Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1969 Aug;99(2):576–589. doi: 10.1128/jb.99.2.576-589.1969

Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Biosynthesis of Macromolecules and Wall During Successive Steps1

Otto E Landman a,2, Arnold Forman a
PMCID: PMC250058  PMID: 4980068

Abstract

Protoplasts of Bacillus subtilis plated on SDG medium formed L colonies in quantative yield and propagated in the L-form indefinitely. Protoplasts or L bodies placed in 25% gelatin medium formed bacillary colonies. Details of the reversion of these naked bodies to the walled form are reported here. Protoplasts prepared in minimal medium reverted fairly synchronously 3 to 4 hr after inoculation into gelatin, but protoplasts preincubated in casein hydrolysate (CH)-enriched minimal medium were primed to revert within 1 hr in the gelatin. Preincubation for 1.5 hr in 0.44% CH was required for good priming. Cells must be subjected to this preincubation (step 1) in the naked state; it is effective for L bodies as well as protoplasts. Priming was blocked by chloramphenicol, puromycin, and actinomycin D but was not affected by penicillin, lysozyme, or inhibition of deoxyribonucleic acid (DNA) synthesis. It is concluded that protein and ribonucleic acid (RNA) synthesis are required during step 1, that DNA synthesis is not required, and that wall mucopeptide is not made. The reversion of well-primed protoplasts in the gelatin (step 2) proceeded undisturbed in thymine-starved cells with chromosomes arrested at the terminus. It was scarcely slowed by chloramphenicol in the gelatin but was delayed about 3 hr by both puromycin and actinomycin D. Escape from inhibition occurred while the inhibitors were still actively blocking growth. Penicillin and cycloserine inhibited and lysozyme reversed reversion. Momentary melting of the gelatin delayed reversion. It is concluded that mucopeptide synthesis occurs in step 2, that concomitant RNA, DNA, or protein synthesis is not essential, but that physical immobilization of excreted cell products at the protoplast surface is necessary early in step 2. Newly reverted cells were misshapen and osmotically sensitive. Processes which confer osmotic stability after reversion (step 3) did not occur in the presence of chloramphenicol or actinomycin D.

Full text

PDF
589

Selected References

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

  1. ALTENBERN R. A. Reversion of L forms and spheroplasts of Proteus mirabilis. J Bacteriol. 1963 Feb;85:269–272. doi: 10.1128/jb.85.2.269-272.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anraku N., Landman O. E. Control of the synthesis of macromolecules during amino acid and thymine starvation in Bacillus subtilis. J Bacteriol. 1968 May;95(5):1813–1827. doi: 10.1128/jb.95.5.1813-1827.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bumsted R. M., Dahl J. L., Söll D., Strominger J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. X. Further study of the glycyl transfer ribonucleic acids active in peptidoglycan synthesis in Staphylococcus aureus. J Biol Chem. 1968 Feb 25;243(4):779–782. [PubMed] [Google Scholar]
  4. Clark D. J. Regulation of deoxyribonucleic acid replication and cell division in Escherichia coli B-r. J Bacteriol. 1968 Oct;96(4):1214–1224. doi: 10.1128/jb.96.4.1214-1224.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. HANCOCK R., PARK J. T. Cell-wall synthesis by Staphylococcus aureus in the presence of chloramphenicol. Nature. 1958 Apr 12;181(4615):1050–1052. doi: 10.1038/1811050a0. [DOI] [PubMed] [Google Scholar]
  6. Hash J. H., Davies M. C. Electron Microscopy of Staphylococcus aureus Treated with Tetracycline. Science. 1962 Nov 16;138(3542):828–829. doi: 10.1126/science.138.3542.828. [DOI] [PubMed] [Google Scholar]
  7. Hirota Y., Jacob F. Production de bactéries sans DNA. C R Acad Sci Hebd Seances Acad Sci D. 1966 Nov 21;263(21):1619–1621. [PubMed] [Google Scholar]
  8. Kawakami M., Landman O. E. Retention of episomes during protoplasting and during propagation in the L state. J Bacteriol. 1966 Aug;92(2):398–404. doi: 10.1128/jb.92.2.398-404.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. LANDMAN O. E., HALLE S. ENZYMICALLY AND PHYSICALLY INDUCED INHERITANCE CHANGES IN BACILLUS SUBTILIS. J Mol Biol. 1963 Dec;7:721–738. doi: 10.1016/s0022-2836(63)80119-9. [DOI] [PubMed] [Google Scholar]
  10. LARDY H. A., WELLMAN H. The catalytic effect of 2,4-dinitrophenol on adenosinetriphosphate hydrolysis by cell particles and soluble enzymes. J Biol Chem. 1953 Mar;201(1):357–370. [PubMed] [Google Scholar]
  11. Landman O. E., Ryter A., Fréhel C. Gelatin-induced reversion of protoplasts of Bacillus subtilis to the bacillary form: electron-microscopic and physical study. J Bacteriol. 1968 Dec;96(6):2154–2170. doi: 10.1128/jb.96.6.2154-2170.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lark K. G. Regulation of chromosome replication and segregation in bacteria. Bacteriol Rev. 1966 Mar;30(1):3–32. doi: 10.1128/br.30.1.3-32.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. NECAS O. Physical conditions as important factors for the regeneration of naked yeast protoplasts. Nature. 1961 Nov 11;192:580–581. doi: 10.1038/192580a0. [DOI] [PubMed] [Google Scholar]
  14. ROSENKRANZ H. S., BENDICH A. J. STUDIES ON THE BACTERIOSTATIC ACTION OF HYDROXYLAMINE. Biochim Biophys Acta. 1964 May 18;87:40–53. doi: 10.1016/0926-6550(64)90045-3. [DOI] [PubMed] [Google Scholar]
  15. RYTER A., LANDMAN O. E. ELECTRON MICROSCOPE STUDY OF THE RELATIONSHIP BETWEEN MESOSOME LOSS AND THE STABLE L STATE (OR PROTOPLAST STATE) IN BACILLUS SUBTILIS. J Bacteriol. 1964 Aug;88:457–467. doi: 10.1128/jb.88.2.457-467.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ryter A. Relationship between synthesis of the cytoplasmic membrane and nuclear segregation in Bacillus subtilis. Folia Microbiol (Praha) 1967;12(3):283–290. doi: 10.1007/BF02868745. [DOI] [PubMed] [Google Scholar]
  17. Shockman G. D. Symposium on the fine structure and replication of bacteria and their parts. IV. Unbalanced cell-wall synthesis: autolysis and cell-wall thickening. Bacteriol Rev. 1965 Sep;29(3):345–358. doi: 10.1128/br.29.3.345-358.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES