Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1980 Feb;33(2):780–788. doi: 10.1128/jvi.33.2.780-788.1980

Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

M A DeWyngaert, D C Hinkle
PMCID: PMC288604  PMID: 6997508

Abstract

The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein.

Full text

PDF
780

Images in this article

785Image
on p.785

Selected References

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

  1. BURGI E., HERSHEY A. D. Sedimentation rate as a measure of molecular weight of DNA. Biophys J. 1963 Jul;3:309–321. doi: 10.1016/s0006-3495(63)86823-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernstein C., Bernstein H. Coiled rings of DNA released from cells infected with bacteriophages T7 or T4 or from uninfected Escherichia coli. J Virol. 1974 Jun;13(6):1346–1355. doi: 10.1128/jvi.13.6.1346-1355.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Center M. S. Replicative intermediates of bacteriophage T7 deoxyribonucleic acid. J Virol. 1972 Jul;10(1):115–123. doi: 10.1128/jvi.10.1.115-123.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Center M. S. Role of gene 2 in bacteriophage T7 DNA synthesis. J Virol. 1975 Jul;16(1):94–100. doi: 10.1128/jvi.16.1.94-100.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chamberlin M. Isolation and characterization of prototrophic mutants of Escherichia coli unable to support the intracellular growth of T7. J Virol. 1974 Sep;14(3):509–516. doi: 10.1128/jvi.14.3.509-516.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeWyngaert M. A., Hinkle D. C. Bacterial mutants affecting phage T7 DNA replication produce RNA polymerase resistant to inhibition by the T7 gene 2 protein. J Biol Chem. 1979 Nov 25;254(22):11247–11253. [PubMed] [Google Scholar]
  7. Fröhlich B., Powling A., Knippers R. Formation of concatemeric DNA in bacteriophage T7-infected bacteria. Virology. 1975 Jun;65(2):455–468. doi: 10.1016/0042-6822(75)90051-3. [DOI] [PubMed] [Google Scholar]
  8. Hesselbach B. A., Nakada D. "Host shutoff" function of bacteriophage T7: involvement of T7 gene 2 and gene 0.7 in the inactivation of Escherichia coli RNA polymerase. J Virol. 1977 Dec;24(3):736–745. doi: 10.1128/jvi.24.3.736-745.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hesselbach B. A., Nakada D. I protein: bacteriophage T7-coded inhibitor of Escherichia coli RNA polymerase. J Virol. 1977 Dec;24(3):746–760. doi: 10.1128/jvi.24.3.746-760.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hiebsch R., Center M. S. Intracellular organization of bacteriophage T7 DNA: analysis of parenteral bacteriophage T7 DNA-membrane and DNA-protein complexes. J Virol. 1977 May;22(2):540–547. doi: 10.1128/jvi.22.2.540-547.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hinkle D. C., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Requirements for deoxyribonucleic acid synthesis and characterization of the product. J Biol Chem. 1974 May 10;249(9):2974–2980. [PubMed] [Google Scholar]
  12. Kelly T. J., Jr, Thomas C. A., Jr An intermediate in the replication of bacteriophage T7 DNA molecules. J Mol Biol. 1969 Sep 28;44(3):459–475. doi: 10.1016/0022-2836(69)90373-8. [DOI] [PubMed] [Google Scholar]
  13. Langman L., Paetkau V., Scraba D., Miller R. C., Jr, Roeder G. S., Sadowski P. D. The structure and maturation of intermediates in bacteriophage T7 DNA replication. Can J Biochem. 1978 Jun;56(6):508–516. doi: 10.1139/o78-078. [DOI] [PubMed] [Google Scholar]
  14. LeClerc J. E., Richardson C. C. Gene 2 protein of bacteriophage T7: purification and requirement for packaging of T7 DNA in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4852–4856. doi: 10.1073/pnas.76.10.4852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mark D. F., Richardson C. C. Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1976 Mar;73(3):780–784. doi: 10.1073/pnas.73.3.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McAllister W. T., Barrett C. L. Roles of the early genes of bacteriophage T7 in shutoff of host macromolecular synthesis. J Virol. 1977 Sep;23(3):543–553. doi: 10.1128/jvi.23.3.543-553.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McConaughy B. L., Laird C. D., McCarthy B. J. Nucleic acid reassociation in formamide. Biochemistry. 1969 Aug;8(8):3289–3295. doi: 10.1021/bi00836a024. [DOI] [PubMed] [Google Scholar]
  18. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  19. Modrich P., Richardson C. C. Bacteriophage T7 Deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity. J Biol Chem. 1975 Jul 25;250(14):5508–5514. [PubMed] [Google Scholar]
  20. Modrich P., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits. J Biol Chem. 1975 Jul 25;250(14):5515–5522. [PubMed] [Google Scholar]
  21. Paetkau V., Langman L., Bradley R., Scraba D., Miller R. C., Jr Folded, concatenated genomes as replication intermediates of bacteriophage T7 DNA. J Virol. 1977 Apr;22(1):130–141. doi: 10.1128/jvi.22.1.130-141.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ponta H., Rahmsdorf H. J., Pai S. H., Hirsch-Kauffmann M., Herrlich P., Schweiger M. Control of gene expression in bacteriophage T7: transcriptional controls. Mol Gen Genet. 1974;134(4):281–297. doi: 10.1007/BF00337463. [DOI] [PubMed] [Google Scholar]
  23. Roeder G. S., Sadowski P. D. Bacteriophage T7 morphogenesis: phage-related particles in cells infected with wild-type and mutant T7 phage. Virology. 1977 Jan;76(1):263–285. doi: 10.1016/0042-6822(77)90302-6. [DOI] [PubMed] [Google Scholar]
  24. Schlegel R. A., Thomas C. A., Jr Some special structural features of intracellular bacteriophage T7 concatemers. J Mol Biol. 1972 Jul 21;68(2):319–345. doi: 10.1016/0022-2836(72)90216-1. [DOI] [PubMed] [Google Scholar]
  25. Serwer P. Fast sedimenting bacteriophage T7 DNA from T7-infected Escherichia coli. Virology. 1974 May;59(1):70–88. doi: 10.1016/0042-6822(74)90207-4. [DOI] [PubMed] [Google Scholar]
  26. Strätling W., Krause E., Knippers R. Fast sedimenting deoxyribonucleic acid in bacteriophage T7-infected cells. Virology. 1973 Jan;51(1):109–119. doi: 10.1016/0042-6822(73)90371-1. [DOI] [PubMed] [Google Scholar]
  27. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  28. Studier F. W. Genetic analysis of non-essential bacteriophage T7 genes. J Mol Biol. 1973 Sep 15;79(2):227–236. doi: 10.1016/0022-2836(73)90002-8. [DOI] [PubMed] [Google Scholar]
  29. Studier F. W. The genetics and physiology of bacteriophage T7. Virology. 1969 Nov;39(3):562–574. doi: 10.1016/0042-6822(69)90104-4. [DOI] [PubMed] [Google Scholar]
  30. Yamada Y., Silnutzer J., Nakada D. Mutant of Escherichia coli which blocks T7 bacteriophage assembly: accumulation of short T7 DNA. J Mol Biol. 1978 May 5;121(1):95–111. doi: 10.1016/0022-2836(78)90264-4. [DOI] [PubMed] [Google Scholar]
  31. Zillig W., Fujiki H., Blum W., Janeković D., Schweiger M., Rahmsdorf H., Ponta H., Hirsch-Kauffmann M. In vivo and in vitro phosphorylation of DNA-dependent RNA polymerase of Escherichia coli by bacteriophage-T7-induced protein kinase. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2506–2510. doi: 10.1073/pnas.72.7.2506. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES