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. 1980 Feb;141(2):914–927. doi: 10.1128/jb.141.2.914-927.1980

Separation and properties of the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus.

P E Orndorff, M Dworkin
PMCID: PMC293705  PMID: 6767694

Abstract

We have developed methods for separating the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus. The total membrane fraction from ethylenediaminetetraacetic acid-lysozyme-treated cells was resolved into three major fractions by isopycnic density centrifugation. Between 85 and 90% of the succinate dehydrogenase and cyanide-sensitive reduced nicotinamide adenine dinucleotide oxidase activity was found in the first (I) fraction (rho = 1.221 g/ml) and 80% of the membrane-associated 2-keto-3-deoxyoctonate was found in the third (III) fraction (rho = 1.166 g/ml). The middle (II) fraction (rho = 1.185 g/ml) appeared to be a hybrid membrane fraction and contained roughly 10 to 20% of the activity of the enzyme markers and 2-keto-3-deoxyoctonate. No significant amounts of deoxyribonucleic acid or ribonucleic acid were present in the three isolated fractions, although 26% of the total cellular deoxyribonucleic acid and 3% of the total ribonucleic acid were recovered with the total membrane fraction. Phosphatidylethanolamine made up the bulk (60 to 70%) of the phospholipids in the membrane fractions. However, virtually all of the phosphatidylserine and cardiolipin were found in fraction I. Fraction III appeared to contain elevated amounts of lysophospholipids and contained almost three times the amount of total phospholipid as compared with fraction I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved approximately 40 polypeptides in the total membrane fraction. Two-thirds of these polypeptides were enriched in fraction I, and the remainder was enriched in fraction III. Fraction II contained a banding pattern similar to the total membrane fraction. Electron microscopy revealed that vegetative cells of M. xanthus possessed an envelope similar to that of other gram-negative bacteria; however, the vesicular appearance of the isolated membranes was somewhat different from those reported for Escherichia coli and Salmonella typhimurium. The atypically low bouyant density of the outer membrane of M. xanthus is discussed with regard to the high phospholipid content of the outer membrane.

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Selected References

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

  1. Agabian N., Unger B. Caulobacter crescentus cell envelope: effect of growth conditions on murein and outer membrane protein composition. J Bacteriol. 1978 Feb;133(2):987–994. doi: 10.1128/jb.133.2.987-994.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. BROCKERHOFF H. BREAKDOWN OF PHOSPHOLIPIDS IN MILD ALKALINE HYDROLYSIS. J Lipid Res. 1963 Jan;4:96–99. [PubMed] [Google Scholar]
  5. Birdsell D. C., Cota-Robles E. H. Production and ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme spheroplasts of Escherichia coli. J Bacteriol. 1967 Jan;93(1):427–437. doi: 10.1128/jb.93.1.427-437.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Booth B. R., Curtis N. A. Separation of the cytoplasmic and outer membrane of Pseudomonas aeruginosa PAQ. Biochem Biophys Res Commun. 1977 Feb 7;74(3):1168–1176. doi: 10.1016/0006-291x(77)91641-2. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Bretscher A. P., Kaiser D. Nutrition of Myxococcus xanthus, a fruiting myxobacterium. J Bacteriol. 1978 Feb;133(2):763–768. doi: 10.1128/jb.133.2.763-768.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Card G. L. Metabolism of phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin of Bacillus stearothermophilus. J Bacteriol. 1973 Jun;114(3):1125–1137. doi: 10.1128/jb.114.3.1125-1137.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Collins M. L., Niederman R. A. Membranes of Rhodospirillum rubrum: isolation and physicochemical properties of membranes from aerobically grown cells. J Bacteriol. 1976 Jun;126(3):1316–1325. doi: 10.1128/jb.126.3.1316-1325.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Costerton J. W., Ingram J. M., Cheng K. J. Structure and function of the cell envelope of gram-negative bacteria. Bacteriol Rev. 1974 Mar;38(1):87–110. doi: 10.1128/br.38.1.87-110.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DiRienzo J. M., Nakamura K., Inouye M. The outer membrane proteins of Gram-negative bacteria: biosynthesis, assembly, and functions. Annu Rev Biochem. 1978;47:481–532. doi: 10.1146/annurev.bi.47.070178.002405. [DOI] [PubMed] [Google Scholar]
  13. Doi O., Oki M., Nojima S. Two kinds of phospholipase A and lysophospholipase in Escherichia coli. Biochim Biophys Acta. 1972 Feb 21;260(2):244–258. [PubMed] [Google Scholar]
  14. Dworkin M., Sadler W. Induction of cellular morphogenesis in Myxococcus xanthus. I. General description. J Bacteriol. 1966 Apr;91(4):1516–1519. doi: 10.1128/jb.91.4.1516-1519.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dworkin M. Sensitivity of gliding bacteria to actinomycin D. J Bacteriol. 1969 May;98(2):851–852. doi: 10.1128/jb.98.2.851-852.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hancock R. E., Nikaido H. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J Bacteriol. 1978 Oct;136(1):381–390. doi: 10.1128/jb.136.1.381-390.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hasin M., Rottem S., Razin S. The outer membrane of Proteus mirabilis. I. Isolation and characterization of the outer and cytoplasmic membrane fractions. Biochim Biophys Acta. 1975 Feb 14;375(3):381–394. doi: 10.1016/0005-2736(75)90354-5. [DOI] [PubMed] [Google Scholar]
  18. Hodgkin J., Kaiser D. Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2938–2942. doi: 10.1073/pnas.74.7.2938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inouye M., Inouye S., Zusman D. R. Biosynthesis and self-assembly of protein S, a development-specific protein of Myxococcus xanthus. Proc Natl Acad Sci U S A. 1979 Jan;76(1):209–213. doi: 10.1073/pnas.76.1.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Inouye M., Inouye S., Zusman D. R. Gene expression during development of Myxococcus xanthus: pattern of protein synthesis. Dev Biol. 1979 Feb;68(2):579–591. doi: 10.1016/0012-1606(79)90228-8. [DOI] [PubMed] [Google Scholar]
  21. Irschik H., Reichenbach H. Intracellular location of flexirubins in Flexibacter elegans (Cytophagales). Biochim Biophys Acta. 1978 Jun 16;510(1):1–10. doi: 10.1016/0005-2736(78)90125-6. [DOI] [PubMed] [Google Scholar]
  22. Johnston K. H., Gotschlich E. C. Isolation and characterization of the outer membrane of Neisseria gonorrhoeae. J Bacteriol. 1974 Jul;119(1):250–257. doi: 10.1128/jb.119.1.250-257.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones N. C., Osborn M. J. Interaction of Salmonella typhimurium with phospholipid vesicles. Incorporation of exogenous lipids into intact cells. J Biol Chem. 1977 Oct 25;252(20):7398–7404. [PubMed] [Google Scholar]
  24. Kleinig H. Membranes from Myxococcus fulvus (Myxobacterales) containing carotenoid glucosides. I. Isolation and composition. Biochim Biophys Acta. 1972 Aug 9;274(2):489–498. doi: 10.1016/0005-2736(72)90194-0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Kulpa C. F., Jr, Leive L. Mode of insertion of lipopolysaccharide into the outer membrane of escherichia coli. J Bacteriol. 1976 Apr;126(1):467–477. doi: 10.1128/jb.126.1.467-477.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. LEPAGE M. THE SEPARATION AND IDENTIFICATION OF PLANT PHOSPHOLIPIDS AND GLYCOLIPIDS BY TWO-DIMENSIONAL THIN-LAYER CHROMATOGRAPHY. J Chromatogr. 1964 Jan;13:99–103. doi: 10.1016/s0021-9673(01)95078-2. [DOI] [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Leive L. The barrier function of the gram-negative envelope. Ann N Y Acad Sci. 1974 May 10;235(0):109–129. doi: 10.1111/j.1749-6632.1974.tb43261.x. [DOI] [PubMed] [Google Scholar]
  30. Matsushita K., Adachi O., Shinagawa E., Ameyama M. Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes. J Biochem. 1978 Jan;83(1):171–181. doi: 10.1093/oxfordjournals.jbchem.a131888. [DOI] [PubMed] [Google Scholar]
  31. Nicolaidis A. A., Holland I. B. Evidence for the specific association of the chromosomal origin with outer membrane fractions isolated from Escherichia coli. J Bacteriol. 1978 Jul;135(1):178–189. doi: 10.1128/jb.135.1.178-189.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nikaido H. Outer membrane of Salmonella typhimurium. Transmembrane diffusion of some hydrophobic substances. Biochim Biophys Acta. 1976 Apr 16;433(1):118–132. doi: 10.1016/0005-2736(76)90182-6. [DOI] [PubMed] [Google Scholar]
  33. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  34. Rosenberg E., Keller K. H., Dworkin M. Cell density-dependent growth of Myxococcus xanthus on casein. J Bacteriol. 1977 Feb;129(2):770–777. doi: 10.1128/jb.129.2.770-777.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Scott C. C., Makula S. R., Finnerty W. R. Isolation and characterization of membranes from a hydrocarbon-oxidizing Acinetobacter sp. J Bacteriol. 1976 Jul;127(1):469–480. doi: 10.1128/jb.127.1.469-480.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shaw N. The detection of lipids on thin-layer chromatograms with the periodate-Schiff reagents. Biochim Biophys Acta. 1968 Oct 22;164(2):435–436. doi: 10.1016/0005-2760(68)90171-9. [DOI] [PubMed] [Google Scholar]
  39. Silberstein S., Inouye M. The effects of lysozyme on DNA--membrane association in Escherichia coli. Biochim Biophys Acta. 1974 Oct 11;366(2):149–158. [PubMed] [Google Scholar]
  40. Smith D. K., Winkler H. H. Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions. J Bacteriol. 1979 Feb;137(2):963–971. doi: 10.1128/jb.137.2.963-971.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Steck T. L., Straus J. H., Wallach D. F. A model for the behavior of vesicles in density gradients: implications for fractionation. Biochim Biophys Acta. 1970 Jun 2;203(3):385–393. doi: 10.1016/0005-2736(70)90179-3. [DOI] [PubMed] [Google Scholar]
  42. Sudo S. Z., Dworkin M. Resistance of vegetative cells and microcysts of Myxococcus xanthus. J Bacteriol. 1969 Jun;98(3):883–887. doi: 10.1128/jb.98.3.883-887.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tsukagoshi N., Fox C. F. Hybridization of membranes by sonic irradiation. Biochemistry. 1971 Aug 17;10(17):3309–3313. doi: 10.1021/bi00793a023. [DOI] [PubMed] [Google Scholar]
  44. Vaskovsky V. E., Kostetsky E. Y. Modified spray for the detection of phospholipids on thin-layer chromatograms. J Lipid Res. 1968 May;9(3):396–396. [PubMed] [Google Scholar]
  45. 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]
  46. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  47. White D. A., Lennarz W. J., Schnaitman C. A. Distribution of lipids in the wall and cytoplasmic membrane subfractions of the cell envelope of Escherichia coli. J Bacteriol. 1972 Feb;109(2):686–690. doi: 10.1128/jb.109.2.686-690.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. White D. C., Tucker A. N. Phospholipid metabolism during bacterial growth. J Lipid Res. 1969 Mar;10(2):220–233. [PubMed] [Google Scholar]
  49. Wireman J. W., Dworkin M. Morphogenesis and developmental interactions in myxobacteria. Science. 1975 Aug 15;189(4202):516–523. doi: 10.1126/science.806967. [DOI] [PubMed] [Google Scholar]
  50. Wu H. C. Isolation and characterization of an Escherichia coli mutant with alteration in the outer membrane porteins of the cell envelope. Biochim Biophys Acta. 1972 Dec 1;290(1):274–289. doi: 10.1016/0005-2736(72)90070-3. [DOI] [PubMed] [Google Scholar]
  51. Yamato I., Anraku Y., Hirosawa K. Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes. J Biochem. 1975 Apr;77(4):705–718. doi: 10.1093/oxfordjournals.jbchem.a130774. [DOI] [PubMed] [Google Scholar]
  52. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]
  53. Zusman D. R., Krotoski D. M., Cumsky M. Chromosome replication in Myxococcus xanthus. J Bacteriol. 1978 Jan;133(1):122–129. doi: 10.1128/jb.133.1.122-129.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

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