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. 1970 Dec;104(3):1338–1353. doi: 10.1128/jb.104.3.1338-1353.1970

Separation and Localization of Cell Wall Layers of a Gram-Negative Bacterium

C W Forsberg a,1, J W Costerton a,2, Robert A MacLeod a
PMCID: PMC248297  PMID: 16559113

Abstract

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na+, K+, and Mg++ at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 × g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.

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Selected References

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

  1. BIRCH-ANDERSEN A., MAALØE O., SJOSTRAND F. S. High-resolution electron micrographs of sections of E. coli. Biochim Biophys Acta. 1953 Nov;12(3):395–400. doi: 10.1016/0006-3002(53)90157-7. [DOI] [PubMed] [Google Scholar]
  2. BLADEN H. A., MERGENHAGEN S. E. ULTRASTRUCTURE OF VEILLONELLA AND MORPHOLOGICAL CORRELATION OF AN OUTER MEMBRANE WITH PARTICLES ASSOCIATED WITH ENDOTOXIC ACTIVITY. J Bacteriol. 1964 Nov;88:1482–1492. doi: 10.1128/jb.88.5.1482-1492.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benson J. V., Jr, Patterson J. A. Accelerated chromatographic analysis of amino acids commonly found in physiological fluids on a spherical resin of specific design. Anal Biochem. 1965 Nov;13(2):265–280. doi: 10.1016/0003-2697(65)90196-x. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Buckmire F. L., MacLeod R. A. Nutrition and metabolism of marine bacteria. XIV. On the mechanism of lysis of a marine bacterium. Can J Microbiol. 1965 Aug;11(4):677–691. doi: 10.1139/m65-091. [DOI] [PubMed] [Google Scholar]
  6. Buckmire F. L., MacLeod R. A. Penetrability of a marine pseudomonad by inulin, sucrose, and glycerol and its relation to the mechanism of lysis. Can J Microbiol. 1970 Feb;16(2):75–81. doi: 10.1139/m70-014. [DOI] [PubMed] [Google Scholar]
  7. CESSI C., PILIEGO F. The determination of amino sugars in the presence of amino acids and glucose. Biochem J. 1960 Dec;77:508–510. doi: 10.1042/bj0770508. [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. DEPETRIS S. ULTRASTRUCTURE OF THE CELL WALL OF ESCHERICHIA COLI. J Ultrastruct Res. 1965 Apr;12:247–262. doi: 10.1016/s0022-5320(65)80098-3. [DOI] [PubMed] [Google Scholar]
  10. De Petris S. Ultrastructure of the cell wall of Escherichia coli and chemical nature of its constituent layers. J Ultrastruct Res. 1967 Jul;19(1):45–83. doi: 10.1016/s0022-5320(67)80059-5. [DOI] [PubMed] [Google Scholar]
  11. De Voe I. W., Oginsky E. L. Antagonistic effect of monovalent cations in maintenance of cellular integrity of a marine bacterium. J Bacteriol. 1969 Jun;98(3):1355–1367. doi: 10.1128/jb.98.3.1355-1367.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Voe I. W., Thompson J., Costerton J. W., MacLeod R. A. Stability and comparative transport capacity of cells, mureinoplasts, and true protoplasts of a gram-negative bacterium. J Bacteriol. 1970 Mar;101(3):1014–1026. doi: 10.1128/jb.101.3.1014-1026.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Forsberg C. W., Costerton J. W., Macleod R. A. Quantitation, chemical characteristics, and ultrastructure of the three outer cell wall layers of a gram-negative bacterium. J Bacteriol. 1970 Dec;104(3):1354–1368. doi: 10.1128/jb.104.3.1354-1368.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hofschneider P. H., Martin H. H. Diversity of surface layers in L-forms of Proteus mirabilis. J Gen Microbiol. 1968 Apr;51(1):23–33. doi: 10.1099/00221287-51-1-23. [DOI] [PubMed] [Google Scholar]
  15. Imaeda T., Kanetsuna F., Galindo B. Ultrastructure of cell walls of genus Mycobacterium. J Ultrastruct Res. 1968 Oct;25(1):46–63. doi: 10.1016/s0022-5320(68)80059-0. [DOI] [PubMed] [Google Scholar]
  16. KELLENBERGER E., RYTER A. Cell wall and cytoplasmic membrane of Escherichia coli. J Biophys Biochem Cytol. 1958 May 25;4(3):323–326. doi: 10.1083/jcb.4.3.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. MURRAY R. G., STEED P., ELSON H. E. THE LOCATION OF THE MUCOPEPTIDE IN SECTIONS OF THE CELL WALL OF ESCHERICHIA COLI AND OTHER GRAM-NEGATIVE BACTERIA. Can J Microbiol. 1965 Jun;11:547–560. doi: 10.1139/m65-072. [DOI] [PubMed] [Google Scholar]
  19. Martin H. H. Bacterial protoplasts--a review. J Theor Biol. 1963 Jul;5(1):1–34. doi: 10.1016/0022-5193(63)90034-1. [DOI] [PubMed] [Google Scholar]
  20. Pate J. L., Ordal E. J. The fine structure of Chondrococcus columnaris. 3. The surface layers of Chondrococcus columnaris. J Cell Biol. 1967 Oct;35(1):37–51. doi: 10.1083/jcb.35.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. RYTER A., KELLENBERGER E. L'inclusion au polyester pour l'ultramicrotomie. J Ultrastruct Res. 1958 Dec;2(2):200–214. doi: 10.1016/s0022-5320(58)90018-2. [DOI] [PubMed] [Google Scholar]
  23. SCHRAM E., MOORE S., BIGWOOD E. J. Chromatographic determination of cystine as cysteic acid. Biochem J. 1954 May;57(1):33–37. doi: 10.1042/bj0570033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thompson J., Costerton J. W., MacLeon R. A. K plus-dependent deplasmolysis of a marine pseudomonad plasmolyzed in a hypotonic solution. J Bacteriol. 1970 Jun;102(3):843–854. doi: 10.1128/jb.102.3.843-854.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Thornley M. J., Glauert A. M. Fine structure and radiation resistance in Acinetobacter: studies on a resistant strain. J Cell Sci. 1968 Jun;3(2):273–294. doi: 10.1242/jcs.3.2.273. [DOI] [PubMed] [Google Scholar]
  26. WEIDEL W., FRANK H., MARTIN H. H. The rigid layer of the cell wall of Escherichia coli strain B. J Gen Microbiol. 1960 Feb;22:158–166. doi: 10.1099/00221287-22-1-158. [DOI] [PubMed] [Google Scholar]
  27. Watson S. W., Remsen C. C. Macromolecular subunits in the walls of marine nitrifying bacteria. Science. 1969 Feb 14;163(3868):685–686. doi: 10.1126/science.163.3868.685. [DOI] [PubMed] [Google Scholar]

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