Abstract
With procedures developed previously in this laboratory, the various layers of the cell wall of a gram-negative bacterium, a marine pseudomonad (ATCC 19855), were removed completely giving rise to true protoplasts. Membranes were isolated from the protoplasts formed. After treatment with ribonuclease, deoxyribonuclease, and washing, the membranes isolated were shown by electron microscopy and chemical analysis to be essentially free from both wall material and cytoplasmic constituents. The membranes gave rise to a single compact band in a sucrose density gradient. All of the lipid and protein were found to be associated in the membrane band. Analysis showed the membranes to contain 30.5% lipid (78% of which was phospholipid), 62.8% protein, and 2% carbohydrate. The predominant phospholipid present was phosphatidylethanolamine with a lesser amount of diphosphatidylglycerol and traces of unidentified compounds.
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Selected References
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- Allen R. J. The estimation of phosphorus. Biochem J. 1940 Jun;34(6):858–865. doi: 10.1042/bj0340858. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bangham A. D., Standish M. M., Weissmann G. The action of steroids and streptolysin S on the permeability of phospholipid structures to cations. J Mol Biol. 1965 Aug;13(1):253–259. doi: 10.1016/s0022-2836(65)80094-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Bodman H., Welker N. E. Isolation of spheroplast membranes and stability of spheroplasts of Bacillus stearothermophilus. J Bacteriol. 1969 Feb;97(2):924–935. doi: 10.1128/jb.97.2.924-935.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- CARSON K. J., EAGON R. G. CELLULAR DISINTEGRATION WITH CONCOMITANT RELEASE OF SLIME AND PRODUCTION OF EXTRACELLULAR MANNAN BY PSEUDOMONAS AERUGINOSA: TWO SEPARATE PHENOMENA. Can J Microbiol. 1964 Jun;10:467–472. doi: 10.1139/m64-056. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- FITZ-JAMES P. FATE OF THE MESOSOMES OF BACILLUS MEGATERIUM DURING PROTOPLASTING. J Bacteriol. 1964 Jun;87:1483–1491. doi: 10.1128/jb.87.6.1483-1491.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Gordon R. C., MacLeod R. A. MG++ phospholipids in cell envelopes of a marine and a terrestrial pseudomonad. Biochem Biophys Res Commun. 1966 Sep 8;24(5):684–690. doi: 10.1016/0006-291x(66)90378-0. [DOI] [PubMed] [Google Scholar]
- HUSTON C. K., ALBRO P. W. LIPIDS OF SARCINA LUTEA. I. FATTY ACID COMPOSITION OF THE EXTRACTABLE LIPIDS. J Bacteriol. 1964 Aug;88:425–432. doi: 10.1128/jb.88.2.425-432.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Kaback H. R., Deuel F. Proline uptake by disrupted membrane preparations from Escherichia coli. Arch Biochem Biophys. 1969 Jun;132(1):118–129. doi: 10.1016/0003-9861(69)90343-9. [DOI] [PubMed] [Google Scholar]
- Kahane I., Razin S. Synthesis and turnover of membrane protein and lipid in Mycoplasma laidlawii. Biochim Biophys Acta. 1969 Jun 3;183(1):79–89. doi: 10.1016/0005-2736(69)90131-x. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- RAZIN S., ARGAMAN M., AVIGAN J. CHEMICAL COMPOSITION OF MYCOPLASMA CELLS AND MEMBRANES. J Gen Microbiol. 1963 Dec;33:477–487. doi: 10.1099/00221287-33-3-477. [DOI] [PubMed] [Google Scholar]
- Razin S., Ne'eman Z., Ohad I. Selective reaggregation of solubilized Mycoplasma-membrane proteins and the kinetics of membrane reformation. Biochim Biophys Acta. 1969;193(2):277–293. doi: 10.1016/0005-2736(69)90189-8. [DOI] [PubMed] [Google Scholar]
- Reaveley D. A., Rogers H. J. Some enzymic activities and chemical properties of the mesosomes and cytoplasmic membranes of Bacillus licheniformis 6346. Biochem J. 1969 Jun;113(1):67–79. doi: 10.1042/bj1130067. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhodes M. E., Payne W. J. Substrate binding by crude membranes and solubilized membrane extracts from Pseudomonas natriegens. Antonie Van Leeuwenhoek. 1968;34(3):298–312. doi: 10.1007/BF02046451. [DOI] [PubMed] [Google Scholar]
- Salton M. R., Freer J. H. Composition of the membranes isolated from several Gram-positive bacteria. Biochim Biophys Acta. 1965 Oct 18;107(3):531–538. doi: 10.1016/0304-4165(65)90197-2. [DOI] [PubMed] [Google Scholar]
- Salton M. R. Structure and function of bacterial cell membranes. Annu Rev Microbiol. 1967;21:417–442. doi: 10.1146/annurev.mi.21.100167.002221. [DOI] [PubMed] [Google Scholar]
- 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]
- WEIBULL C. Characterization of the protoplasmic constituents of bacillus megaterium. J Bacteriol. 1953 Dec;66(6):696–702. doi: 10.1128/jb.66.6.696-702.1953. [DOI] [PMC free article] [PubMed] [Google Scholar]