broken with a sonic disintegrator in water. Cell envelopes were recovered by differential centrifugation, suspended in 2 ml of water, and extracted with an equal volume of 4% Triton X-100-20 mM Tris-
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hydrochloride (pH 8.0)-20 mM MgCl2 at 37°C for 30 min, based on the method of Schnaitman (16). Insoluble residues were recovered by centrifugation, and protein profiles were analyzed by urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis as described previously (10, 18). For quantitative determination of individual proteins, stained gels were scanned with a Joyce-Loebl densitometer, and the peaks of major outer membrane proteins 0-8, 0-9, and 0-10 plus 011 were cut out and weighed (for the nomenclature of 0-10 and 0-11, see reference 4). The amount of protein in envelope preparations was determined by the method of Lowry et al. (6). Sugars and dextrans of small molecular weights. Sucrose and lactose were obtained from Katayama Chemical Ind.; raffinose, maltose and galactose were obtained from Yoneyama Chemical Co.; mannitol came from Wako Chemicals; and stachyose came from Sigma Chemical Co. a-1,6-Glucans with number average molecular weights of 2,100, 1,300, 1,100, and 700 were gifts from Meito Sangyo Co. In this paper they are referred to as dextran-2100, dextran-1300, dextran-1100 and dextran-700, respectively. The contents as molar percentages of the sum of mono-, di-, and trisaccharides in these preparations were about 2, 10, 20, and 40%, respectively. The mixture of isomaltose and isomaltotriose (1:1 in weight ratio) was prepared from an enzymatic hydrolysate of dextran (DXase-D), a gift from Meito Sangyo Co., as follows. DXase-D (20 g) was applied to a column of Norit A-celite (1:1 in weight ratio; 1 liter) and washed with 1.5 liters of water. The mixture of isomaltose and isomaltotriose was eluted from the column with 5% ethanol and concentrated under reduced pressure. All preparations derived from dextran were successively treated with Amberlite IR120 (H+) and Dowex 1-X8 (OH-) to remove possible contamination of ionic substances. Spheroplast-stabilizing activity of sugars and dextrans. E. coli W4626Phe- was grown in medium A, and spheroplasts were prepared from the cells as described previously (10). When the spheroplast stabilizing activity of lactose, maltose, and galactose was studied, cells were grown in medium A supplemented with 0.05% of the individual sugars. Spheroplast suspensions were diluted with a 10-fold volume of 25 mM Tris-hydrochloride (pH 7.8) containing different concentrations of sugars or dextrans and incubated at 37°C. The lysis of spheroplasts was followed by measuring the turbidity decrease at 660 nm.
RESULTS The ratio of 0-8 to 0-9 in the outer membrane of E. coli W4626Phe- grown in medium A
strictly depended on the sucrose concentration of the medium, whereas the relative amounts of 0-10 and 0-11, two other major outer membrane proteins, were almost constant irrespective of the sucrose concentration (Fig. 1). When cells grown in medium A were transferred to the same medium supplemented with 0.6 M sucrose, the synthesis of 0-9 was immediately replaced by that of 0-8. Conversely, transfer from the me-
J. BACTERIOL. 0, c
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A
A
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FIG. 1. Influence of sucrose in the growth medium on relative amounts of major outer membrane proteins 0-8, 0-9, 0-10, and 0-11 as percentages of the sum of the four proteins. Cells were grown in medium A supplemented with the indicated concentrations of sucrose, and outer membrane proteins were analyzed on urea-sodium dodecyl sulfate polyacrylamide gel. Symbols: E, 0-8; *, 0-9; A, the sum of 0-10 and 011.
dium with 0.6 M sucrose to that without sucrose resulted in the immediate cessation of 0-8 synthesis with a concomitant initiation of 0-9 synthesis (data not shown). An inhibition experiment with chloramphenicol and a pulse-chase experiment with ["4C]leucine revealed that this phenomenon is due to the switching of the net synthesis of these proteins. These results, being consistent with our previous observations (3), confirmed the results of van Alphen and Lugtenberg (19). Since sucrose is not taken up by E. coli cells, these results strongly suggest that the phenomenon was caused by the increased osmotic strength of the medium rather than a specific action of sucrose. Pursuing this, we examined the effect of sugars of different molecular weights on the switching phenomenon. None of the sugars examined supported the growth of this strain. They were all able to stabilize spheroplasts of W4626Phe-. More than 80% of the spheroplasts were protected against osmotic lysis during the 30-min incubation with 0.6 M sugars, indicating that the rate of uptake of these sugars by cells, if any, is slow. However, the degree of stabilization was slightly different among the sugars, galactose, mannitol, and maltose being less potent than the others. In any case, the results indicate that these sugars exert osmotic pressure on the outer surface of the cytoplasmic membrane. Without exception, the effect on the switching of the mono-, di-, and trisaccharides examined was the same as that of sucrose regardless of molecular weight (Fig. 2), indicating that the phenomenon was caused by the osmotic strength of the medium. On the other hand, stachyose, a tetrasaccharide, was
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0.6
Sugars in Medium (M)
FIG. 2. Influence of different sugars in the growth medium on relative amounts ofouter membrane proteins 0-8 and 0-9 as percentages of the sum of 0-8, 0-9, 0-10, and 0-11. Cells were grown in medium A supplemented with the indicated concentrations of sugars, and outer membrane proteins were analyzed on urea-sodium dodecyl sulfate polyacrylamide gel. Symbols: Open, 0-8; closed, 0-9. (A) A A, Lactose (molecular weight [MW] 342); 0 , raffinose (MW 504); 0 *, stachyose (MW 666). (B). V , Galactose (MW 180); A A, mannitol (MW 182); 0 I, maltose (MW 342). The broken lines in (B) are a reproduction of the curves for stachyose in (A).
more effective than the other sugars, suggesting that the mode of action of this sugar may be somewhat different from that of smaller sugars. To study this possibility further, a-1,6-glucans of different molecular sizes were prepared from dextran, and their effect on the switching was examined (Fig. 3). None of the glucans used was utilized by this strain, and all were active in stabilizing the spheroplasts of this strain. The mixture of di- and trisaccharides, isomaltose and isomaltotriose, respectively, was as effective on the switching as the other mono-, di- and trisaccharides examined (see Fig. 1 and 2). On the other hand, the effect of dextran-2100, dextran1300, and dextran-1100 was the same irrespective of the differences in molecular weight, but this effect was appreciably greater than that of the smaller sugars. The effect of dextran-700 was slightly weaker than that of higher-molecularweight dextrans but still appreciably greater than that of the mixture of isomaltose and isomaltotriose. In Fig. 4 we summarize the effects of sugars and dextrans on the switching phenomenon. The molarities of individual sugars that caused 50% reduction of the 0-9 content were plotted against their molecular weights. It is clear that the sugars and dextrans examined can be divided into two groups. All substances whose molecular weights were larger than 700 showed the same effect regardless of molecular weight. The effect of mono-, di-, and trisaccharides was also the same for all irrespective of molecular weight. However, the amount of these sugars required, for the 50% reduction of 0-9 was about three times as much as that of the dextrans of higher molecular weights. Oligosaccharides of molecu-
lar weights 600 to 700 showed an intermediate effect.
DISCUSSION In the present work, we examined the influence of sugars and dextrans that function as osmotic stabilizers for E. coli spheroplasts on the synthesis of major outer membrane proteins 60 0 10
0
30
-
E
0
0.4 0.2 d-1.6- Glucans in Medium (M)
FIG. 3. Influence of a-1,6-glucans of different molecular weights in the growth medium on relative amounts of outer membrane proteins 0-8 and 0-9 as percentages of the sum of 0-8, 0-9, 0-10, and 0-11. Cells were grown in medium A supplemented with the indicated concentrations of a-1,6-glucans, and the outer membrane proteins were analyzed on urea-sodium dodecyl sulfate polyacrylamide gel. Symbols: Open, 0-8; closed, 0-9. V V, Mixture of isomaltose and isomaltotriose (1:1 in weight ratio, average molecular weight was about 420); A A, O *, ], and 0 0 were dextran-700, dextran-1100, dextran-1300, and dextran-2100, respectively.
846
KAWAJI, MIZUNO, AND MIZUSHIMA Lac
Rot
Gal Mal
0.3
-
_
A-XJ
\
Suc
.2 0220
EaSta
O01 _
~Dex7 t ex.. Dexi Dexl 3
C
2 5 10 Molecular Weights ( 10
20
2)
FIG. 4. Relationships between molecular weight and effect of sugars and dextrans on the switching phenomenon. All data were taken from Fig. 1, 2, and 3. Molar concentrations of individual carbohydrates that caused 50% reduction of the 0-9 content were plotted against molecular weights. Gal, galactose; Mtl, mannitol; Mal, maltose; Lac, lactose; Suc, sucrose; Raf, raffinose; Sta, stachyose; Dex2.3, mixture of isomaltose and isomaltotriose; Dex7, dextran- 700; Dexll, dextran-1100; Dexl3, dextran-1300; Dex2l, dextran-2100.
J. BACTERIOL.
permeable to water and unable to support a pressure gradient, the periplasm and the cytoplasm are most probably isoosmotic, although substances responsible for the periplasmic osmotic strength have not been determined yet (17). On the other hand, the outer membranepeptidoglycan layer is believed to be resistant to the osmotic pressure exerted by the periplasm. Stock et al. reported that the osmotic pressure across the outer membrane is around 0.15 osM (17). The presence of an impermeable solute at 0.1 M will reduce it to 0.05 osM, i.e., to one-third of the original value. To achieve the same degree of reduction with a carbohydrate that can freely pass through the outer membrane, the volume of the periplasmic space has to be expanded by a factor of 3, which seems to require a carbohydrate of around 0.5 M (17). Contrary to the case of the outer membrane, the difference of osmotic pressure between both sides of the cytoplasmic membrane should always be negligible, provided that the periplasm and the cytoplasm are isoosmotic. With these considerations, it is probable that the outer membrane-peptidoglycan layer recognizes the difference of osmotic pressure exerted on both its sides to regulate the biosynthesis of 0-8 and 0-9. It is unlikely that the increase in osmotic strength itself influences the synthesis of 0-8 and 0-9, since the intracellular osmotic strength raised by carbohydrates that can penetrate the outer membrane should be larger than that raised by carbohydrates that cannot penetrate this membrane. Finally, it should also be asked why E. coli cells switch the synthesis of 0-9 to that of 0-8 in a medium of high osmotic strength. Analyses of mutants which lack one or both of 0-8 and 0-9 suggest that the two proteins are involved in the entry of different molecules (8, 21). Although the specificity of the individual proteins in the entry of sugars has not been determined yet, the switching might be related to a specificity difference. Alternatively, the rigidity of the outer membrane-peptidoglycan layer might be different depending on whether this layer contains 08 or 0-9, and the switching might be related to this possible difference.
0-8 and 0-9. Supplementation of the growth medium with high concentrations of sugars or dextrans resulted in a decrease in the 0-9 content, accompanied by a roughly equal increase in the 0-8 content. Irrespective of molecular weight and species, all mono-, di-, and trisaccharides examined caused the switching of synthesis of 0-9 to that of 0-8 to almost the same extent, indicating that the switching was due to the osmotic strength of the medium caused by them. Carbohydrates whose molecular weights were larger than 600 to 700 also caused the switching, the extent of which was also almost the same for all irrespective of molecular weight. However, they were appreciably more effective than sugars of smaller molecular weight. The outer membranes of E. coli and Salmonella typhimurium possess aqueous pores by which hydrophilic molecules of molecular weights smaller than 600 can pass through the outer ACKNOWLEDGMENTS membrane (2). In E. coli K-12, 0-8 and 0-9 have We thank Meito Sangyo Co. for providing us with dextrana. been demonstrated to be mainly responsible for This work was supported by grants from the Ministry of this pore function (8, 11, 20, 21). Taking this fact into consideration, we can probably conclude Education, Science and Culture of Japan. that carbohydrates that cannot pass through the LITERATURE CITED outer membrane switch the synthesis of 0-9 to 1. Bassford, P. J., Jr., D. L. Diedrich, C. A. Schnaitman, that of 0-8 more effectively than those that can and P. Reeves. 1977. Outer membrane proteins of Escherichia coli. VI. Protein alteration in bacteriopenetrate the outer membrane. A possible explaphage-resistant mutants. J. Bacteriol. 131:608-622. nation for these differential effects of carbohy- 2. Decad, G. M., and H. Nikaido. 1976. Outer membrane drates is as follows. of gram-negative bacteria. XII. Molecular sieving funcis membrane Provided that the cytoplasmic tion of cell wall. J. Bacteriol. 128:325-336.
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MEDIUM OSMOLARITY AND OUTER MEMBRANE PROTEINS
3. Hasegawa, Y., H. Yamada, and S. Mizushima. 1976. Interactions of outer membrane proteins 0-8 and 0-9 with peptidoglycan sacculus of Escherichia coli K-12. J. Biochem. (Tokyo) 80:1401-1409. 4. Ichihara, S., and S. Mizushima. 1977. Strain specificity of outer membrane proteins in Escherichia coli. J. Biochem. (Tokyo) 81:1525-1530. 5. Ichihara, S., and S. Mizushima. 1978. Characterization of major outer membrane proteins 0-8 and 0-9 of Escherichia coli K-12. Evidence that structural genes for the two proteins are different. J. Biochem. (Tokyo) 83:1095-1100. 6. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 7. Lugtenberg, B., J. MeiJers, R. Peters, P. van der Hoek, and L. van Alphen. 1975. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 58:254258. 8. Lutkenhaus, J. F. 1977. Role of a major outer membrane protein in Escherichia coli. J. Bacteriol. 131:631-637. 9. Miyoshi, Y., and H. Yamagata. 1976. Sucrose-dependent, spectinomycin-resistant mutants of Escherichia coli. J. Bacteriol. 125:142-148. 10. Mizushima, S., and H. Yamada. 1975. Isolation and characterization of two outer membrane preparations from Escherichia coli. Biochim. Biophys. Acta 375:4453. 11. Nakae, T. 1976. Identification of the outer membrane protein of Escherichia coli that produces transmembrane channels in reconstituted vesicle membrane. Biochem. Biophys. Res. Commun. 71:877-884. 12. Nakae, T., J. Ishii, and M. Tokunaga. 1979. Subunit structure of functional porin oligomers that form permeability channels in the outer membrane of Escherichia coli. J. Biol. Chem. 254:1457-1461. 13. Nakamura, K., and S. Mizushima. 1976. Effects of heating in dodecyl sulfate solution on the conformation and electrophoretic mobility of isolated major outer membrane proteins from Escherichia coli K-12. J. Bio-
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chem. (Tokyo) 80:1411-1422. 14. Rosenbusch, J. P. 1974. Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidogylcan and unusual dodecyl sulfate binding. J. Biol. Chem. 249:8019-8029. 15. Schmitges, C. J., and U. Henning. 1976. The major protein of the Escherichia coli outer cell envelope membrane. Heterogeneity of protein I. Eur. J. Biochem. 63:47-52. 16. Schnaitman, C. A. 1971. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J. Bacteriol. 108:545-552. 17. Stock, J. B., B. Rauch, and S. Roseman. 1977. Periplasmic space in Salmonella typhimurium and Escherichia coli. J. Biol. Chem. 252:7850-7861. 18. Uemura, J., and S. Mizushima. 1975. Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel. Biochimn. Biophys. Acta 413:163-176. 19. van Alphen, W., and B. Lugtenberg. 1977. Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli. J. Bacteriol. 131:623-630. 20. van Alphen, W., R. van Boxtel, N. van Selm, and B. Lugtenberg. 1978. Pores in the outer membrane of E. coli K-12. Involvement of proteins b and c in the permeation of cephaloridine and ampicillin. FEMS Microbiol. Lett. 3:103-106. 21. van Alphen, W., N. van Selm, and B. Lugtenberg. 1978. Pores in the outer membrane of Escherichia coli K12. Involvement of proteins b and e in the functioning of pores for nucleotides. Mol. Gen. Genet. 159:7543. 22. Yamada, H., and S. Mizushima. 1978. Reconstitution of an ordered structure from major outer membrane constitutents and the lipoprotein-bearing peptidoglycan sacculus of Escherichia coli. J. Bacteriol. 135:10241031. 23. Yu, F., S. Ichihara, and S. Mizushima. 1979. A major outer membrane protein, 0-8, of Escherichia coli K-12 exists as a trimer in sodium dodecyl sulfate solution. FEBS Lett. 100:71-74.
