Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Oct;178(19):5573–5578. doi: 10.1128/jb.178.19.5573-5578.1996

Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis.

A L Douglas 1, T P Hatch 1
PMCID: PMC178393  PMID: 8824599

Abstract

On the basis of position from the transcription start site, the P2 promoter of the gene encoding the major outer membrane protein (ompA) of Chlamydia trachomatis consists of a -35 hexamer region of -42 aaaaaga TATACAaa -28 and an unusual, GC-rich -10 hexamer region of -13 tTATCGCt -6. The P2 promoter was analyzed by in vitro transcription of templates containing deletions and site-specific mutations. The 5' extent of P2 was located at bp -42. Replacement of wild-type sequence with two G's at positions -41 and 40, -35 and 34, and -29 and 28 resulted in severely decreased transcription. Additionally, the spacing between the -35 and -10 hexamers could not be shortened without adversely affecting in vitro activity. Substitution of G at position -13, -10, -7, or -6 had little or no effect on transcription, whereas substitution of G at -11 or -12 significantly decreased promoter strength. Triple point mutations which changed the -10 hexamer from TATCGC to TATTAT,TATATT, or TATAAT had little effect on promoter activity. Unlike the partially purified C. trachomatis sigma66-RNA polymerase used in this study, purified Escherichia coli sigma70-RNA polymerase did not recognize the wild-type P2 promoter. Mutant P2 templates with -10 hexamers that resembled the consensus recognition site were transcribed by E. coli holoenzyme in vitro, suggesting that C. trachomatis sigma66-RNA polymerase has special promoter recognition properties not found in E. coli sigma70-holoenzyme.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Busby S., Ebright R. H. Promoter structure, promoter recognition, and transcription activation in prokaryotes. Cell. 1994 Dec 2;79(5):743–746. doi: 10.1016/0092-8674(94)90063-9. [DOI] [PubMed] [Google Scholar]
  2. Crenshaw R. W., Fahr M. J., Wichlan D. G., Hatch T. P. Developmental cycle-specific host-free RNA synthesis in Chlamydia spp. Infect Immun. 1990 Oct;58(10):3194–3201. doi: 10.1128/iai.58.10.3194-3201.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Douglas A. L., Hatch T. P. Functional analysis of the major outer membrane protein gene promoters of Chlamydia trachomatis. J Bacteriol. 1995 Nov;177(21):6286–6289. doi: 10.1128/jb.177.21.6286-6289.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Douglas A. L., Saxena N. K., Hatch T. P. Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC. J Bacteriol. 1994 May;176(10):3033–3039. doi: 10.1128/jb.176.10.3033-3039.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Engel J. N., Ganem D. A polymerase chain reaction-based approach to cloning sigma factors from eubacteria and its application to the isolation of a sigma-70 homolog from Chlamydia trachomatis. J Bacteriol. 1990 May;172(5):2447–2455. doi: 10.1128/jb.172.5.2447-2455.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Everett K. D., Andersen A. A., Plaunt M., Hatch T. P. Cloning and sequence analysis of the major outer membrane protein gene of Chlamydia psittaci 6BC. Infect Immun. 1991 Aug;59(8):2853–2855. doi: 10.1128/iai.59.8.2853-2855.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fahr M. J., Douglas A. L., Xia W., Hatch T. P. Characterization of late gene promoters of Chlamydia trachomatis. J Bacteriol. 1995 Aug;177(15):4252–4260. doi: 10.1128/jb.177.15.4252-4260.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gu L., Wenman W. M., Remacha M., Meuser R., Coffin J., Kaul R. Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis. J Bacteriol. 1995 May;177(9):2594–2601. doi: 10.1128/jb.177.9.2594-2601.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Ishihama A. Role of the RNA polymerase alpha subunit in transcription activation. Mol Microbiol. 1992 Nov;6(22):3283–3288. doi: 10.1111/j.1365-2958.1992.tb02196.x. [DOI] [PubMed] [Google Scholar]
  12. Koehler J. E., Burgess R. R., Thompson N. E., Stephens R. S. Chlamydia trachomatis RNA polymerase major sigma subunit. Sequence and structural comparison of conserved and unique regions with Escherichia coli sigma 70 and Bacillus subtilis sigma 43. J Biol Chem. 1990 Aug 5;265(22):13206–13214. [PubMed] [Google Scholar]
  13. 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]
  14. Lonetto M., Gribskov M., Gross C. A. The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol. 1992 Jun;174(12):3843–3849. doi: 10.1128/jb.174.12.3843-3849.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Malakooti J., Ely B. Principal sigma subunit of the Caulobacter crescentus RNA polymerase. J Bacteriol. 1995 Dec;177(23):6854–6860. doi: 10.1128/jb.177.23.6854-6860.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Malakooti J., Wang S. P., Ely B. A consensus promoter sequence for Caulobacter crescentus genes involved in biosynthetic and housekeeping functions. J Bacteriol. 1995 Aug;177(15):4372–4376. doi: 10.1128/jb.177.15.4372-4376.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mathews S. A., Douglas A., Sriprakash K. S., Hatch T. P. In vitro transcription in Chlamydia psittaci and Chlamydia trachomatis. Mol Microbiol. 1993 Mar;7(6):937–946. doi: 10.1111/j.1365-2958.1993.tb01185.x. [DOI] [PubMed] [Google Scholar]
  18. Mathews S. A., Sriprakash K. S. The RNA polymerase of Chlamydia trachomatis has a flexible sequence requirement at the -10 and -35 boxes of its promoters. J Bacteriol. 1994 Jun;176(12):3785–3789. doi: 10.1128/jb.176.12.3785-3789.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nickerson C. A., Achberger E. C. Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters. J Bacteriol. 1995 Oct;177(20):5756–5761. doi: 10.1128/jb.177.20.5756-5761.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Perez Melgosa M., Kuo C. C., Campbell L. A. Sequence analysis of the major outer membrane protein gene of Chlamydia pneumoniae. Infect Immun. 1991 Jun;59(6):2195–2199. doi: 10.1128/iai.59.6.2195-2199.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stephens R. S., Wagar E. A., Edman U. Developmental regulation of tandem promoters for the major outer membrane protein gene of Chlamydia trachomatis. J Bacteriol. 1988 Feb;170(2):744–750. doi: 10.1128/jb.170.2.744-750.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yuan Y., Zhang Y. X., Manning D. S., Caldwell H. D. Multiple tandem promoters of the major outer membrane protein gene (omp1) of Chlamydia psittaci. Infect Immun. 1990 Sep;58(9):2850–2855. doi: 10.1128/iai.58.9.2850-2855.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES