Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1972 Apr 1;53(1):1–23. doi: 10.1083/jcb.53.1.1

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS

IV. Chemical and Cytological Characterization and Biosynthetic Capabilities of Fragments Obtained by Mild Procedures

R Scharff 1, R W Hendler 1, N Nanninga 1, A H Burgess 1
PMCID: PMC2108700  PMID: 4335249

Abstract

Membrane-envelope fragments have been isolated from Escherichia coli by comparatively mild techniques. The use of DNAase, RNAase, detergents, sonication, lysozyme, and ethylenediaminetetraacetate were avoided in the belief that rather delicate, but metabolically important, associations may exist between the plasma membrane and various cytoplasmic components. The membrane-envelope fragments have been characterized in terms of their content of major chemical components as well as their electron microscope appearance. Fractions containing membrane-envelope fragments were found to possess appreciable DNA- and protein-synthesizing activities. The fragments were rich in membrane content as determined by reduced nicotinamide adenine dinucleotide (NADH) oxidase activity and deficient in soluble components as measured by NADH dehydrogenase activity. The particulate fraction obtained between 20,000 g and 105,000 g and usually considered a ribosomal fraction was rich in membrane content and had a relatively high capacity for DNA synthesis. Envelope fragments sedimenting at 20,000 g attained very high levels of incorporation of amino acids into protein.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

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

  1. AMES B. N., DUBIN D. T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem. 1960 Mar;235:769–775. [PubMed] [Google Scholar]
  2. AMINOFF D. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J. 1961 Nov;81:384–392. doi: 10.1042/bj0810384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aronson A. Membrane-bound messenger RNA and polysomes in sporulating bacteria. J Mol Biol. 1965 Aug;13(1):92–104. doi: 10.1016/s0022-2836(65)80082-1. [DOI] [PubMed] [Google Scholar]
  4. BOLLUM F. J. Thermal conversion of nonpriming deoxyribonucleic acid to primer. J Biol Chem. 1959 Oct;234:2733–2734. [PubMed] [Google Scholar]
  5. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barondes S. H., Nirenberg M. W. Fate of a Synthetic Polynucleotide Directing Cell-Free Protein Synthesis I. Characteristics of Degradation. Science. 1962 Nov 16;138(3542):810–813. doi: 10.1126/science.138.3542.810. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Costerton J. W., Forsberg C., Matula T. I., Buckmire F. L., MacLeod R. A. Nutrition and metabolism of marine bacteria. XVI. Formation of protoplasts, spheroplasts, and related forms from a gram-negative marine bacterium. J Bacteriol. 1967 Nov;94(5):1764–1777. doi: 10.1128/jb.94.5.1764-1777.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De Lucia P., Cairns J. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature. 1969 Dec 20;224(5225):1164–1166. doi: 10.1038/2241164a0. [DOI] [PubMed] [Google Scholar]
  10. ELBEIN A. D., HEATH E. C. THE BIOSYNTHESIS OF CELL WALL LIPOPOLYSACCHARIDE IN ESCHERICHIA COLI. I. THE BIOCHEMICAL PROPERTIES OF A URIDINE DIPHOSPHATE GALACTOSE 4-EPIMERASELESS MUTANT. J Biol Chem. 1965 May;240:1919–1925. [PubMed] [Google Scholar]
  11. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  12. Fuchs E., Hanawalt P. Isolation and characterization of the DNA replication complex from Escherichia coli. J Mol Biol. 1970 Sep 14;52(2):301–322. doi: 10.1016/0022-2836(70)90032-x. [DOI] [PubMed] [Google Scholar]
  13. GILVARG C. The enzymatic synthesis of diaminopimelic acid. J Biol Chem. 1958 Dec;233(6):1501–1504. [PubMed] [Google Scholar]
  14. Ganesan A. T. Studies on in vitro replication of Bacillus subtilis DNA. Cold Spring Harb Symp Quant Biol. 1968;33:45–57. doi: 10.1101/sqb.1968.033.01.010. [DOI] [PubMed] [Google Scholar]
  15. Ganesan A. T. Studies on the in vitro synthesis of transforming DNA. Proc Natl Acad Sci U S A. 1968 Nov;61(3):1058–1065. doi: 10.1073/pnas.61.3.1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. HENDLER R. W., BANFIELD W. G., TANI J., KUFFEL ON THE CYTOLOGICAL UNIT FOR PROTEIN SYNTHESIS IN VIVO IN E. COLI. III. ELECTRON MICROSCOPIC AND ULTRACENTRIFUGAL EXAMINATION OF INTACT CELLS AND FRACTIONS. Biochim Biophys Acta. 1964 Feb 17;80:307–314. [PubMed] [Google Scholar]
  17. HENDLER R. W. Some properties and the possible metabolic significance of amino acid-lipid complexes. Biochim Biophys Acta. 1961 May 13;49:297–307. doi: 10.1016/0006-3002(61)90129-9. [DOI] [PubMed] [Google Scholar]
  18. HENDLER R. W. Studies on the nature of the amino acid incorporation process of hen oviduct tissue. J Biol Chem. 1956 Dec;223(2):831–842. [PubMed] [Google Scholar]
  19. HENDLER R. W., TANI J. ON THE CYTOLOGICAL UNIT FOR PROTEIN SYNTHESIS IN VIVO IN E. COLI. II. STUDIES WITH INTACT CELLS OF TYPE B. Biochim Biophys Acta. 1964 Feb 17;80:294–306. doi: 10.1016/0926-6550(64)90101-x. [DOI] [PubMed] [Google Scholar]
  20. Hendler R. W., Burgess A. H., Scharff R. Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. I. Oxidative activities with soluble substrates. J Cell Biol. 1969 Sep;42(3):715–732. doi: 10.1083/jcb.42.3.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hendler R. W., Nanninga N. Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. 3. Electron microscopy and analysis of the cytochromes. J Cell Biol. 1970 Jul;46(1):114–129. doi: 10.1083/jcb.46.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jacob F., Ryter A., Cuzin F. On the association between DNA and membrane in bacteria. Proc R Soc Lond B Biol Sci. 1966 Mar 22;164(995):267–278. doi: 10.1098/rspb.1966.0029. [DOI] [PubMed] [Google Scholar]
  23. KELLENBERGER E., RYTER A., SECHAUD J. Electron microscope study of DNA-containing plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J Biophys Biochem Cytol. 1958 Nov 25;4(6):671–678. doi: 10.1083/jcb.4.6.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaback H. R., Stadtman E. R. Proline uptake by an isolated cytoplasmic membrane preparation of Escherichia coli. Proc Natl Acad Sci U S A. 1966 Apr;55(4):920–927. doi: 10.1073/pnas.55.4.920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kaback H. R. The role of the phosphoenolpyruvate-phosphotransferase system in the transport of sugars by isolated membrane preparations of Escherichia coli. J Biol Chem. 1968 Jul 10;243(13):3711–3724. [PubMed] [Google Scholar]
  26. Klenow H., Henningsen I. Effect of monovalent cations on the activity of the DNA polymerase of Escherichia coli B. Eur J Biochem. 1969 May 1;9(1):133–141. doi: 10.1111/j.1432-1033.1969.tb00586.x. [DOI] [PubMed] [Google Scholar]
  27. Knippers R., Strätling W. The DNA replicating capacity of isolated E. coli cell wall-membrane complexes. Nature. 1970 May 23;226(5247):713–717. doi: 10.1038/226713a0. [DOI] [PubMed] [Google Scholar]
  28. LENGYEL P., SPEYER J. F., OCHOA S. Synthetic polynucleotides and the amino acid code. Proc Natl Acad Sci U S A. 1961 Dec 15;47:1936–1942. doi: 10.1073/pnas.47.12.1936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Leibowitz M. J., Soffer R. L. A soluble enzyme from Escherichia coli which catalyzes the transfer of leucine and phenylalanine from tRNA to acceptor proteins. Biochem Biophys Res Commun. 1969 Jul 7;36(1):47–53. doi: 10.1016/0006-291x(69)90647-0. [DOI] [PubMed] [Google Scholar]
  31. Leibowitz M. J., Soffer R. L. Enzymatic modification of proteins. 3. Purification and properties of a leucyl, phenylalanyl transfer ribonucleic acid protein transferase from Escherichia coli. J Biol Chem. 1970 Apr 25;245(8):2066–2073. [PubMed] [Google Scholar]
  32. MANS R. J., NOVELLI G. D. A convenient, rapid and sensitive method for measuring the incorporation of radioactive amino acids into protein. Biochem Biophys Res Commun. 1960 Nov;3:540–543. doi: 10.1016/0006-291x(60)90171-6. [DOI] [PubMed] [Google Scholar]
  33. MATSUBARA K., WATANABE I. Studies of amino acid incorporation with purified ribosomes and soluble enzymes from Escherichia coli. Biochem Biophys Res Commun. 1961 May 15;5:22–26. doi: 10.1016/0006-291x(61)90073-0. [DOI] [PubMed] [Google Scholar]
  34. Miura T., Mizushima S. Separation and properties of outer and cytoplasmic membranes in Escherichia coli. Biochim Biophys Acta. 1969;193(2):268–276. doi: 10.1016/0005-2736(69)90188-6. [DOI] [PubMed] [Google Scholar]
  35. Miura T., Mizushima S. Separation by density gradient centrifugation of two types of membranes from spheroplast membrane of Escherichia coli K12. Biochim Biophys Acta. 1968 Jan 3;150(1):159–161. doi: 10.1016/0005-2736(68)90020-5. [DOI] [PubMed] [Google Scholar]
  36. Nagata Y., Shibuya I., Maruo B. Preparation and properties of an active membrane system from Escherichia coli. J Biochem. 1967 May;61(5):623–632. doi: 10.1093/oxfordjournals.jbchem.a128592. [DOI] [PubMed] [Google Scholar]
  37. Nanninga N. Ultrastructure of the cell envelope of Escherichia coli B after freeze-etching. J Bacteriol. 1970 Jan;101(1):297–303. doi: 10.1128/jb.101.1.297-303.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Osborn M. J. Structure and biosynthesis of the bacterial cell wall. Annu Rev Biochem. 1969;38:501–538. doi: 10.1146/annurev.bi.38.070169.002441. [DOI] [PubMed] [Google Scholar]
  39. Quigley J. W., Cohen S. S. The isolation of envelopes of Escherichia coli spheroplasts in the zonal ultracentrifuge. J Biol Chem. 1969 May 10;244(9):2450–2458. [PubMed] [Google Scholar]
  40. RAPPORT M. M., ALONZO N. Photometric determination of fatty acid ester groups in phospholipides. J Biol Chem. 1955 Nov;217(1):193–198. [PubMed] [Google Scholar]
  41. Rothfield L., Pearlman-Kothencz M. Synthesis and assembly of bacterial membrane components. A lipopolysaccharide-phospholipid-protein complex excreted by living bacteria. J Mol Biol. 1969 Sep 28;44(3):477–492. doi: 10.1016/0022-2836(69)90374-x. [DOI] [PubMed] [Google Scholar]
  42. Rouvière J., Lederberg S., Granboulan P., Gros F. Structural sites of RNA synthesis in Escherichia coli. J Mol Biol. 1969 Dec 28;46(3):413–430. doi: 10.1016/0022-2836(69)90185-5. [DOI] [PubMed] [Google Scholar]
  43. SUIT J. C. Ribonucleic acid in a "membrane" fraction of Escherichia coli and its relation to cell-wall synthesis. J Bacteriol. 1962 Nov;84:1061–1070. doi: 10.1128/jb.84.5.1061-1070.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Smith D. W., Hanawalt P. C. Properties of the growing point region in the bacterial chromosome. Biochim Biophys Acta. 1967 Dec 19;149(2):519–531. doi: 10.1016/0005-2787(67)90180-3. [DOI] [PubMed] [Google Scholar]
  46. Sueoka N., Quinn W. G. Membrane attachment of the chromosome replication origin in Bacillus subtilis. Cold Spring Harb Symp Quant Biol. 1968;33:695–705. doi: 10.1101/sqb.1968.033.01.078. [DOI] [PubMed] [Google Scholar]
  47. TANI J., HENDLER R. W. ON THE CYTOLOGICAL UNIT FOR PROTEIN SYNTHESIS IN VIVO IN E. COLI. I. STUDIES WITH SPHEROPLASTS OF TYPE K-12. Biochim Biophys Acta. 1964 Feb 17;80:279–293. doi: 10.1016/0926-6550(64)90100-8. [DOI] [PubMed] [Google Scholar]
  48. Tissieres A., Schlessinger D., Gros F. AMINO ACID INCORPORATION INTO PROTEINS BY ESCHERICHIA COLI RIBOSOMES. Proc Natl Acad Sci U S A. 1960 Nov;46(11):1450–1463. doi: 10.1073/pnas.46.11.1450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tonomura B., Rabinowitz J. C. An investigation of the induction of beta-galactosidase in a broken spheroplast preparation of Escherichia coli. J Mol Biol. 1967 Mar 14;24(2):177–202. doi: 10.1016/0022-2836(67)90325-7. [DOI] [PubMed] [Google Scholar]
  50. Tremblay G. Y., Daniels M. J., Schaechter M. Isolation of a cell membrane-DNA-nascent RNA complex from bacteria. J Mol Biol. 1969 Feb 28;40(1):65–76. doi: 10.1016/0022-2836(69)90296-4. [DOI] [PubMed] [Google Scholar]
  51. WORK E., DENMAN R. F. The use of a bacterial culture fluid as a source of alpha-diaminopimelic acid. Biochim Biophys Acta. 1953 Jan;10(1):183–183. doi: 10.1016/0006-3002(53)90226-1. [DOI] [PubMed] [Google Scholar]
  52. YUDKIN M. D., DAVIS B. NATURE OF THE RNA ASSOCIATED WITH THE PROTOPLAST MEMBRANE OF BACILLUS MEGATERIUM. J Mol Biol. 1965 May;12:193–204. doi: 10.1016/s0022-2836(65)80293-5. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES