Vol. 129, No. 3 Printed in U.S.A.
JOURNAL OF BACTZRIOLOGY, Mar. 1977, p. 1651-1652 Copyright 0 1977 American Society for Microbiology
Action of a Major Outer Cell Envelope Membrane Protein in Conjugation of Escherichia coli K-12 MARGARETE SCHWEIZER AND ULF HENNING* Max-Planck-Institut fur Biologie, D 7400 Tubingen, West Germany Received for publication 27 October 1976
Protein II*, a major outer cell envelope membrane protein, was found together with lipopolysaccharide to stoichiometrically inhibit conjugation in Escherichia coli K-12. Loci formerly called tolG (5), con (13), and tut (9) have recently been found to be identical and to map at about min 21 on the Escherichia coli K-12 chromosome (2). It has been agreed to call
pair formation. Other data (1, 7, 8) may, by itself, argue against such a role of this protein. Particularly, it has been shown that mutations causing loss of the protein appear to be pleio-
TABLE 1. Inhibition of conjugation by protein 11* and cell envelope extractsa Expt
Protein II* (jLg/ml)
LPSml) (.gI
Cell(,zg/ml envelope extract [strain])
1
2 3 4 5 6 7 8 9 10 11 12
100 50-80 10 50-90 0.1-0.5 0.1-0.5
100
3,000b 500b 500b 16 1.6 0.4c
Recombinants (% of expt 1)
100 100 100 100
0.1-0.5 10
-d -
1 (P400) (P400) 5-10 (P400 TuII*-R 1.3) 100 10 (P400 TuII*-R 1.3) 0.1 a HfrH cells (108) in 1-ml antibiotic no. 3 medium (Difco) were incubated, with or without the additions listed, for 15 min at 37°C. P400 (thi, arg, pro, thr, leu, str; reference 13) were added and incubation was continued for a further 15 min. Selection on minimal medium was for thr+ leu+ str. Protein II* was isolated from P400 by a modified procedure according to Hindennach and Henning (10). LPS was isolated fromE. coli B/r and was free from protein (amino acid analysis). Lyophilized protein II* was suspended (10 mg/ml) in 0.3% Triton X-100-2 mM NaHCO3, and LPS (10 mg/ml) was dissolved in the same solvent. Cell envelope extracts were prepared from envelopes (3) by suspending 500 mg (wet weight) of them in 20 ml of 10 mM tris (hydroxymethyl)aminomethane-hydrochloride (pH 7.5) containing 5 mM ethylenediaminetetraacetate, 0.05% 2-mercaptoethanol, and 2% sodium dodecyl sulfate. Solubilized material was precipitated with acetone (final concentration, 90%), washed several times with acetone, and lyophilized. The substance contains 50 to 60% protein, the remainder being LPS and phospholipid. P400TuII*-R1-3 is a mutant resistant to phage TuII* (9) and missing protein II*; the same result has been obtained with two other such mutants, and all such mutants have a Con phenotype. LPS from strain P400 under the conditions of these experiments had the same effect as the LPS from E. coli B/r. b Most of the protein was not in solution. c 1.18 x 10-" M. d Addition of LPS was without effect.
100 25 100 100
this locus ompA (4, 11). It represents the structural gene for a major outer cell envelope membrane protein (4, 9, 11), protein 11* (6), which is identical with protein 3A (12). Mutants that lack this protein were first shown by Skurray et al. (13) to exhibit the Con phenotype, specifically, to be defective as recipients in mating-
tropic since a number of additional cell envelope proteins are missing or decrease in such a mutant (1). Table 1 shows that the isolated protein, together with lipopolysaccharide (LPS), acts as a powerful and apparently highly specific inhibitor of conjugation. The action of the protein
1651
1652
NOTES
J. BACTERIOL.
appears to be stoichiometric and not catalytic: lar component the protein or a protein-LPS preincubation of donor cells with the protein (1 complex reacts and exactly which step in conju,ug/ml) for 2 min resulted in the same inhibi- gation is inhibited. tory effect as a 15-min preincubation. Addition LITERATURE CITED of the protein to donor and recipient cells that the reduce not did 5 for min mated had been 1. Ames, G. F.-L., and K. Nikaido. 1976. Two-dimensional number of recombinants which appeared in the gel electrophoresis of membrane proteins. Biochemistry 15:616-623. control without the protein during 15 min of B. J., K. B. Low, and A. L. Taylor. 1976. further incubation of the cells. Crude-envelope 2. Bachmann, Recalibrated linkage map of Escherichi coli K-12. did the protein extracts from strains lacking Bacteriol. Rev. 40:116-167. not inhibit conjugation (Table 1), and such an 3. Braun, V., H. Gnirke, U. Henning, and K. Rehn. 1973. Model for the structure of the shape maintaining extract contains a large number of different layer of the Escherichia coli cell envelope. J. Bacteenvelope proteins (plus LPS). riol. 114:1264-1270. Preincubation of recipient cells with the pro- 4. Datta, D. B., C. Kramer, and U. Henning. 1976. Diptein had the same inhibitory effect as found in loidy for a structural gene specifying a major protein of the outer cell envelope membrane from Escherichia the experiments of Table 1, possibly because coli K-12. J. Bacteriol. 128:834-841. the reaction rate of the protein with cells is 5. Foulds, J. 1974. Chromosomal location of the toiG locus diffusion controlled and thus much faster than for tolerance to bacteriocin JF246 in Escherichia coli cell-cell interaction. We therefore have not yet K-12. J. Bacteriol. 117:1354-1355. Henning. 1975. The identified the target of the inhibitor. Neverthe- 6. Garten, W., I. Hindennach,E.and U.outer cell envelope coli major proteins of the less, it is tempting to assume that the protein membrane. Characterization of proteins II* and III, mutational because cells reacts with the donor comparison of all proteins. Eur. J. Biochem. 59:215221. loss of protein II* leads to the Con phenotype L. M., and W. P. M. Hoekstra. 1976. Charac(13) in recipient cells only, and such HfrH mu- 7. Havekes, terization of an Escherichia coli K-12 F-Con- Mutant. tants, as expected, were found to exhibit unalJ. Bacteriol. 126:593-600. tered fertility. 8. Havekes, L. M., B. J. J. Lugtenberg, and W. P. M. All data available indicate that protein II* Hoekstra. 1976. Conjugation deficient E. coli K-12 Fmutants with heptose-less lipopolysaccharide. Mol. acts as a receptor in conjugation. An argument Gen. Genet. 146:43-50. against this claim would be that the isolated 9. Henning, U., I. Hindennach, and I. Haller. 1976. The protein, protein is contaminated with another major proteins of the E. coli outer cell envelope membrane: evidene for the structural gene of protein II*. and that this other protein is also missing in FEBS Lett. 61:46-48. mutant extracts without protein II* (see above: 10. Hindennach, I., and U. Henning. 1975. The major propleiotropy). As judged by protein-chemical data teins of the E. coli outer cell envelope membrane. a above exist (6), such a contamination does not Preparative isolation of all major membrane proteins. Eur. J. Biochem. 59:207-213. 1 to 5% level. In view of the very low concentraP. A., A. Puspurs, and P. Reeves. 1976. tion of protein required for conjugation inhibi- 11. Manning,membrane ofEscherichia coli K-12: isolation of Outer tion it appears rather unlikely, but cannot yet mutants with altered protein 3A using host range than protein definitely ruled out, that another mutants of bacteriophage K3. J. Bacteriol. 127:10801084. II* is involved. 1974. Outer membrane proteins of The role of LPS is open to speculation. One, 12. Schnaitman, C. A. III. Evidence that the major protein Escherichia coli. and perhaps the only one, of its actions is that of of Escherichia coli 0111 outer membranes consists of increases a detergent because it visibly greatly four distinct polypeptide species. J. Bacteriol. 118:442-453. the solubility of the isolated protein in the matR. E. W. Hancock, and P. Reeves. ing medium. This detergent action may, of 13. Skurray, R. A., 1974. Con- Mutants: class of mutants in Escherichia course, also be specific in that LPS could influcoli K-12 lacking a major cell wall protein and defecence the conformation of the protein (see refertive in conjugation and adsorption of a bacteriophage. J. Bacteriol. 119:726-735. ence 8). It remains to be seen with which cellu-
JOURNAL OF BACTZRIOLOGY, Mar. 1977, p. 1651-1652 Copyright 0 1977 American Society for Microbiology
Action of a Major Outer Cell Envelope Membrane Protein in Conjugation of Escherichia coli K-12 MARGARETE SCHWEIZER AND ULF HENNING* Max-Planck-Institut fur Biologie, D 7400 Tubingen, West Germany Received for publication 27 October 1976
Protein II*, a major outer cell envelope membrane protein, was found together with lipopolysaccharide to stoichiometrically inhibit conjugation in Escherichia coli K-12. Loci formerly called tolG (5), con (13), and tut (9) have recently been found to be identical and to map at about min 21 on the Escherichia coli K-12 chromosome (2). It has been agreed to call
pair formation. Other data (1, 7, 8) may, by itself, argue against such a role of this protein. Particularly, it has been shown that mutations causing loss of the protein appear to be pleio-
TABLE 1. Inhibition of conjugation by protein 11* and cell envelope extractsa Expt
Protein II* (jLg/ml)
LPSml) (.gI
Cell(,zg/ml envelope extract [strain])
1
2 3 4 5 6 7 8 9 10 11 12
100 50-80 10 50-90 0.1-0.5 0.1-0.5
100
3,000b 500b 500b 16 1.6 0.4c
Recombinants (% of expt 1)
100 100 100 100
0.1-0.5 10
-d -
1 (P400) (P400) 5-10 (P400 TuII*-R 1.3) 100 10 (P400 TuII*-R 1.3) 0.1 a HfrH cells (108) in 1-ml antibiotic no. 3 medium (Difco) were incubated, with or without the additions listed, for 15 min at 37°C. P400 (thi, arg, pro, thr, leu, str; reference 13) were added and incubation was continued for a further 15 min. Selection on minimal medium was for thr+ leu+ str. Protein II* was isolated from P400 by a modified procedure according to Hindennach and Henning (10). LPS was isolated fromE. coli B/r and was free from protein (amino acid analysis). Lyophilized protein II* was suspended (10 mg/ml) in 0.3% Triton X-100-2 mM NaHCO3, and LPS (10 mg/ml) was dissolved in the same solvent. Cell envelope extracts were prepared from envelopes (3) by suspending 500 mg (wet weight) of them in 20 ml of 10 mM tris (hydroxymethyl)aminomethane-hydrochloride (pH 7.5) containing 5 mM ethylenediaminetetraacetate, 0.05% 2-mercaptoethanol, and 2% sodium dodecyl sulfate. Solubilized material was precipitated with acetone (final concentration, 90%), washed several times with acetone, and lyophilized. The substance contains 50 to 60% protein, the remainder being LPS and phospholipid. P400TuII*-R1-3 is a mutant resistant to phage TuII* (9) and missing protein II*; the same result has been obtained with two other such mutants, and all such mutants have a Con phenotype. LPS from strain P400 under the conditions of these experiments had the same effect as the LPS from E. coli B/r. b Most of the protein was not in solution. c 1.18 x 10-" M. d Addition of LPS was without effect.
100 25 100 100
this locus ompA (4, 11). It represents the structural gene for a major outer cell envelope membrane protein (4, 9, 11), protein 11* (6), which is identical with protein 3A (12). Mutants that lack this protein were first shown by Skurray et al. (13) to exhibit the Con phenotype, specifically, to be defective as recipients in mating-
tropic since a number of additional cell envelope proteins are missing or decrease in such a mutant (1). Table 1 shows that the isolated protein, together with lipopolysaccharide (LPS), acts as a powerful and apparently highly specific inhibitor of conjugation. The action of the protein
1651
1652
NOTES
J. BACTERIOL.
appears to be stoichiometric and not catalytic: lar component the protein or a protein-LPS preincubation of donor cells with the protein (1 complex reacts and exactly which step in conju,ug/ml) for 2 min resulted in the same inhibi- gation is inhibited. tory effect as a 15-min preincubation. Addition LITERATURE CITED of the protein to donor and recipient cells that the reduce not did 5 for min mated had been 1. Ames, G. F.-L., and K. Nikaido. 1976. Two-dimensional number of recombinants which appeared in the gel electrophoresis of membrane proteins. Biochemistry 15:616-623. control without the protein during 15 min of B. J., K. B. Low, and A. L. Taylor. 1976. further incubation of the cells. Crude-envelope 2. Bachmann, Recalibrated linkage map of Escherichi coli K-12. did the protein extracts from strains lacking Bacteriol. Rev. 40:116-167. not inhibit conjugation (Table 1), and such an 3. Braun, V., H. Gnirke, U. Henning, and K. Rehn. 1973. Model for the structure of the shape maintaining extract contains a large number of different layer of the Escherichia coli cell envelope. J. Bacteenvelope proteins (plus LPS). riol. 114:1264-1270. Preincubation of recipient cells with the pro- 4. Datta, D. B., C. Kramer, and U. Henning. 1976. Diptein had the same inhibitory effect as found in loidy for a structural gene specifying a major protein of the outer cell envelope membrane from Escherichia the experiments of Table 1, possibly because coli K-12. J. Bacteriol. 128:834-841. the reaction rate of the protein with cells is 5. Foulds, J. 1974. Chromosomal location of the toiG locus diffusion controlled and thus much faster than for tolerance to bacteriocin JF246 in Escherichia coli cell-cell interaction. We therefore have not yet K-12. J. Bacteriol. 117:1354-1355. Henning. 1975. The identified the target of the inhibitor. Neverthe- 6. Garten, W., I. Hindennach,E.and U.outer cell envelope coli major proteins of the less, it is tempting to assume that the protein membrane. Characterization of proteins II* and III, mutational because cells reacts with the donor comparison of all proteins. Eur. J. Biochem. 59:215221. loss of protein II* leads to the Con phenotype L. M., and W. P. M. Hoekstra. 1976. Charac(13) in recipient cells only, and such HfrH mu- 7. Havekes, terization of an Escherichia coli K-12 F-Con- Mutant. tants, as expected, were found to exhibit unalJ. Bacteriol. 126:593-600. tered fertility. 8. Havekes, L. M., B. J. J. Lugtenberg, and W. P. M. All data available indicate that protein II* Hoekstra. 1976. Conjugation deficient E. coli K-12 Fmutants with heptose-less lipopolysaccharide. Mol. acts as a receptor in conjugation. An argument Gen. Genet. 146:43-50. against this claim would be that the isolated 9. Henning, U., I. Hindennach, and I. Haller. 1976. The protein, protein is contaminated with another major proteins of the E. coli outer cell envelope membrane: evidene for the structural gene of protein II*. and that this other protein is also missing in FEBS Lett. 61:46-48. mutant extracts without protein II* (see above: 10. Hindennach, I., and U. Henning. 1975. The major propleiotropy). As judged by protein-chemical data teins of the E. coli outer cell envelope membrane. a above exist (6), such a contamination does not Preparative isolation of all major membrane proteins. Eur. J. Biochem. 59:207-213. 1 to 5% level. In view of the very low concentraP. A., A. Puspurs, and P. Reeves. 1976. tion of protein required for conjugation inhibi- 11. Manning,membrane ofEscherichia coli K-12: isolation of Outer tion it appears rather unlikely, but cannot yet mutants with altered protein 3A using host range than protein definitely ruled out, that another mutants of bacteriophage K3. J. Bacteriol. 127:10801084. II* is involved. 1974. Outer membrane proteins of The role of LPS is open to speculation. One, 12. Schnaitman, C. A. III. Evidence that the major protein Escherichia coli. and perhaps the only one, of its actions is that of of Escherichia coli 0111 outer membranes consists of increases a detergent because it visibly greatly four distinct polypeptide species. J. Bacteriol. 118:442-453. the solubility of the isolated protein in the matR. E. W. Hancock, and P. Reeves. ing medium. This detergent action may, of 13. Skurray, R. A., 1974. Con- Mutants: class of mutants in Escherichia course, also be specific in that LPS could influcoli K-12 lacking a major cell wall protein and defecence the conformation of the protein (see refertive in conjugation and adsorption of a bacteriophage. J. Bacteriol. 119:726-735. ence 8). It remains to be seen with which cellu-
