Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jul;177(13):3743–3751. doi: 10.1128/jb.177.13.3743-3751.1995

Purification and characterization of the bacteriophage P4 delta protein.

B Julien 1, R Calendar 1
PMCID: PMC177091  PMID: 7601839

Abstract

The bacteriophage P4 delta protein is a transcriptional activator of the late genes of P4 as well as the late genes of its helpers, such as bacteriophage P2. delta was purified, using a variation of the MalE fusion system. With this method we purified two forms of delta: a fusion of MalE and delta and a unfused form. The fusion by itself is not active in vivo or in vitro, but the mixture of the fusion and the unfused delta is active in both. Using nitrocellulose filtration and gel mobility shift assays, we show that delta binds DNA, and using DNase I footprinting, we show that delta binds to sequences centered at approximately -55 in the two late promoters of P4 as well as the four late promoters of its helper P2. In addition, the P4 sid promoter contains a second delta binding site centered at -18.

Full Text

The Full Text of this article is available as a PDF (547.0 KB).

Selected References

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

  1. Artz S. W., Broach J. R. Histidine regulation in Salmonella typhimurium: an activator attenuator model of gene regulation. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3453–3457. doi: 10.1073/pnas.72.9.3453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ayers D. J., Sunshine M. G., Six E. W., Christie G. E. Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein. J Bacteriol. 1994 Dec;176(24):7430–7438. doi: 10.1128/jb.176.24.7430-7438.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birkeland N. K., Lindquist B. H. Coliphage P2 late control gene ogr. DNA sequence and product identification. J Mol Biol. 1986 Apr 5;188(3):487–490. doi: 10.1016/0022-2836(86)90170-1. [DOI] [PubMed] [Google Scholar]
  4. Bochner B. R., Huang H. C., Schieven G. L., Ames B. N. Positive selection for loss of tetracycline resistance. J Bacteriol. 1980 Aug;143(2):926–933. doi: 10.1128/jb.143.2.926-933.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christie G. E., Calendar R. Bacteriophage P2 late promoters. II. Comparison of the four late promoter sequences. J Mol Biol. 1985 Feb 5;181(3):373–382. doi: 10.1016/0022-2836(85)90226-8. [DOI] [PubMed] [Google Scholar]
  6. Christie G. E., Calendar R. Bacteriophage P2 late promoters. Transcription initiation sites for two late mRNAs. J Mol Biol. 1983 Jul 15;167(4):773–790. doi: 10.1016/s0022-2836(83)80110-7. [DOI] [PubMed] [Google Scholar]
  7. Christie G. E., Calendar R. Interactions between satellite bacteriophage P4 and its helpers. Annu Rev Genet. 1990;24:465–490. doi: 10.1146/annurev.ge.24.120190.002341. [DOI] [PubMed] [Google Scholar]
  8. Christie G. E., Haggård-Ljungquist E., Feiwell R., Calendar R. Regulation of bacteriophage P2 late-gene expression: the ogr gene. Proc Natl Acad Sci U S A. 1986 May;83(10):3238–3242. doi: 10.1073/pnas.83.10.3238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collado-Vides J., Magasanik B., Gralla J. D. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. doi: 10.1128/mr.55.3.371-394.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dale E. C., Christie G. E., Calendar R. Organization and expression of the satellite bacteriophage P4 late gene cluster. J Mol Biol. 1986 Dec 20;192(4):793–803. doi: 10.1016/0022-2836(86)90029-x. [DOI] [PubMed] [Google Scholar]
  11. Dehó G., Zangrossi S., Ghisotti D., Sironi G. Alternative promoters in the development of bacteriophage plasmid P4. J Virol. 1988 May;62(5):1697–1704. doi: 10.1128/jvi.62.5.1697-1704.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dibbens J. A., Egan J. B. Control of gene expression in the temperate coliphage 186. IX. B is the sole phage function needed to activate transcription of the phage late genes. Mol Microbiol. 1992 Sep;6(18):2629–2642. doi: 10.1111/j.1365-2958.1992.tb01440.x. [DOI] [PubMed] [Google Scholar]
  13. Dunbar B. S., Kimura H., Timmons T. M. Protein analysis using high-resolution two-dimensional polyacrylamide gel electrophoresis. Methods Enzymol. 1990;182:441–459. doi: 10.1016/0076-6879(90)82036-2. [DOI] [PubMed] [Google Scholar]
  14. Eaton D., Rodriguez H., Vehar G. A. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry. 1986 Jan 28;25(2):505–512. doi: 10.1021/bi00350a035. [DOI] [PubMed] [Google Scholar]
  15. Ghisotti D., Finkel S., Halling C., Dehò G., Sironi G., Calendar R. Nonessential region of bacteriophage P4: DNA sequence, transcription, gene products, and functions. J Virol. 1990 Jan;64(1):24–36. doi: 10.1128/jvi.64.1.24-36.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grambow N. J., Birkeland N. K., Anders D. L., Christie G. E. Deletion analysis of a bacteriophage P2 late promoter. Gene. 1990 Oct 30;95(1):9–15. doi: 10.1016/0378-1119(90)90407-i. [DOI] [PubMed] [Google Scholar]
  17. Halling C., Calendar R. Bacteriophage P2 ogr and P4 delta genes act independently and are essential for P4 multiplication. J Bacteriol. 1990 Jul;172(7):3549–3558. doi: 10.1128/jb.172.7.3549-3558.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Halling C., Sunshine M. G., Lane K. B., Six E. W., Calendar R. A mutation of the transactivation gene of satellite bacteriophage P4 that suppresses the rpoA109 mutation of Escherichia coli. J Bacteriol. 1990 Jul;172(7):3541–3548. doi: 10.1128/jb.172.7.3541-3548.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ishihama A. Protein-protein communication within the transcription apparatus. J Bacteriol. 1993 May;175(9):2483–2489. doi: 10.1128/jb.175.9.2483-2489.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kahn M. L., Ziermann R., Dehò G., Ow D. W., Sunshine M. G., Calendar R. Bacteriophage P2 and P4. Methods Enzymol. 1991;204:264–280. doi: 10.1016/0076-6879(91)04013-e. [DOI] [PubMed] [Google Scholar]
  21. Keener J., Dale E. C., Kustu S., Calendar R. In vitro transcription from the late promoter of bacteriophage P4. J Bacteriol. 1988 Aug;170(8):3543–3546. doi: 10.1128/jb.170.8.3543-3546.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kumar A., Grimes B., Fujita N., Makino K., Malloch R. A., Hayward R. S., Ishihama A. Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation. J Mol Biol. 1994 Jan 14;235(2):405–413. doi: 10.1006/jmbi.1994.1001. [DOI] [PubMed] [Google Scholar]
  23. Lee H. S., Berger D. K., Kustu S. Activity of purified NIFA, a transcriptional activator of nitrogen fixation genes. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2266–2270. doi: 10.1073/pnas.90.6.2266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee T. C., Christie G. E. Purification and properties of the bacteriophage P2 ogr gene product. A prokaryotic zinc-binding transcriptional activator. J Biol Chem. 1990 May 5;265(13):7472–7477. [PubMed] [Google Scholar]
  25. Li M., Moyle H., Susskind M. M. Target of the transcriptional activation function of phage lambda cI protein. Science. 1994 Jan 7;263(5143):75–77. doi: 10.1126/science.8272867. [DOI] [PubMed] [Google Scholar]
  26. Linderoth N. A., Julien B., Flick K. E., Calendar R., Christie G. E. Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion. Virology. 1994 May 1;200(2):347–359. doi: 10.1006/viro.1994.1199. [DOI] [PubMed] [Google Scholar]
  27. Lindqvist B. H., Dehò G., Calendar R. Mechanisms of genome propagation and helper exploitation by satellite phage P4. Microbiol Rev. 1993 Sep;57(3):683–702. doi: 10.1128/mr.57.3.683-702.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lindqvist B. H., Six E. W. Replication of bacteriophage P4 DNA in a nonlysogenic host. Virology. 1971 Jan;43(1):1–7. doi: 10.1016/0042-6822(71)90218-2. [DOI] [PubMed] [Google Scholar]
  29. Lisser S., Margalit H. Compilation of E. coli mRNA promoter sequences. Nucleic Acids Res. 1993 Apr 11;21(7):1507–1516. doi: 10.1093/nar/21.7.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Makino K., Amemura M., Kim S. K., Nakata A., Shinagawa H. Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli. Genes Dev. 1993 Jan;7(1):149–160. doi: 10.1101/gad.7.1.149. [DOI] [PubMed] [Google Scholar]
  31. Nagai K., Perutz M. F., Poyart C. Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7252–7255. doi: 10.1073/pnas.82.21.7252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Quinlan R. A., Moir R. D., Stewart M. Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation. J Cell Sci. 1989 May;93(Pt 1):71–83. doi: 10.1242/jcs.93.1.71. [DOI] [PubMed] [Google Scholar]
  34. Sasaki I., Bertani G. Growth abnormalities in Hfr derivatives of Escherichia coli strain C. J Gen Microbiol. 1965 Sep;40(3):365–376. doi: 10.1099/00221287-40-3-365. [DOI] [PubMed] [Google Scholar]
  35. Schweizer H., Boos W. Transfer of the delta (argF-lac)U169 mutation between Escherichia coli strains by selection for a closely linked Tn10 insertion. Mol Gen Genet. 1983;192(1-2):293–294. doi: 10.1007/BF00327683. [DOI] [PubMed] [Google Scholar]
  36. Sedmak J. J., Grossberg S. E. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem. 1977 May 1;79(1-2):544–552. doi: 10.1016/0003-2697(77)90428-6. [DOI] [PubMed] [Google Scholar]
  37. Siegele D. A., Hu J. C., Walter W. A., Gross C. A. Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J Mol Biol. 1989 Apr 20;206(4):591–603. doi: 10.1016/0022-2836(89)90568-8. [DOI] [PubMed] [Google Scholar]
  38. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  39. Srivastava S. K., Cannistraro V. J., Kennell D. Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli. J Bacteriol. 1992 Jan;174(1):56–62. doi: 10.1128/jb.174.1.56-62.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sternberg N. L., Maurer R. Bacteriophage-mediated generalized transduction in Escherichia coli and Salmonella typhimurium. Methods Enzymol. 1991;204:18–43. doi: 10.1016/0076-6879(91)04004-8. [DOI] [PubMed] [Google Scholar]
  41. Sunshine M. G., Sauer B. A bacterial mutation blocking P2 phage late gene expression. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2770–2774. doi: 10.1073/pnas.72.7.2770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Van Bokkelen G. B., Dale E. C., Halling C., Calendar R. Mutational analysis of a bacteriophage P4 late promoter. J Bacteriol. 1991 Jan;173(1):37–45. doi: 10.1128/jb.173.1.37-45.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  45. Waldburger C., Gardella T., Wong R., Susskind M. M. Changes in conserved region 2 of Escherichia coli sigma 70 affecting promoter recognition. J Mol Biol. 1990 Sep 20;215(2):267–276. doi: 10.1016/s0022-2836(05)80345-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES