Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1981 May;146(2):542–551. doi: 10.1128/jb.146.2.542-551.1981

Interactions of Cations with Membrane Fractions of Smooth and Rough Strains of Salmonella typhimurium and Other Gram-Negative Bacteria

H Stan-Lotter 1,†, Kenneth E Sanderson 1
PMCID: PMC216997  PMID: 7012132

Abstract

Addition of cations (20 to 50 mM for Mg2+ or Ca2+ or 100 to 500 mM for Na+) to N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer during preparation of membranes from smooth and rough strains of Salmonella typhimurium LT2, Salmonella minnesota, and Escherichia coli O8 had two effects on the composition of the membranes isolated. First, in rough strains of chemotypes Ra to Re the “total membranes” (pellets from high-speed centrifugation) were deficient in the proteins of the outer membrane. The missing proteins were found to have been sedimented in a prior low-speed centrifugation in a fraction we call “cation-aggregated membranes.” Since these membranes were enriched for lipopolysaccharide and for outer membrane proteins, deficient in succinic dehydrogenase, and contained primarily the dense peak after sucrose gradient centrifugation, it appears to be relatively pure outer membrane. About 10% of the membrane protein of smooth strains and up to 50% that of rough strains were cation-aggregated membranes, appearing to contain most of the outer membrane of rough strains. Thus, cation aggregation may be a useful means of preparation of outer membrane samples. The second effect was that with cation addition, several high-molecular-weight proteins not seen when membranes were prepared without cation addition were found in the total membranes of both smooth and rough strains after high-speed centrifugation. These proteins were bound by cations to the inner membranes, since they were soluble in Triton X-100 and separated into the less dense peak upon sucrose gradient centrifugation. They originated from the cytoplasm or the periplasm, since they corresponded to soluble proteins found in the supernatant after high-speed centrifugation and were depleted from this supernatant when preparation was done in the presence of cations.

Full text

PDF
542

Images in this article

544Image
on p.544
545Image
on p.545
547Image
on p.547
547Image
on p.547
549Image
on p.549

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ames G. F., Nikaido K. Two-dimensional gel electrophoresis of membrane proteins. Biochemistry. 1976 Feb 10;15(3):616–623. doi: 10.1021/bi00648a026. [DOI] [PubMed] [Google Scholar]
  2. Ames G. F. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem. 1974 Jan 25;249(2):634–644. [PubMed] [Google Scholar]
  3. Ames G. F., Spudich E. N., Nikaido H. Protein composition of the outer membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J Bacteriol. 1974 Feb;117(2):406–416. doi: 10.1128/jb.117.2.406-416.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bragg P. D., Hou C. Organization of proteins in the native and reformed outer membrane of Escherichia coli. Biochim Biophys Acta. 1972 Aug 9;274(2):478–488. doi: 10.1016/0005-2736(72)90193-9. [DOI] [PubMed] [Google Scholar]
  5. Chatterjee A. K., Sanderson K. E., Ross H. Influence of temperature on growth of lipopolysaccharide-deficient (rough) mutants of Salmonella typhimurium and Salmonella minnesota. Can J Microbiol. 1976 Oct;22(10):1540–1548. doi: 10.1139/m76-226. [DOI] [PubMed] [Google Scholar]
  6. Irvin R. T., MacAlister T. J., Chan R., Costerton J. W. Citrate-tris(hydroxymethyl)aminomethane-mediated release of outer membrane sections from the cell envelope of a deep-rough (heptose-deficient lipopolysaccharide) strain of Escherichia coli O8. J Bacteriol. 1981 Mar;145(3):1386–1396. doi: 10.1128/jb.145.3.1386-1396.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Koplow J., Goldfine H. Alterations in the outer membrane of the cell envelope of heptose-deficient mutants of Escherichia coli. J Bacteriol. 1974 Feb;117(2):527–543. doi: 10.1128/jb.117.2.527-543.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. McMichael J. C., Ou J. T. Metal ion dependence of a heat-modifiable protein from the outer membrane of Escherichia coli upon sodium dodecyl sulfate-gel electrophoresis. J Bacteriol. 1977 Oct;132(1):314–320. doi: 10.1128/jb.132.1.314-320.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nakae T., Nikaido H. Outer membrane as a diffusion barrier in Salmonella typhimurium. Penetration of oligo- and polysaccharides into isolated outer membrane vesicles and cells with degraded peptidoglycan layer. J Biol Chem. 1975 Sep 25;250(18):7359–7365. [PubMed] [Google Scholar]
  11. Nikaido H., Levinthal M., Nikaido K., Nakane K. Extended deletions in the histidine-rough-B region of the Salmonella chromosome. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1825–1832. doi: 10.1073/pnas.57.6.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nikaido H., Nakae T. The outer membrane of Gram-negative bacteria. Adv Microb Physiol. 1979;20:163–250. doi: 10.1016/s0065-2911(08)60208-8. [DOI] [PubMed] [Google Scholar]
  13. Nikaido H. Permeability of the outer membrane of bacteria. Angew Chem Int Ed Engl. 1979 May;18(5):337–350. doi: 10.1002/anie.197903373. [DOI] [PubMed] [Google Scholar]
  14. Nikaido H., Song S. A., Shaltiel L., Nurminen M. Outer membrane of Salmonella XIV. Reduced transmembrane diffusion rates in porin-deficient mutants. Biochem Biophys Res Commun. 1976 May 23;76(2):324–330. doi: 10.1016/0006-291x(77)90728-8. [DOI] [PubMed] [Google Scholar]
  15. Nurminen M., Lounatmaa K., Sarvas M., Mäkelä P. H., Nakae T. Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins. J Bacteriol. 1976 Aug;127(2):941–955. doi: 10.1128/jb.127.2.941-955.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  17. OSBORN M. J. STUDIES ON THE GRAM-NEGATIVE CELL WALL. I. EVIDENCE FOR THE ROLE OF 2-KETO- 3-DEOXYOCTONATE IN THE LIPOPOLYSACCHARIDE OF SALMONELLA TYPHIMURIUM. Proc Natl Acad Sci U S A. 1963 Sep;50:499–506. doi: 10.1073/pnas.50.3.499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Reithmeier R. A., Bragg P. D. Molecular characterization of a heat-modifiable protein from the outer membrane of Escherichia coli. Arch Biochem Biophys. 1977 Jan 30;178(2):527–534. doi: 10.1016/0003-9861(77)90223-5. [DOI] [PubMed] [Google Scholar]
  19. SUBBAIAH T. V., STOCKER B. A. ROUGH MUTANTS OF SALMONELLA TYPHIMURIUM. I. GENETICS. Nature. 1964 Mar 28;201:1298–1299. doi: 10.1038/2011298a0. [DOI] [PubMed] [Google Scholar]
  20. Sanderson K. E., Van Wyngaarden J., Lüderitz O., Stocker B. A. Rough mutants of Salmonella typhimurium with defects in the heptose region of the lipopolysaccharide core. Can J Microbiol. 1974 Aug;20(8):1127–1134. doi: 10.1139/m74-175. [DOI] [PubMed] [Google Scholar]
  21. Schindler P. R., Teuber M. Ultrastructural study of Salmonella typhimurium treated with membrane-active agents: specific reaction dansylchloride with cell envelope components. J Bacteriol. 1978 Jul;135(1):198–206. doi: 10.1128/jb.135.1.198-206.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schmidt G., Jann B., Jann K. Immunochemistry of R lipopolysaccharides of Escherichia coli. Studies on R mutants with an incomplete core, derived from E. coli O8:K27. Eur J Biochem. 1970 Oct;16(2):382–392. doi: 10.1111/j.1432-1033.1970.tb01092.x. [DOI] [PubMed] [Google Scholar]
  23. Schmidt G., Lüderitz O. Untersuchungen zur Typisierung von Salmonella-R-Formen. II. Typisierung von S. minnesota-Mutanten durch Phagen. Zentralbl Bakteriol Orig. 1969 Jul;210(3):381–387. [PubMed] [Google Scholar]
  24. Schnaitman C. A. Effect of ethylenediaminetetraacetic acid, Triton X-100, and lysozyme on the morphology and chemical composition of isolate cell walls of Escherichia coli. J Bacteriol. 1971 Oct;108(1):553–563. doi: 10.1128/jb.108.1.553-563.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schnaitman C. A. Examination of the protein composition of the cell envelope of Escherichia coli by polyacrylamide gel electrophoresis. J Bacteriol. 1970 Nov;104(2):882–889. doi: 10.1128/jb.104.2.882-889.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schnaitman C. A. Outer membrane proteins of Escherichia coli. 3. Evidence that the major protein of Escherichia coli O111 outer membrane consists of four distinct polypeptide species. J Bacteriol. 1974 May;118(2):442–453. doi: 10.1128/jb.118.2.442-453.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schnaitman C. A. Outer membrane proteins of Escherichia coli. I. Effect of preparative conditions on the migration of protein in polyacrylamide gels. Arch Biochem Biophys. 1973 Aug;157(2):541–552. doi: 10.1016/0003-9861(73)90673-5. [DOI] [PubMed] [Google Scholar]
  28. Schnaitman C. A. Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J Bacteriol. 1970 Nov;104(2):890–901. doi: 10.1128/jb.104.2.890-901.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schnaitman C. A. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol. 1971 Oct;108(1):545–552. doi: 10.1128/jb.108.1.545-552.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smit J., Kamio Y., Nikaido H. Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J Bacteriol. 1975 Nov;124(2):942–958. doi: 10.1128/jb.124.2.942-958.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stan-Lotter H., Gupta M., Sanderson K. E. The influence of cations on the permeability of the outer membrane of Salmonella typhimurium and other gram-negative bacteria. Can J Microbiol. 1979 Apr;25(4):475–485. doi: 10.1139/m79-070. [DOI] [PubMed] [Google Scholar]
  32. Uemura J., Mizushima S. Isolation of outer membrane proteins of Escherchia coli and their characterization on polyacrylamide gel. Biochim Biophys Acta. 1975 Dec 1;413(2):163–176. doi: 10.1016/0005-2736(75)90101-7. [DOI] [PubMed] [Google Scholar]
  33. WEISSBACH A., HURWITZ J. The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. I. Identification. J Biol Chem. 1959 Apr;234(4):705–709. [PubMed] [Google Scholar]
  34. Wilkinson R. G., Gemski P., Jr, Stocker B. A. Non-smooth mutants of Salmonella typhimurium: differentiation by phage sensitivity and genetic mapping. J Gen Microbiol. 1972 May;70(3):527–554. doi: 10.1099/00221287-70-3-527. [DOI] [PubMed] [Google Scholar]
  35. Wolf-Watz H., Normark S., Bloom G. D. Rapid method for isolation of large quantities of outer membrane from Escherichia coli K-12 and its application to the study of envelope mutants. J Bacteriol. 1973 Sep;115(3):1191–1197. doi: 10.1128/jb.115.3.1191-1197.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES