Abstract
Spheroplast production by lysozyme and ethylenediaminetetraacetate (EDTA) was examined as a means of obtaining osmotically sensitive cells for studies of enzyme localization. Physiologically young cells plasmolyzed with 0.5 m sucrose in 0.01 m tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7, 8, or 9) were quantitatively converted to plasmolyzed osmotically sensitive rods after lysozyme treatment. Although such cells were osmotically sensitive, a 1:1 dilution in Tris buffer was necessary for conversion of rods into spheroplasts. Addition of EDTA resulted in a rapid conversion of the plasmolyzed spheroplasts into spherical structures devoid of a plasmolysis vacuole. These structures, which we call EDTA-lysozyme spheroplasts, contained a number of attached membranes. We believe that this conversion results from a weakening of the outer trilaminar component of the cell wall by EDTA, resulting in the collapse of the plasmolysis vacuole. Dilution of sucrose below 0.15 m also resulted in the collapse of the plasmolysis vacuole. Both the lysozyme spheroplasts and the EDTA-lysozyme spheroplasts were osmotically sensitive. Thin sections of the EDTA-lysozyme spheroplasts demonstrated that the outer trilaminar component of the cell wall was broken, exposing large areas of the cytoplasmic membrane to the environment.
Full text
PDF![427](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/de6884e55a03/jbacter00590-0463.png)
![428](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/fdaa6b3304e3/jbacter00590-0464.png)
![429](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/269da5e4b5de/jbacter00590-0465.png)
![430](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/f40dd61c4854/jbacter00590-0466.png)
![431](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/3d7233ae5a1d/jbacter00590-0467.png)
![432](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/72ff4625a599/jbacter00590-0468.png)
![433](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/af718b99e011/jbacter00590-0469.png)
![434](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/64b6da3306c0/jbacter00590-0470.png)
![435](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/3cb41e367bb4/jbacter00590-0471.png)
![436](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/e6a9ad9dc0fc/jbacter00590-0472.png)
![437](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7fe4/315014/71550bc0bee6/jbacter00590-0473.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Asbell M. A., Eagon R. G. The role of multivalent cations in the organization and structure of bacterial cell walls. Biochem Biophys Res Commun. 1966 Mar 22;22(6):664–671. doi: 10.1016/0006-291x(66)90198-7. [DOI] [PubMed] [Google Scholar]
- COTA-ROBLES E. H., DUNCAN P. H. The effect of D-glutamic acid upon spheroplast formation in Escherichia coli B. Exp Cell Res. 1962 Nov;28:342–349. doi: 10.1016/0014-4827(62)90288-4. [DOI] [PubMed] [Google Scholar]
- Carson K. J., Eagon R. G. Lysozyme sensitivity of the cell wall of Pseudomonas aeruginosa. Further evidence for the role of the non-peptidoglycan components in cell wall rigidity. Can J Microbiol. 1966 Feb;12(1):105–108. doi: 10.1139/m66-015. [DOI] [PubMed] [Google Scholar]
- Cordonnier C., Bernardi G. Localization of E. coli endonuclease I. Biochem Biophys Res Commun. 1965 Sep 8;20(5):555–559. doi: 10.1016/0006-291x(65)90434-1. [DOI] [PubMed] [Google Scholar]
- DRESDEN M., HOAGLAND M. B. POLYRIBOSOMES FROM ESCHERICHIA COLI: ENZYMATIC METHOD FOR ISOLATION. Science. 1965 Aug 6;149(3684):647–649. doi: 10.1126/science.149.3684.647. [DOI] [PubMed] [Google Scholar]
- FRASER D., JERREL E. A. The amino acid composition of T3 bacteriophage. J Biol Chem. 1953 Nov;205(1):291–295. [PubMed] [Google Scholar]
- GUTHRIE G. D., SINSHEIMER R. L. Observations on the infection of bacterial protoplasts with the deoxyribonucleic acid of bacteriophage phi X174. Biochim Biophys Acta. 1963 Jun 25;72:290–297. [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]
- LEDERBERG J., ST CLAIR J. Protoplasts and L-type growth of Escherichia coli. J Bacteriol. 1958 Feb;75(2):143–160. doi: 10.1128/jb.75.2.143-160.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leive L. Release of lipopolysaccharide by EDTA treatment of E. coli. Biochem Biophys Res Commun. 1965 Nov 22;21(4):290–296. doi: 10.1016/0006-291x(65)90191-9. [DOI] [PubMed] [Google Scholar]
- MALAMY M. H., HORECKER B. L. PURIFICATION AND CRYSTALLIZATION OF THE ALKALINE PHOSPHATASE OF ESCHERICHIA COLI. Biochemistry. 1964 Dec;3:1893–1897. doi: 10.1021/bi00900a018. [DOI] [PubMed] [Google Scholar]
- MALAMY M. H., HORECKER B. L. RELEASE OF ALKALINE PHOSPHATASE FROM CELLS OF ESCHERICHIA COLI UPON LYSOZYME SPHEROPLAST FORMATION. Biochemistry. 1964 Dec;3:1889–1893. doi: 10.1021/bi00900a017. [DOI] [PubMed] [Google Scholar]
- MEYER F., MACKAL R. P., TAO M., EVANS E. A., Jr Infectious deoxyribonucleic acid from gamma bacteriophage. J Biol Chem. 1961 Apr;236:1141–1143. [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]
- NEU H. C., HEPPEL L. A. THE RELEASE OF RIBONUCLEASE INTO THE MEDIUM WHEN ESCHERICHIA COLI CELLS ARE CONVERTED TO SPEROPLASTS. J Biol Chem. 1964 Nov;239:3893–3900. [PubMed] [Google Scholar]
- NOLLER E. C., HARTSELL S. E. Bacteriolysis of Enterobacteriaceae. I. Lysis by four lytic systems utilizing lysozyme. J Bacteriol. 1961 Mar;81:482–491. doi: 10.1128/jb.81.3.482-491.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neu H. C., Heppel L. A. On the surface localization of enzymes in E. coli. Biochem Biophys Res Commun. 1964 Oct 14;17(3):215–219. doi: 10.1016/0006-291x(64)90386-9. [DOI] [PubMed] [Google Scholar]
- Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
- Protass J. J., Korn D. Impairment of Temperate Bacteriophage Adsorption by Brief Treatment of Escherichia coli with Dilute Solutions of Ethylenediaminetetraacetate. J Bacteriol. 1966 Jan;91(1):143–147. doi: 10.1128/jb.91.1.143-147.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ryter A., Jacob F. Etude morphologique de la liaison du noyau à la membrane chez E. coli et chez les protoplastes de B. subtilis. Ann Inst Pasteur (Paris) 1966 Jun;110(6):801–812. [PubMed] [Google Scholar]
- SISTROM W. R. On the physical state of the intracellularly accumulates substrates of beta-galactoside-permease in Escherichia coli. Biochim Biophys Acta. 1958 Sep;29(3):579–587. doi: 10.1016/0006-3002(58)90015-5. [DOI] [PubMed] [Google Scholar]
- VENABLE J. H., COGGESHALL R. A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY. J Cell Biol. 1965 May;25:407–408. doi: 10.1083/jcb.25.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- VOSS J. G. LYSOZYME LYSIS OF GRAM-NEGATIVE BACTERIA WITHOUT PRODUCTION OF SPHEROPLASTS. J Gen Microbiol. 1964 May;35:313–317. doi: 10.1099/00221287-35-2-313. [DOI] [PubMed] [Google Scholar]