Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1980 Dec;144(3):957–966. doi: 10.1128/jb.144.3.957-966.1980

Genetic transformation with cell wall-associated deoxyribonucleic acid in Bacillus subtilis.

R J Doyle, U N Streips, S Imada, V S Fan, W C Brown
PMCID: PMC294758  PMID: 6777372

Abstract

Cell walls from bacillus subtilis 168 were prepared by conventional methods and found to contain deoxyribonucleic acid (DNA). In transformation assays, after autolysis, it was found that two major regions of the chromosome were selectively enriched in the wall preparations. One region clustered around the replication origin and is represented by the markers purA16, ts8132, thiC5, sacA321, and hisA1. The other region included the replication terminus with representative loci metB10, citK5, gltA292, and pyrA1. All other (internal) loci which were examined showed no statistical enrichment. The two areas of enrichment were similar to but more extensive than those reported for membrane-DNA complexes. The wall preparations also contained protein and lipid, indicating a possible membrane involvement. Analyses of the cell walls revealed that the fatty acid composition of the membrane component was not typical of the for B. subtilis protoplast membranes or for lipoteichoic acids. In addition, radioiodination of cell wall autolysates, followed by gel electrophoresis and autoradiography, demonstrated the presence of proteins not readily detectable in bulk protoplast membranes or on the surfaces of intact cells. These data suggest that a unique component of the membrane and regions of the B. subtilis genome involved in DNA replication events are tightly associated with cell walls. The binding of DNA-membrane complexes to the "rigid" cell wall and the replication of the wall could be a mechanism by which the segregation of growing chromosomes occurs.

Full text

PDF
962

Images in this article

963Image
on p.963

Selected References

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

  1. Abraham G. N., Scaletta C., Vaughan J. H. Modified diphenylamine reaction for increased sensitivity. Anal Biochem. 1972 Oct;49(2):547–549. doi: 10.1016/0003-2697(72)90460-5. [DOI] [PubMed] [Google Scholar]
  2. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  3. Bishop D. G., Rutberg L., Samuelsson B. The chemical composition of the cytoplasmic membrane of Bacillus subtilis. Eur J Biochem. 1967 Nov;2(4):448–453. doi: 10.1111/j.1432-1033.1967.tb00158.x. [DOI] [PubMed] [Google Scholar]
  4. Boyd A., Holland I. B. Protein d, an iron-transport protein induced by filtration of cultures of Escherichia coli. FEBS Lett. 1977 Apr 1;76(1):20–24. doi: 10.1016/0014-5793(77)80112-9. [DOI] [PubMed] [Google Scholar]
  5. Boyd A., Holland I. B. Regulation of the synthesis of surface protein in the cell cycle of E. coli B/r. Cell. 1979 Oct;18(2):287–296. doi: 10.1016/0092-8674(79)90048-5. [DOI] [PubMed] [Google Scholar]
  6. Boylan R. J., Mendelson N. H., Brooks D., Young F. E. Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid. J Bacteriol. 1972 Apr;110(1):281–290. doi: 10.1128/jb.110.1.281-290.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Braun V. Covalent lipoprotein from the outer membrane of Escherichia coli. Biochim Biophys Acta. 1975 Oct 31;415(3):335–377. doi: 10.1016/0304-4157(75)90013-1. [DOI] [PubMed] [Google Scholar]
  8. Brown W. C., Doyle R. J., Streips U. N. Comparison of various procedures for removing proteins and nucleic acids from cell walls of Bacillus subtilis. Prep Biochem. 1976;6(6):479–488. doi: 10.1080/00327487608069130. [DOI] [PubMed] [Google Scholar]
  9. Brown W. C., Fraser D. K., Young F. E. Problems in purification of a Bacillus subtilis autolytic enzyme caused by association with teichoic acid. Biochim Biophys Acta. 1970 Feb 11;198(2):308–315. doi: 10.1016/0005-2744(70)90063-x. [DOI] [PubMed] [Google Scholar]
  10. Burdett I. D., Higgins M. L. Study of pole assembly in Bacillus subtilis by computer reconstruction of septal growth zones seen in central, longitudinal thin sections of cells. J Bacteriol. 1978 Feb;133(2):959–971. doi: 10.1128/jb.133.2.959-971.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crabb W. D., Streips U. N., Doyle R. J. Selective enrichment for genetic markers in DNA released by competent cultures of Bacillus subtilis. Mol Gen Genet. 1977 Oct 20;155(2):179–183. doi: 10.1007/BF00393157. [DOI] [PubMed] [Google Scholar]
  12. Doyle R. J., Streips U. N., Fan V. S., Brown W. C., Mobley H., Mansfield J. M. Cell wall protein in Bacillus subtilis. J Bacteriol. 1977 Jan;129(1):547–549. doi: 10.1128/jb.129.1.547-549.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dworsky P. Membrane attachment of folded chromosome of Escherichia coli. Biochem J. 1976 Jan 15;154(1):239–241. doi: 10.1042/bj1540239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fan D. P., Beckman M. M. New centrifugation technique for isolating enzymes from large cell structures: isolation and characterization of two Bacillus subtilis autolysins. J Bacteriol. 1972 Mar;109(3):1258–1265. doi: 10.1128/jb.109.3.1258-1265.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fan D. P., Pelvit M. C., Cunningham W. P. Structural difference between walls from ends and sides of the rod-shaped bacterium Bacillus subtilis. J Bacteriol. 1972 Mar;109(3):1266–1272. doi: 10.1128/jb.109.3.1266-1272.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Firshein W. The DNA-membrane fraction of Pneumococcus contains a DNA replication complex. J Mol Biol. 1972 Oct 14;70(3):383–397. doi: 10.1016/0022-2836(72)90547-5. [DOI] [PubMed] [Google Scholar]
  17. Ghosh B. K. The mesosome--a clue to the evolution of the plasma membrane. Subcell Biochem. 1974 Dec;3(4):311–367. [PubMed] [Google Scholar]
  18. Glauert A. M., Thornley M. J. The topography of the bacterial cell wall. Annu Rev Microbiol. 1969;23:159–198. doi: 10.1146/annurev.mi.23.100169.001111. [DOI] [PubMed] [Google Scholar]
  19. Gudas L. J., James R., Paradee A. B. Evidence of the involvement of an outer membrane protein in DNA initiation. J Biol Chem. 1976 Jun 10;251(11):3470–3479. [PubMed] [Google Scholar]
  20. Gudas L. J., Pardee A. B. Deoxyribonucleic acid synthesis during the division cycle of Escherichia coli: a comparison of strains B-r, K-12, 15, and 15T- under conditions of slow growth. J Bacteriol. 1974 Mar;117(3):1216–1223. doi: 10.1128/jb.117.3.1216-1223.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Harford N. Bidirectional chromosome replication in Bacillus subtilis 168. J Bacteriol. 1975 Mar;121(3):835–847. doi: 10.1128/jb.121.3.835-847.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heidrich H. G., Olsen W. L. Deoxyribonucleic acid-envelope complexes from Escherichia coli. A complex-specific protein and its possible function for the stability of the complex. J Cell Biol. 1975 Nov;67(2PT1):444–460. doi: 10.1083/jcb.67.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Horowitz S., Doyle R. J., Young F. E., Streips U. N. Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis. J Bacteriol. 1979 Jun;138(3):915–922. doi: 10.1128/jb.138.3.915-922.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Leibowitz P. J., Schaechter M. The attachment of the bacterial chromosome to the cell membrane. Int Rev Cytol. 1975;41:1–28. doi: 10.1016/s0074-7696(08)60964-x. [DOI] [PubMed] [Google Scholar]
  26. Lepesant-Kejzlarová J., Lepesant J. A., Walle J., Billault A., Dedonder R. Revision of the linkage map of Bacillus subtilis 168: indications for circularity of the chromosome. J Bacteriol. 1975 Mar;121(3):823–834. doi: 10.1128/jb.121.3.823-834.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mauck J., Glaser L. On the mode of in vivo assembly of the cell wall of Bacillus subtilis. J Biol Chem. 1972 Feb 25;247(4):1180–1187. [PubMed] [Google Scholar]
  28. Mendelson N. H., Haag S. M., Cole R. M. Cellular organization of Bacillus subtilis: sodium dodecyl sulfate-induced cell partitioning into zebra structures. J Bacteriol. 1976 Jun;126(3):1285–1296. doi: 10.1128/jb.126.3.1285-1296.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nanninga N. The mesosome of Bacillus subtilis as affected by chemical and physical fixation. J Cell Biol. 1971 Jan;48(1):219–224. doi: 10.1083/jcb.48.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nester E W, Schafer M, Lederberg J. Gene Linkage in DNA Transfer: A Cluster of Genes Concerned with Aromatic Biosynthesis in Bacillus Subtilis. Genetics. 1963 Apr;48(4):529–551. doi: 10.1093/genetics/48.4.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Olsen W. L., Heidrich H. G., Hannig K., Hofschneider P. H. Deoxyribonucleic acid-envelope complexes isolated from Escherichia coli by free-flow electrophoresis: biochemical and electron microscope characterization. J Bacteriol. 1974 May;118(2):646–653. doi: 10.1128/jb.118.2.646-653.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schwarz U., Ryter A., Rambach A., Hellio R., Hirota Y. Process of cellular division in Escherichia coli: differention of growth zones in the Sacculus. J Mol Biol. 1975 Nov 15;98(4):749–759. doi: 10.1016/s0022-2836(75)80008-8. [DOI] [PubMed] [Google Scholar]
  33. Sjöquist J., Movitz J., Johansson I. B., Hjelm H. Localization of protein A in the bacteria. Eur J Biochem. 1972 Oct 17;30(1):190–194. doi: 10.1111/j.1432-1033.1972.tb02086.x. [DOI] [PubMed] [Google Scholar]
  34. Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. doi: 10.1073/pnas.44.10.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Streips U. N., Young F. E. Transformation in Bacillus subtilis using excreted DNA. Mol Gen Genet. 1974;133(1):47–55. doi: 10.1007/BF00268676. [DOI] [PubMed] [Google Scholar]
  36. Sueoka N., Hammers J. M. Isolation of DNA-membrane complex in Bacillus subtilis. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4787–4791. doi: 10.1073/pnas.71.12.4787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Sueoka N., Yoshikawa H. The chromosome of Bacillus subtilis. I. Theory of marker frequency analysis. Genetics. 1965 Oct;52(4):747–757. doi: 10.1093/genetics/52.4.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Toyoda H., Yamaguchi K., Yoshikawa H. Chromosome-membrane association in Bacillus subtilis. IV. Further purification of DNA-membrane complex by using a combination of centrifugation and electrophoresis. J Bacteriol. 1978 Nov;136(2):799–802. doi: 10.1128/jb.136.2.799-802.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Uehara H., Yoneda Y., Yamane K., Maruo B. Regulation of neutral protease productivity in Bacillus subtilis: transformation of high protease productivity. J Bacteriol. 1974 Jul;119(1):82–91. doi: 10.1128/jb.119.1.82-91.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Worcel A., Burgi E. Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J Mol Biol. 1974 Jan 5;82(1):91–105. doi: 10.1016/0022-2836(74)90576-2. [DOI] [PubMed] [Google Scholar]
  42. Yamaguchi K., Yoshikawa H. Association of the replication terminus of the Bacillus subtilis chromosome to the cell membrane. J Bacteriol. 1975 Nov;124(2):1030–1033. doi: 10.1128/jb.124.2.1030-1033.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yamaguchi K., Yoshikawa H. Topography of chromosome membrane junction in Bacillus subtilis. Nat New Biol. 1973 Aug 15;244(137):204–206. doi: 10.1038/newbio244204a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES