Chapter 7 : Cell Fractionation

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This chapter presents techniques for the fractionation of cellular components, including organelles and appendages, beginning with external surfaces and ending with internal components. It should be appreciated, however, that many of the techniques described in the chapter, from cell breakage to centrifugation theory, will have similar applications. The factors pertinent to cell fractionation, often dictating the approach needed, are the differences in wall structures and the bonding forces within walls (and associated surface materials) responsible for maintaining cell shape. Purification of the murein-outer membrane complex from the crude envelope fraction by density gradient separation in sucrose is described. Lipopolysaccharide (LPS) isolated by the phenol-water method preserves the reactivity of pseudomonad LPS with antibodies in Western immunoblots and is better in this regard than LPS isolated by the Darveau-Hancock method. In general, the murein portion of the cell wall is first removed to form protoplasts or spheroplasts, which are then lysed to provide either the plasma membrane or the plasma plus outer membranes. A structurally intact, intracytoplasmic membrane array may best be isolated by lysis of osmotically sensitive spheroplasts. The effect of lysis and physiological conditions on the nucleoid is discussed by Korch.

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7
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Isolation of cytoplasmic membranes by discontinuous sucrose gradient centrifugation. A membrane fraction from spheroplast lysate in 33% sucrose was loaded on 40 to 50% sucrose steps and centrifuged at 4°C for 48 h (110,000 × , max) in a Beckman SW27 swinging-bucket rotor. A thin section of the upper band revealed closed vesicles bounded by the double-track membrane, typical of cytoplasmic membranes. See reference 135 for further details.

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7
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Isolation of envelopes and related fractions from gram-negative bacteria.

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7
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1. Altman, E.,, J.-R. Brisson,, and M. B. Perry. 1986. Structural studies of the capsular polysaccharide from Haemophilus pleuropneumoniae serotype 1. Biochem. Cell Biol. 64: 707 716.
2. Altman, E.,, M. B. Perry,, and J.-R. Brisson. 1989. Structure of the lipopolysaccharide antigenic O-chain produced by Actinobacillus pleuropneumoniae serotype 4 (ATCC 33378). Carbohydr. Res. 191: 295 303.
3. Ames, G. F.-L.,, C. Prody,, and S. Kustu. 1984. Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J. Bacteriol. 160: 1181 1183.
4. Archer, D. B.,, and N. R. King. 1984. Isolation of gas vesicles from Methanosarcina barkeri. J. Gen. Microbiol. 130: 167 172.
5. Austin, E. A.,, J.F. Graves,, L. A. Hite,, C. T. Parker,, and C. A. Schnaitman. 1990. Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12. J. Bacteriol. 172: 5312 5325.
6. Austin, J. W.,, and R. G. E. Murray. 1987. The perforate component of the regularly-structured (RS) layer of Lampropedia hyalina. Can. J. Microbiol. 33: 1039 1045.
7. Bakau, B.,, J. M. Brass,, and W. Boos. 1985. Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of bindingprotein- dependent transport. J. Bacteriol. 163: 61 68.
8. Barber, E. J. 1976. Viscosity and density tables, p. 415 418. In G. D. Fasman (ed.), Handbook of Biochemistry and Molecular Biology, 3rd ed., vol. 1. Physical and Chemical Data. CRC Press, Inc., Cleveland, OH.
9. Barrett, A. J. 1980. Introduction: the classification of proteinases. Ciba Found. Symp. 75: 1 13.
10. Beaman, T. C.,, and P. Gerhardt. 1986. Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation. Appl. Environ. Microbiol. 52: 1242 1246.
11. Beaman, T. C.,, J. T. Greenamyre,, T. R. Corner,, H. S. Pankratz,, and P. Gerhardt. 1982. Bacterial spore heat resistance correlated with water content, wet density, and protoplast/sporoplast volume ratio. J. Bacteriol. 150: 870 877.
12. Beveridge, T. J. 1979. Surface arrays on the wall of Sporosarcina urea. J. Bacteriol. 139: 1039 1048.
13. Beveridge, T. J.,, G. B. Patel,, B. J. Harris,, and G. D. Sprott. 1986. The ultrastructure of Methanothrix concilii, a mesophilic aceticlastic methanogen. Can. J. Microbiol. 32: 703 710.
14. Beveridge, T. J.,, M. Stewart,, R. J. Doyle,, and G. D. Sprott. 1985. Unusual stability of the Methanospirillum hungatei sheath. J. Bacteriol. 162: 728 737.
15. Beynon, L. M.,, M. Moreau,, J. C. Richards,, and M. B. Perry. 1991. Structure of the O-antigen of Actinobacillus pleuropneumoniae serotype 7 lipopolysaccharide. Carbohydr. Res. 209: 225 238.
16. Bingle, W. H.,, J. L. Doran,, and W. J. Page. 1984. Regular surface layer of Azotobacter vinelandii. J. Bacteriol. 159: 251 259.
17. Blakemore, R. P. 1982. Magnetotactic bacteria. Annu. Rev. Microbiol. 36: 217 238.
18. Bohrmann, B.,, W. Villiger,, R. Johansen,, and E. Kellenberger. 1991. Coralline shape of the bacterial nucleoid after cryofixation. J. Bacteriol. 173: 3149 3158.
19. Brinton, C. C.,, A. Buzzell,, and M. A. Lauffer. 1954. Electrophoresis and phage susceptibility on a filamentproducing variant of the E. coli B bacterium. Biochim. Biophys. Acta 15: 533 542.
20. Bush, J. W. 1985. Enzymatic lysis of the pseudomureincontaining methanogen Methanobacterium formicicum. J. Bacteriol. 163: 27 36.
21. Chen, W.,, K. Ohmiya,, and S. Shimizu. 1986. Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium. Appl. Environ. Microbiol. 52: 612 616.
22. Clark, J. E.,, H. Beegen,, and H. G. Wood. 1985. Isolation of polyphosphate from Propionibacterium shermanii. Fed. Proc. 44: 1079.
23. Coulton, J. W.,, and R. G. E. Murray. 1977. Membraneassociated components of the bacterial flagellar apparatus. Biochim. Biophys. Acta 465: 290 310.
24. Cunha, S.,, T. Odijk,, E. Suleymanoglu,, and C. L. Woldringh. 2001. Isolation of the Escherichia coli nucleoid. Biochimie 83: 149 154.
25. Cunningham, T. M.,, E. M. Walker,, J. N. Miller,, and M. A. Lovett. 1988. Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114. J. Bacteriol. 170: 5789 5796.
26. Darveau, R. P.,, and R. E. W. Hancock. 1983. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J. Bacteriol. 155: 831 838.
27. Davis, R. P.,, and J. E. Harris. 1985. Spontaneous protoplast formation by Methanosarcina barkeri. J. Gen. Microbiol. 131: 1481 1486.
28. De Pamphilis, M. L.,, and J. Adler. 1971. Purification of intact flagella from Escherichia coli and Bacillus subtilis. J. Bacteriol. 105: 376 383.
29. Dubreuil, J. D.,, S. M. Logan,, S. Cubbage,, D. NiEidhin,, W. D. McCubbin,, C. M. Kay,, T. J. Beveridge,, F. G. Ferris,, and T. J. Trust. 1988. Structural and biochemical analysis of a surface array protein of Campylobacter fetus. J. Bacteriol. 170: 4165 4173.
30. Dunn, R. M.,, M. J. Munster,, R. J. Sharp,, and B. N. Dancer. 1987. A novel method for regenerating the protoplasts of thermophilic bacilli. Arch. Microbiol. 146: 323 326.
31. Dupont, C.,, and A. J. Clarke. 1991. Dependence of lysozyme-catalysed solubilization of Proteus mirabilis peptidoglycan on the extent of O-acetylation. Eur. J. Biochem. 195: 763 769.
32. Easterbrook, K. B., 1989. Spinate bacteria, p. 1991 1993. In J.G. Holt (ed.), Bergey’s Manual of Systematic Bacteriology, vol. 3. The Williams and Wilkins Co., Baltimore, MD.
33. Easterbrook, K. B.,, and R. W. Coombs. 1976. Spinin: the subunit protein of bacterial spinae. Can. J. Microbiol. 22: 438 440.
34. Easterbrook, K. B.,, and S. Sperker. 1982. Physiological controls of bacterial spinae production in complex medium and their value as indicators of spina function. Can. J. Microbiol. 28: 130 136.
35. Elhardt, D.,, and A. Böck. 1982. An in vitro polypeptide synthesizing system from methanogenic bacteria: sensitivity to antibiotics. Mol. Gen. Genet. 188: 128 134.
36. Filip, C.,, G. Fletcher,, J. L. Wulff,, and C. F. Earhart. 1977. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium lauryl sarcosinate. J. Bacteriol. 115: 717 722.
37. Ford, T. C.,, and J. M. Graham. 1991. An Introduction to Centrifugation. BIOS Scientific Publishers Ltd., Oxford, United Kingdom.
38. Galanos, C.,, and O. Luderitz. 1975. Electrodialysis of lipopolysaccharides and their conversion to uniform salt form. Eur. J. Biochem. 54: 603 610.
39. Galanos, C.,, O. Lüderitz,, and O. Westphal. 1969. A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9: 245 249.
40. Gilleland, H. E., Jr.,, J. D. Stinnett,, I. L. Roth,, and R. G. Eagon. 1973. Freeze-etch study of Pseudomonas aeruginosa: localization within the cell wall of an ethylenediaminetetraacetate- extractable component. J. Bacteriol. 113: 417 432.
41. Gorby, Y. A.,, T. J. Beveridge,, and R. P. Blakemore. 1988. Characterization of the bacterial magnetosome membrane. J. Bacteriol. 170: 834 841.
42. Gould, G. W. 1971. Methods for studying bacterial spores. Methods Microbiol. 6A: 361 381.
43. Gray, G. W.,, and S. G. Wilkinson. 1965. The effect of ethylenediaminetetraacetic acid on the cell walls of some gram-negative bacteria. J. Gen. Microbiol. 39: 385 399.
44. Gruter, L.,, and R. Laufs. 1991. Protoplast transformation of Staphylococcus epidermidis. J. Microbiol. Methods 13: 299 304.
45. Hamilton, M. G. 1971. Isodensity equilibrium centrifugation of ribosomal particles; the calculation of the protein content of ribosomes and other ribonucleoproteins from buoyant density measurements. Methods Enzymol. 20: 512 521.
46. Hancock, I. C.,, and I. R. Poxton,. 1988. Isolation and purification of cell walls, p. 55 65. In I. C. Hancock, and I. R. Poxton (ed.), Bacterial Cell Surface Techniques. John Wiley & Sons Ltd., Toronto, Canada.
47. Hancock, I. C.,, and I. R. Poxton,. 1988. Teichoic acids and other accessory carbohydrates from Gram-positive bacteria, p. 79 88. In I. C. Hancock, and I. R. Poxton (ed.), Bacterial Cell Surface Techniques. John Wiley & Sons Ltd., Toronto, Canada.
48. Hancock, I. C.,, and I. R. Poxton,. 1988. Isolation of exopolysaccharides, p. 121 125. In I. C. Hancock, and I. R. Poxton (ed.), Bacterial Cell Surface Techniques. John Wiley & Sons Ltd., Toronto, Canada.
49. Hancock, R. E. W.,, and H. Nikaido. 1978. Outer membranes of Gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J. Bacteriol. 136: 381 390.
50. Harold, F. M. 1963. Accumulation of inorganic polyphosphate in Aerobacter aerogenes. I. Relationship to growth and nucleic acid synthesis. J. Bacteriol. 86: 216 221.
51. Harwood, C. R.,, and S. M. Cutting. 1990. Molecular Biological Methods for Bacillus. John Wiley & Sons, Inc., New York, NY.
52. Hash, J. H.,, and M. V. Rothlauf. 1967. The N,Odiacetylmuramidase of Chalaropsis species. J. Biol. Chem. 242: 5586 5590.
53. Heckels, J. E.,, and M. Virji,. 1988. Detection and preparation of surface appendages, p. 67 72. In I. C. Hancock, and I. R. Poxton (ed.), Bacterial Cell Surface Techniques. John Wiley & Sons Ltd., Toronto, Canada.
54. Helenius, A.,, D. R. McCaslin,, E. Fries,, and C. Tanford. 1979. Properties of detergents. Methods Enzymol. 56: 734 749.
55.. Heppel, L. A., 1971. The concept of periplasmic enzymes, p. 223 247. In L. I. Rothfield (ed.), Structure and Function of Biological Membranes. Academic Press, Inc., New York, NY.
56. Hitchcock, P. J.,, and T. M. Brown. 1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154: 269 277.
57. Hobot, J. A.,, E. Carlemalm,, W. Villiger,, and E. Kellenberger. 1984. Periplasmic gel: new concept resulting from the reinvestigation of bacterial cell envelope ultrastructure by new methods. J. Bacteriol. 160: 143 152.
58. Holloway, P. W. 1973. A simple procedure for the removal of Triton X-100 from protein samples. Anal. Biochem. 53: 304 308.
59. Hughes, D. E.,, J. W. T. Wimpenny,, and D. Lloyd,. 1971. The disintegration of microorganisms, p. 1 54. In J. R. Norris, and D. W. Ribbons (ed.), Methods in Microbiology, vol. 5B. Academic Press, Inc., New York, NY.
60. Jann, B.,, and K. Jann. 1990. Structure and biosynthesis of the capsular antigens of Escherichia coli, p. 19-42. Curr. Top. Microbiol. Immunol. 150: 19 42.
61. Jarrell, K. F.,, J. R. Colvin,, and G. D. Sprott. 1982. Spontaneous protoplast formation in Methanobacterium bryantii. J. Bacteriol. 149: 346 353.
62. Jarrell, K. F.,, D. Faguy,, A. M. Hebert,, and M. L. Kalmokoff. 1992. A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria). Can. J. Microbiol. 38: 65 68.
63. Jarrell, K. F.,, and G. D. Sprott. 1984. Formation and regeneration of Halobacterium protoplasts. Curr. Microbiol. 10: 147 152.
64. Jennings, H. J. 1990. Capsular polysaccharides as vaccine candidates. Curr. Top. Microbiol. Immunol. 150: 97 127.
65. Johannsen, L.,, H. Labischinski,, B. Reinicke,, and P. Giesbrecht. 1983. Changes in the chemical structure of walls of Staphylococcus aureus grown in the presence of chloramphenicol. FEMS Microbiol. Lett. 16: 313 316.
66. Jollès, P. 1969. Lysozymes: a chapter of molecular biology. Angew. Chem. Int. Ed. 8: 227 294.
67. Jussofie, A.,, F. Mayer,, and G. Gottschalk. 1986. Methane formation from methanol and molecular hydrogen by protoplasts of new methanogen isolates and inhibition by dicyclohexylcarbodiimide. Arch. Microbiol. 146: 245 249.
68. Kaback, H. R. 1971. Bacterial membranes. Methods Enzymol. 22: 99 120.
69. Kalmokoff, M. L.,, K. F. Jarrell,, and S. F. Koval. 1988. Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114. J. Bacteriol. 170: 1752 1758.
70. Kamekura, M.,, D. Oesterhelt,, R. Wallace,, P. Anderson,, and D. J. Kushner. 1988. Lysis of halobacteria in Bacto- Peptone by bile acids. Appl. Environ. Microbiol. 54: 990 995.
71. Kandler, O.,, and H. König. 1985. Cell envelopes of archaebacteria. Bacteria 8: 413 457.
72. Karch, H.,, H. Leying,, K.-H. Buscher,, H.-P. Kroll,, and W. Opferkuch. 1985. Isolation and separation of physiochemically distinct fimbrial types expressed on a single culture of Escherichia coli O7:K1:H6. Infect. Immun. 47: 549 554.
73. Kates, M.,, S. C. Kushwaha,, and G. D. Sprott. 1982. Lipids of purple membrane from extreme halophiles and of methanogenic bacteria. Methods Enzymol. 88: 98 111.
74. Kiener, A.,, H. König,, J. Winter,, and T. Leisinger. 1987. Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum. J. Bacteriol. 169: 1010 1016.
75. Kobayashi, H.,, J. van Brunt,, and F. M. Harold. 1978. ATP-linked calcium transport in cells and membrane vesicles of Streptococcus faecalis. J. Biol. Chem. 253: 2085 2092.
76. Konig, H.,, and K. O. Stetter. 1986. Studies on archaebacterial S-layers. Syst. Appl. Microbiol. 7: 300 309.
77. Korch, C.,, S. Ovreb,, and K. Kleppe. 1976. Envelopeassociated folded chromosomes from Escherichia coli: variations under different physiological conditions. J. Bacteriol. 127: 904 916.
78. Kotzian, S.,, V. Kreis-Kleinschmidt,, T. Krafft,, O. Klimmek,, J. M. Macy,, and A. Kröger. 1996. Properties of a Wolinella succinogenes mutant lacking periplasmic sulfide dehydrogenase (Sud). Arch. Microbiol. 165: 65 68.
79. Koval, S. F. 1988. Paracrystalline protein surface arrays on bacteria. Can. J. Microbiol. 34: 407 414.
80. Koval, S. F.,, and K. F. Jarrell. 1987. Ultrastructure and biochemistry of the cell wall of Methanococcus voltae. J. Bacteriol. 169: 1298 1306.
81. Koval, S. F.,, and R. G. E. Murray. 1984. The isolation of surface array proteins from bacteria. Can. J. Biochem. Cell Biol. 62: 1181 1189.
82. Kulaev, I. S. 1975. Biochemistry of inorganic polyphosphates. Rev. Physiol. Biochem. Pharmacol. 73: 131 157.
83. Kurland, C. G. 1971. Purification of ribosomes from Escherichia coli. Methods Enzymol. 20: 379 381.
84. Kushwaha, S. C.,, M. Kates,, and W. G. Martin. 1975. Characterization and composition of the purple and red membrane from Halobacterium cutirubrum. Can. J. Microbiol. 53: 284 292.
85. Kushwaha, S. C.,, M. Kates,, and W. Stoeckenius. 1976. Comparison of the purple membrane from Halobacterium cutirubrum and Halobacterium halobium. Biochim. Biophys. Acta 426: 703 710.
86. Laddaga, R. A.,, and R. A. MacLeod. 1982. Effects of wash treatments on the ultrastructure and lysozyme penetrability of the outer membrane of various marine and two terrestrial gram-negative bacteria. Can. J. Microbiol. 28: 318 324.
87. Langworthy, T. A., 1978. Membranes and lipids of extremely thermoacidophilic microorganisms, p. 11 30. In S. M. Friedman (ed.), Biochemistry of Thermophily. Academic Press, Inc., New York, NY.
88. Leive, L. 1965. Release of lipopolysaccharide by EDTA treatment of Escherichia coli. Biochem. Biophys. Res. Commun. 21: 290 296.
89. Lesse, A. J.,, A. A. Campagnari,, W. E. Bittner,, and M. A. Apicella. 1990. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Immunol. Methods 126: 109 117.
90. Li, N.,, and M. C. Cannon. 1998. Gas vesicle genes identified in Bacillus megaterium and functional expression in Escherichia coli. J. Bacteriol. 180: 2450 2458.
91. Lindsay, J. A.,, T. C. Beaman,, and P. Gerhardt. 1985. Protoplast water content of bacterial spores determined by buoyant density sedimentation. J. Bacteriol. 163: 735 737.
92. Lu, W.,, A. P. Wood,, and D. P. Kelly. 1983. An enzymatic lysis procedure for the assay of enzymes in Thiobacillus A2. Microbios 38: 171 176.
93. Lugtenberg, B.,, and L. van Alphen. 1983. Molecular architecture and functioning of the outer membrane of Escherichia coli and other Gram-negative bacteria. Biochim. Biophys. Acta 737: 51 115.
94. MacDonald, R. E.,, and J. K. Lanyi. 1975. Light-induced leucine transport in Halobacterium halobium envelope vesicles: a chemiosmotic study. Biochemistry 14: 2882 2889.
95. MacLeod, R. A. 1985. Marine microbiology far from the sea. Annu. Rev. Microbiol. 39: 1 20.
96. Manoharan, R.,, E. Ghiamati,, R. A. Dalterio,, K. A. Britton,, W. H. Nelson,, and J. F. Sperry. 1990. UV resonance Raman spectra of bacteria, bacterial spores, protoplasts and calcium dipicolinate. J. Microbiol. Methods 11: 1 15.
97. Marvin, H. J. P.,, and B. Witholt. 1987. A highly efficient procedure for the quantitative formation of intact and viable-lysozyme spheroplasts from Escherichia coli. Anal. Biochem. 164: 320 330.
98. Matheson, A. T.,, M. Yaguchi,, W. E. Balch,, and R. S. Wolfe. 1980. Sequence homologies in the N-terminal region of the ribosomal “A” proteins from Methanobacterium thermoautotrophicum and Halobacterium cutirubrum. Biochim. Biophys. Acta 626: 162 169.
99. Mayer, F.,, A. Jussofie,, M. Salzmann,, M. Lübben,, M. Rohde,, and G. Gottschalk. 1987. Immunoelectron microscopic demonstration of ATPase on the cytoplasmic membrane of the methanogenic bacterium strain GÖ1. J. Bacteriol. 169: 2307 2309.
100. Meakin, S. A.,, J. H. E. Nash,, W. D. Murray,, K. J. Kennedy,, and G. D. Sprott. 1991. A generally applicable technique for the extraction of restrictable DNA from methanogenic bacteria. J. Microbiol. Methods 14: 119 126.
101. Messner, P.,, and U. B. Sleytr,. 1988. Separation and purification of S-layers from Gram-positive and Gramnegative bacteria, p. 97 104. In I. Hancock, and I. Poxton (ed.), Bacterial Cell Surface Techniques. John Wiley & Sons Ltd., Toronto, Canada.
102. Miura, T.,, and S. Mizushima. 1968. Separation by density gradient centrifugation of two types of membranes from spheroplast membranes of Escherichia coli K12. Biochim. Biophys. Acta 150: 159 161.
103. Moore, P. B. 1979. The preparation of deuterated ribosomal materials for neutron scattering. Methods Enzymol. 59: 639 655.
104. Morii, H.,, and Y. Koga. 1992. An improved assay for a pseudomurein-degrading enzyme from Methanobacterium wolfei and the protoplast formation of Methanobacterium thermoautotrophicum by the enzyme. J. Ferment. Bioeng. 73: 6 10.
105. Nilles, M. L.,, A. W. Williams,, E. Skrzypek,, and S. C. Straley. 1997. Yersinia pestis LcrV forms a stable complex with LcrG and may have a secretion-related regulatory role in the low-Ca 2+ response. J. Bacteriol. 179: 1307 1316.
106. Nossal, N. G.,, and L. A. Heppel. 1966. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J. Biol. Chem. 241: 3055 3062.
107. Offner, S.,, A. Hofacker,, G. Wanner,, and F. Pfeifer. 2000. Eight of fourteen gvp genes are sufficient for formation of gas vesicles in halophilic archaea. J. Bacteriol. 182: 4328 4336.
108. Offner, S.,, G. Wanner,, and F. Pfeifer. 1996. Functional studies of the gvpACNO operon of Halobacterium salinarium reveal that the GvpC protein shapes gas vesicles. J. Bacteriol. 178: 2071 2078.
109. Osborn, M. J.,, and R. Munson. 1974. Separation of the inner (cytoplasmic) and outer membranes of gram negative bacteria. Methods Enzymol . 31A: 642 653.
110. Paranchych, W.,, P. A. Sastry,, L. S. Frost,, M. Carpenter,, G. D. Armstrong,, and T. H. Watts. 1979. Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO. Can. J. Microbiol. 25: 1175 1181.
111. Penn, C. W.,, A. Cockayne,, and M. J. Bailey. 1985. The outer membrane of Treponema pallidum: biological significance and biochemical properties. J. Gen. Microbiol. 131: 2349 2357.
112. Pertoft, H.,, T. C. Laurent,, R. Seljelid,, G. Akerstrom,, L. Kagedal,, and M. Hirtenstein,. 1979. The use of density gradients of PercollR for the separation of biological particles, p. 67 72. In H. Peeters (ed.), Separation of Cells and Subcellular Elements. Pergamon Press, Toronto, Canada.
113. Peterson, G. L. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346 356.
114. Petrov, V. V.,, V. Y. Artzatbanov,, E. N. Ratner,, A. I. Severin,, and I. S. Kulaev. 1991. Isolation, structural and functional characterization of Staphylococcus aureus protoplasts obtained using lysoamidase. Arch. Microbiol. 155: 549 553.
115. Price, C.A. 1982. Centrifugation in Density Gradients. Academic Press, New York, NY.
116. Rheinberger, H.-J.,, U. Geigenmüller,, M. Wedde,, and K. H. Nierhaus. 1988. Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding. Methods Enzymol. 164: 658 670.
117. Rickwood, D. (ed.). 1984. Centrifugation: a Practical Approach, 2nd ed. IRL Press, Oxford, United Kingdom.
118. Ross, J. W. 1963. Continuous-flow mechanical cell disintegrator. Appl. Microbiol. 11: 33 35.
119. Salton, M. R. J. 1957. The properties of lysozyme and its action on microorganisms. Bacteriol. Rev. 21: 82 99.
120. Salton, M. R. J. 1974. Isolation of cell walls from Grampositive bacteria. Methods Enzymol. 31: 653 667.
121. Sambrook, J.,, E. F. Fritsch,, and T. Maniatis. 1989. Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
122. Scherer, P. A.,, and H.-P. Bochem. 1983. Ultrastructural investigation of 12 Methanosarcinae and related species grown on methanol for occurrence of polyphosphate-like inclusion. Can. J. Microbiol. 29: 1190 1199.
123. Schmidt, K. 1980. A comparative study on the composition of chlorosomes (chlorobium vesicles) and cytoplasmic membranes from Chloroflex aurantiacus strain OK-70- f1 and Chlorobium thiosulfatophilum strain 6230. Arch. Microbiol. 124: 21 31.
124. Schnaitman, C. A. 1970. Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli. J. Bacteriol. 104: 890 901.
125. Schnaitman, C. A. 1971. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J. Bacteriol. 108: 545 552.
126. Schüler, D.,, and R. B. Frankel. 1999. Bacterial magnetosomes: microbiology, biomineralization and biotechnological applications. Appl. Microbiol. Biotechnol. 52: 464 473.
127. Schulman, H.,, and E. P. Kennedy. 1979. Localization of membrane-derived oligosaccharides in the outer envelope of Escherichia coli and their occurrence in other gram-negative bacteria. J. Bacteriol. 137: 686 688.
128. Sharrock, W. J.,, and J. C. Rabinowitz. 1979. Fractionation of ribosomal particles from Bacillus subtilis. Methods Enzymol. 59: 371 382.
129. Siegel, J. L.,, S. F. Hurst,, E. S. Liberman,, S. E. Coleman,, and A. S. Bleiweis. 1981. Mutanolysin-induced spheroplasts of Streptococcus mutans are true protoplasts. Infect. Immun. 31: 808 815.
130. Simon, R. D. 1981. Morphology and protein composition of gas vesicles from wild type and gas vacuole defective strains of Halobacterium salinarium strain 5. J. Gen. Microbiol. 125: 103 111.
131. Sleytr, W. B.,, and P. Messner. 1988. Crystalline surface layers in procaryotes. J. Bacteriol. 170: 2891 2897.
132. Spizizen, J. 1962. Preparation and use of protoplasts. Methods Enzymol. 5: 122 134.
133. Sprott, G. D.,, J. R. Colvin,, and R. C. McKellar. 1979. Spheroplasts of Methanospirillum hungatei formed upon treatment with dithiothreitol. Can. J. Microbiol. 25: 730 738.
134. Sprott, G. D.,, and K. F. Jarrell. 1981. K +, Na +, and Mg + content and permeability of Methanospirillum hungatei and Methanobacterium thermoautotrophicum. Can. J. Microbiol. 27: 444 451.
135. Sprott, G. D.,, K. M. Shaw,, and K. F. Jarrell. 1983. Isolation and chemical composition of the cytoplasmic membrane of the archaebacterium Methanospirillum hungatei. J. Biol. Chem. 258: 4026 4031.
136. Staechelin, T.,, and D. R. Maglott. 1971. Preparation of Escherichia coli ribosomal subunits active in polypeptide synthesis. Methods Enzymol. 20: 449 456.
137. Steinberg, W. 1974. Properties and developmental roles of the lysyl- and tryptophanyl-transfer ribonucleic acid synthetase of Bacillus subtilis: common genetic origin of the corresponding spore and vegetative enzymes. J. Bacteriol. 118: 70 82.
138. Stoeckenius, W.,, and R. Rowen. 1967. A morphological study of Halobacterium halobium and its lysis in media of low salt concentration. J. Cell Biol. 34: 365 393.
139. Stonington, O. G.,, and D. E. Pettijohn. 1971. The folded genome of Escherichia coli isolated in a protein-DNARNA complex. Proc. Natl. Acad. Sci. USA 68: 6 9.
140. Sykes, J., 1971. Centrifugation techniques for the isolation and characterization of sub-cellular components from bacteria, p. 55 207. In J. R. Norris, and D. W. Ribbons (ed.), Methods in Microbiology, vol. 5B. Academic Press, Inc., New York, NY.
141. Thom, J. R.,, and L. L. Randall. 1988. Role of the leader peptide of maltose-binding protein in two steps of the export process. J. Bacteriol. 170: 5654 5661.
142. Thomas, N. A.,, S. L. Bardy,, and K. F. Jarrell. 2001. The archaeal flagellum: a different kind of prokaryotic motility structure. FEMS Microbiol. Lett. 25: 147 174.
143. Thompson, B. G.,, R. G. E. Murray,, and J. F. Boyce. 1982. The association of the surface array and the outer membrane of Deinococcus radiodurans. Can. J. Microbiol. 28: 1081 1088.
144. Tisa, L. S.,, T. Koshikawa,, and P. Gerhardt. 1982. Wet and dry bacterial spore densities determined by buoyant sedimentation. Appl. Environ. Microbiol. 43: 1307 1310.
145. Vainshtein, M. B.,, N. E. Suzina,, E. B. Kudryashova,, E. V. Ariskina,, and V. V. Sorokin. 1998. On the diversity of magnetotactic bacteria. Microbiology 67: 670 676.
146. Vandenbergh, P. A.,, R. E. Bawdon,, and R. S. Berk. 1979. Rapid test for determining the intracellular rhodanese activity of various bacteria. Int. J. Syst. Bacteriol. 29: 339 344.
147. Virgilio, R.,, C. Gonzalez,, N. Muñoz,, T. Cabezon,, and S. Mendoza. 1970. Staphylococcus aureus protoplasting induced by D-cycloserine. J. Bacteriol. 104: 1386 1387.
148. Walker, J. E.,, P. K. Hayes,, and A. E. Walsby. 1984. Homology of gas vesicle proteins in Cyanobacteria and Halobacteria. J. Gen. Microbiol. 130: 2709 2715.
149. Walsby, A. E. 1974. The isolation of gas vesicles from blue-green algae. Methods Enzymol. 31: 678 686.
150. Ward, J. B. 1981. Teichoic and teichuronic acids: biosynthesis, assembly, and location. Microbiol. Rev. 45: 211 243.
151. Warth, A. D. 1978. Relationship between the heat resistance of spores and the optimum and maximum growth temperature of Bacillus species. J. Bacteriol. 134: 699 705.
152. Weil, C. F.,, D. S. Cram,, B. A. Sherf,, and J. N. Reeve. 1988. Structure and comparative analysis of the genes encoding component C of methyl coenzyme M reductase in the extremely thermophilic archaebacterium Methanothermus fervidus. J. Bacteriol. 170: 4718 4726.
153. Westphal, O.,, and K. Jann. 1965. Bacterial lipopolysaccharides. Methods Carbohydr. Chem. 5: 83 91.
154. Wilkinson, S. G.,, L. Galbraith,, and G. A. Lightfoot. 1973. Cell walls, lipids, and lipopolysaccharides of Pseudomonas species. Eur. J. Biochem. 33: 158 174.
155. Worcel, A.,, and E. Burgi. 1972. On the structure of the folded chromosome of Escherichia coli. J. Mol. Biol. 71: 127 147.
156. Work, E., 1971. Cell walls, p. 361 418. In J. R. Norris, and D. W. Ribbons (ed.), Methods in Microbiology, vol. 5A. Academic Press, Inc., New York, NY.
157. Yabu, K.,, and S. Takahashi. 1977. Protoplast formation of selected Mycobacterium smegmatis mutants by lysozyme in combination with methionine. J. Bacteriol. 129: 1628 1631.
158. Yamada, H.,, N. Tsukagoshi,, and S. Udaka. 1981. Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47. J. Bacteriol. 148: 322 332.
159. Yokomaku, D.,, N. Yamaguchi,, and M. Nasu. 2000. Improved direct viable count procedure for quantitative estimation of bacterial viability in freshwater environments. Appl. Environ. Microbiol. 66: 5544 5548.
160. Zulauf, M.,, U. Fürstenberger,, M. Grabo,, P. Jäggi,, M. Regenass,, and J. P. Rosenbusch. 1989. Critical micellar concentrations of detergents. Methods Enzymol. 172: 528 538.


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Some commonly used methods to lyse bacteria and archaea to form osmotically sensitive cells in hypotonic solutions

Lysozyme susceptibility follows growth in medium containing penicillin G.

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7
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Buoyant densities of fractions isolated from prokaryotic cells

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7

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