JOURNAL
OF BACTERIOLOGY, Apr. 1979, p. 254-256 0021-9193/79/04-0254/03$02.00/0
Vol. 138, No. 1
Cross-Linking Analysis of the Two Forms of Protein I, a Major Outer Membrane Protein of Escherichia coli E. T. PALVA* AND L. L. RANDALL The Wallenberg Laboratory, University of Uppsala, 75122 Uppsala, Sweden
Received for publication 5 September 1978
The two forms of protein I were cross-linked to molecules of the same species, even when both were present simultaneously. This suggests that they form separate multimers in the outer membrane.
Protein I (3), one of the major proteins in Escherichia coli outer membrane, exists in two closely related fonns, Ia and Ib (15), also called b and c, respectively (5), which can be clearly separated by using high-resolution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (5). The relative amounts of Ia and Ib in the outer membrane seem to vary depending on the strain and growth conditions (6, 16). Recently it was demonstrated that the protein I pores (2, 9) show different specificity, in that protein Ia (b) but not Ib (c) is involved in the permeation of nucleotides through the outer membrane (17). We (10, 11) and Reithmeier and Bragg (13) have previously shown that protein I can be cross-linked to itself to give multimeric complexes. By cross-linking the purified protein we concluded that the basic unit is probably a trimer (11). Reithmeier and Bragg observed that the two forms, named A1 and A2, could be crosslinked to give similar complexes (13) when protein I-peptidoglycan complexes (14) isolated from strains having only one of the two forms at a time are cross-linked. However the SDS-gel system that they used (13) did not resolve the two forms. Thus, they could not question whether heterocomplexes containing both Ia and lb exist. Protein I trimers (11) could consist solely of one species at a time or could be composed of a random mixture of Ia and Ib. We have analyzed the cross-linking of Ia and Ib by using high-resolution SDS-gel electrophoresis (5) to answer this question. We used E. coli K-12 strains having protein Ia (TK69; Fig. 1, slot 2) or protein Ib (TK70; Fig. 1, slot 3) or the parent having both Ia and lb (B14 [1]; Fig. 1, slot 1). Whole cells were crosslinked with a low concentration (2 mM) of the cleavable cross-linking reagent tartryl-di-(glycylazide) (1.3-nm bridge length [7]). This low concentration of the reagent, which produces only dimers of protein I, was used to minimize ambiguities in the mobility of the cross-linked 254
complexes in SDS-gels resulting from different levels of intramolecular cross-linking. Cell envelopes (12) and protein I-peptidoglycan com-
FIG. 1. Protein pattern of isolated cell envelopes (slots 1-3) and isolated protein I-peptidoglycan complexes (slots 4-10) of E. coli K-12 analyzed on 11% SDS-polyacrylamide gels (5). Cells were grown in Luria broth (8) and cross-linked (10, 11) with 2 mM tartryl-di-(glycylazide) for 30 min at room temperature in 100 mM triethanolamine-hydrochloride, pH 8.5, and cell envelopes (12) and protein I-peptidoglycan complexes (11, 14) were isolated. Staining of gels was as described (11). Slots 1-3: isolated cell envelopes not cross-linked of strains B14 (slot 1), TK69 (slot 2), and TK70 (slot 3). Slots 4-10: isolated protein I-peptidoglycan complexes of not-cross-linked controls (slots 4-6) and cross-linked cells (slots 7-10) of strains B14 (slots 4 and 7), TK69 (slots 5 and 8), and TK70 (slots 6 and 9). The material in slot 10 is a 1:1 mixture of that in slots 8 and 9. Arrows at left indicate the position of proteins a (5), Ia([bJ5), Ib([c]5), and HI* ([dl5). Arrows at right indicate the position ofIa and Ib dimers.
VOL. 138, 1979
NOTES first dimension
nv
A
B
N
c
0
255
a lb
U,
c
E
c5 -o C
C
0 0
D
a,
U,
I'll,
NN.
a
lb
'% II* FIG. 2. Symmetrical two-dimensional 11% SDS-polyacrylamide gel patterns of cross-linked and cleaved cell envelopes of E. coli K-12. Cells were grown and cross-linked, and envelopes were isolated as indicated in the legend to Fig. 1. The cleavage of the cross-links after the first dimension run was done with 15 mM NaIO4 for 20 min as described (7). Staining ofgels was as in the legend to Fig. 1. (A) E. coli K-12 TK69; (B) TK70; (C) 1:1 mixture of samples in A and B; (D) B14.
plexes (11, 14) were isolated and analyzed by gel electrophoresis (5). Figure 1 shows the one-dimensional gel analysis of cross-linked Ia and lb. Both Ia (slot 8) and lb (slot 9) are cross-linked to dimers which have a slightly different mobility in the gels. This result confirms the earlier results of Reithmeier and Bragg (13), who used a different cross-linking reagent. If higher concentrations of tartryl-di-(glycylazide) were used, trimers of both Ia and lb (not shown) were also obtained but they had the same mobility. When the strain which had both Ia and Ib (slot 7) was used, both dimers were obtained simultaneously. If the dimners were produced from cross-linking Ia to Ib they would have a mobility between that of Ia dimers and Ib dimers. However, such is not the case; the gel pattem obtained from strain B14 (slot 7) is similar to that of a 1:1 mixture of Ia dimers and Ib dimers (slot 10). These results were further confirmed by analysis of the isolated envelopes of the cross-linked cells on symmetrical two-dimensional SDS-gels (7, 10, 11) that resolve Ia and Ib (5) (Fig. 2). Strain TK69 (Fig. 2A), which produces only Ia, gives only a dimer spot of Ia, whereas the mutant TK70 (Fig. 2B), having only Ib, gives dimers of this form of protein I. The dimer spots of Ia and Ib appear in different positions on the two-di-
mensional gels, as can be seen in Fig. 2C, which is a 1:1 mixture of the material shown in Fig. 2A and B. Both dimers of Ia and Ib are obtained from the wild-type strain B14 (Fig. 2D). Since the dimer spots (Fig. 2D) are not on the same vertical line, but are shifted slightly relative to each other and are in similar positions as the dimers in the mixture (Fig. 20), they are not derived from the same cross-linked complex. These results indicate that the two forms of protein I, even when present simultaneously, tend to be complexed only to molecules of the same form. There is some overlap in the position of the dimers probably due to poor separation on the gels, so presence of minor amounts of heterocomplexes cannot be ruled out. The results presented suggest that Ia and Ib are complexed primarily to themselves and probably form separate multimers in the outer membrane. These multimers may represent two separate species of pores, as has been suggested by van Alphen et al. (17). E.T.P. holds a European Molecular Biology Organization
fellowship. This work was supported by Swedish Natural Sciences Research Council. We thank Hans Fasold for the generous gift of the crosslinking reagent. We are grateful to Ben Lugtenberg for posing
256
J. BACTERIOL.
NOTES
the question which led to this work and for advice about the gel system. 9. 1.
2.
3.
4. 5.
6.
7.
8.
LITERATURE CITED Adler, J. 1973. A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis of Ewcherichia coli. J. Gen. Microbiol. 74: 77-91. Bavoil, P., H. Nikaido, and K. von Meyenburg 1977. Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein. Mol. Gen. Genet. 158:23-33. Garten, W, and U. Henning. 1974. Cell envelope and shape of Escherichia coli K-12. Isolation and preliminary characterization of the major ghost-membrane proteins. Eur. J. Biochem. 47:343-352. Honun, K, D. Hatfield, and M. Schwartz. 1974. maUB region in Eacherichia coli K-12. Characterization of new mutations J. Bacteriol. 117:40-47. Lugtenberg, B., J. Meijers, R. Peters, P. van der Hoek, and L vanAnphen. 1975. Electrophoretic resolution of the 'major outer membrane protein' of Ewcherichia coli K12 into four bands. FEBS Lett. 58:254258. Lugtenberg, B., R. Peters, H. Bernheimer, and W. Berendaen. 1976. Influence of cultural conditions and mutations on the composition of the outer membrane proteins of Ewcherichia coli. Mol. Gen. Genet. 147:251262. Lutter, L C., F. Ortanderl, and H. Fasold. 1974. The use of a new sries of cleavable protein-croslinkers on the Ewcherichia coli ribosome. FEBS Lett. 48:288-292. Miller, J. H. 1972. Experiments in molecular genetics, p.
10.
11. 12. 13. 14.
15.
16.
17.
433. Cold Spring Harbor laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Nakae, T. 1976. Identification of the outer membrane protein of E. coli that produces transmembrane channels in reconstituted vesicle membranes. Biochem. Biophys. Res. Commun. 71:877-884. Palva, E. T., and L L Randall. 1976. Nearest-neighbor analysis of Eacherichia coli outer membrane proteins, Using cleavable cros-links. J. Bacteriol. 127:1558-1560. Palva, E. T., and L L RandalL 1978. Arrangement of protein I in Escherichia coli outer membrane. Crosslinking study. J. Bacteriol. 133:279-286. Palva, E. T. 1978. A major outer membrane protein in SalmoneUa typhimurium induced by maltose. J. Bacteriol. 136:286-294. Relthmeler, R. A. F., and P. D. Bragg. 1977. Crosslinking of the proteins in the outer membrane of Escherichia coli. Biochim. Biophys. Acta 466:245-256. Rosenbuwch, J. P. 1974. Characterization of the major envelope protein from Ewcherichia coli. Regular arrangement on the peptidoglyean and unusual dodecyl sulfate binding. J. Biol. Chem. 249:8019-8029. Shmitges, J., and U. Henning. 1976. The major proteins of the Ewcherichia coli outer cell-envelope membrane. Heterogeneity of protein I. Eur. J. Biochem. 63: 4752. van Aiphen, W., and B. Lugtenberg. 1977. Influence of osmolarity of the growth on the outer membrane protein pattern of Escherichia coli. J. Bacteriol. 131:623630. van Aiphen, 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 fimctioning of pores for nucleotides. Mol. Gen. Genet. 169:75-83.
OF BACTERIOLOGY, Apr. 1979, p. 254-256 0021-9193/79/04-0254/03$02.00/0
Vol. 138, No. 1
Cross-Linking Analysis of the Two Forms of Protein I, a Major Outer Membrane Protein of Escherichia coli E. T. PALVA* AND L. L. RANDALL The Wallenberg Laboratory, University of Uppsala, 75122 Uppsala, Sweden
Received for publication 5 September 1978
The two forms of protein I were cross-linked to molecules of the same species, even when both were present simultaneously. This suggests that they form separate multimers in the outer membrane.
Protein I (3), one of the major proteins in Escherichia coli outer membrane, exists in two closely related fonns, Ia and Ib (15), also called b and c, respectively (5), which can be clearly separated by using high-resolution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (5). The relative amounts of Ia and Ib in the outer membrane seem to vary depending on the strain and growth conditions (6, 16). Recently it was demonstrated that the protein I pores (2, 9) show different specificity, in that protein Ia (b) but not Ib (c) is involved in the permeation of nucleotides through the outer membrane (17). We (10, 11) and Reithmeier and Bragg (13) have previously shown that protein I can be cross-linked to itself to give multimeric complexes. By cross-linking the purified protein we concluded that the basic unit is probably a trimer (11). Reithmeier and Bragg observed that the two forms, named A1 and A2, could be crosslinked to give similar complexes (13) when protein I-peptidoglycan complexes (14) isolated from strains having only one of the two forms at a time are cross-linked. However the SDS-gel system that they used (13) did not resolve the two forms. Thus, they could not question whether heterocomplexes containing both Ia and lb exist. Protein I trimers (11) could consist solely of one species at a time or could be composed of a random mixture of Ia and Ib. We have analyzed the cross-linking of Ia and Ib by using high-resolution SDS-gel electrophoresis (5) to answer this question. We used E. coli K-12 strains having protein Ia (TK69; Fig. 1, slot 2) or protein Ib (TK70; Fig. 1, slot 3) or the parent having both Ia and lb (B14 [1]; Fig. 1, slot 1). Whole cells were crosslinked with a low concentration (2 mM) of the cleavable cross-linking reagent tartryl-di-(glycylazide) (1.3-nm bridge length [7]). This low concentration of the reagent, which produces only dimers of protein I, was used to minimize ambiguities in the mobility of the cross-linked 254
complexes in SDS-gels resulting from different levels of intramolecular cross-linking. Cell envelopes (12) and protein I-peptidoglycan com-
FIG. 1. Protein pattern of isolated cell envelopes (slots 1-3) and isolated protein I-peptidoglycan complexes (slots 4-10) of E. coli K-12 analyzed on 11% SDS-polyacrylamide gels (5). Cells were grown in Luria broth (8) and cross-linked (10, 11) with 2 mM tartryl-di-(glycylazide) for 30 min at room temperature in 100 mM triethanolamine-hydrochloride, pH 8.5, and cell envelopes (12) and protein I-peptidoglycan complexes (11, 14) were isolated. Staining of gels was as described (11). Slots 1-3: isolated cell envelopes not cross-linked of strains B14 (slot 1), TK69 (slot 2), and TK70 (slot 3). Slots 4-10: isolated protein I-peptidoglycan complexes of not-cross-linked controls (slots 4-6) and cross-linked cells (slots 7-10) of strains B14 (slots 4 and 7), TK69 (slots 5 and 8), and TK70 (slots 6 and 9). The material in slot 10 is a 1:1 mixture of that in slots 8 and 9. Arrows at left indicate the position of proteins a (5), Ia([bJ5), Ib([c]5), and HI* ([dl5). Arrows at right indicate the position ofIa and Ib dimers.
VOL. 138, 1979
NOTES first dimension
nv
A
B
N
c
0
255
a lb
U,
c
E
c5 -o C
C
0 0
D
a,
U,
I'll,
NN.
a
lb
'% II* FIG. 2. Symmetrical two-dimensional 11% SDS-polyacrylamide gel patterns of cross-linked and cleaved cell envelopes of E. coli K-12. Cells were grown and cross-linked, and envelopes were isolated as indicated in the legend to Fig. 1. The cleavage of the cross-links after the first dimension run was done with 15 mM NaIO4 for 20 min as described (7). Staining ofgels was as in the legend to Fig. 1. (A) E. coli K-12 TK69; (B) TK70; (C) 1:1 mixture of samples in A and B; (D) B14.
plexes (11, 14) were isolated and analyzed by gel electrophoresis (5). Figure 1 shows the one-dimensional gel analysis of cross-linked Ia and lb. Both Ia (slot 8) and lb (slot 9) are cross-linked to dimers which have a slightly different mobility in the gels. This result confirms the earlier results of Reithmeier and Bragg (13), who used a different cross-linking reagent. If higher concentrations of tartryl-di-(glycylazide) were used, trimers of both Ia and lb (not shown) were also obtained but they had the same mobility. When the strain which had both Ia and Ib (slot 7) was used, both dimers were obtained simultaneously. If the dimners were produced from cross-linking Ia to Ib they would have a mobility between that of Ia dimers and Ib dimers. However, such is not the case; the gel pattem obtained from strain B14 (slot 7) is similar to that of a 1:1 mixture of Ia dimers and Ib dimers (slot 10). These results were further confirmed by analysis of the isolated envelopes of the cross-linked cells on symmetrical two-dimensional SDS-gels (7, 10, 11) that resolve Ia and Ib (5) (Fig. 2). Strain TK69 (Fig. 2A), which produces only Ia, gives only a dimer spot of Ia, whereas the mutant TK70 (Fig. 2B), having only Ib, gives dimers of this form of protein I. The dimer spots of Ia and Ib appear in different positions on the two-di-
mensional gels, as can be seen in Fig. 2C, which is a 1:1 mixture of the material shown in Fig. 2A and B. Both dimers of Ia and Ib are obtained from the wild-type strain B14 (Fig. 2D). Since the dimer spots (Fig. 2D) are not on the same vertical line, but are shifted slightly relative to each other and are in similar positions as the dimers in the mixture (Fig. 20), they are not derived from the same cross-linked complex. These results indicate that the two forms of protein I, even when present simultaneously, tend to be complexed only to molecules of the same form. There is some overlap in the position of the dimers probably due to poor separation on the gels, so presence of minor amounts of heterocomplexes cannot be ruled out. The results presented suggest that Ia and Ib are complexed primarily to themselves and probably form separate multimers in the outer membrane. These multimers may represent two separate species of pores, as has been suggested by van Alphen et al. (17). E.T.P. holds a European Molecular Biology Organization
fellowship. This work was supported by Swedish Natural Sciences Research Council. We thank Hans Fasold for the generous gift of the crosslinking reagent. We are grateful to Ben Lugtenberg for posing
256
J. BACTERIOL.
NOTES
the question which led to this work and for advice about the gel system. 9. 1.
2.
3.
4. 5.
6.
7.
8.
LITERATURE CITED Adler, J. 1973. A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis of Ewcherichia coli. J. Gen. Microbiol. 74: 77-91. Bavoil, P., H. Nikaido, and K. von Meyenburg 1977. Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein. Mol. Gen. Genet. 158:23-33. Garten, W, and U. Henning. 1974. Cell envelope and shape of Escherichia coli K-12. Isolation and preliminary characterization of the major ghost-membrane proteins. Eur. J. Biochem. 47:343-352. Honun, K, D. Hatfield, and M. Schwartz. 1974. maUB region in Eacherichia coli K-12. Characterization of new mutations J. Bacteriol. 117:40-47. Lugtenberg, B., J. Meijers, R. Peters, P. van der Hoek, and L vanAnphen. 1975. Electrophoretic resolution of the 'major outer membrane protein' of Ewcherichia coli K12 into four bands. FEBS Lett. 58:254258. Lugtenberg, B., R. Peters, H. Bernheimer, and W. Berendaen. 1976. Influence of cultural conditions and mutations on the composition of the outer membrane proteins of Ewcherichia coli. Mol. Gen. Genet. 147:251262. Lutter, L C., F. Ortanderl, and H. Fasold. 1974. The use of a new sries of cleavable protein-croslinkers on the Ewcherichia coli ribosome. FEBS Lett. 48:288-292. Miller, J. H. 1972. Experiments in molecular genetics, p.
10.
11. 12. 13. 14.
15.
16.
17.
433. Cold Spring Harbor laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Nakae, T. 1976. Identification of the outer membrane protein of E. coli that produces transmembrane channels in reconstituted vesicle membranes. Biochem. Biophys. Res. Commun. 71:877-884. Palva, E. T., and L L Randall. 1976. Nearest-neighbor analysis of Eacherichia coli outer membrane proteins, Using cleavable cros-links. J. Bacteriol. 127:1558-1560. Palva, E. T., and L L RandalL 1978. Arrangement of protein I in Escherichia coli outer membrane. Crosslinking study. J. Bacteriol. 133:279-286. Palva, E. T. 1978. A major outer membrane protein in SalmoneUa typhimurium induced by maltose. J. Bacteriol. 136:286-294. Relthmeler, R. A. F., and P. D. Bragg. 1977. Crosslinking of the proteins in the outer membrane of Escherichia coli. Biochim. Biophys. Acta 466:245-256. Rosenbuwch, J. P. 1974. Characterization of the major envelope protein from Ewcherichia coli. Regular arrangement on the peptidoglyean and unusual dodecyl sulfate binding. J. Biol. Chem. 249:8019-8029. Shmitges, J., and U. Henning. 1976. The major proteins of the Ewcherichia coli outer cell-envelope membrane. Heterogeneity of protein I. Eur. J. Biochem. 63: 4752. van Aiphen, W., and B. Lugtenberg. 1977. Influence of osmolarity of the growth on the outer membrane protein pattern of Escherichia coli. J. Bacteriol. 131:623630. van Aiphen, 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 fimctioning of pores for nucleotides. Mol. Gen. Genet. 169:75-83.
