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. 1994 May;176(10):3033–3039. doi: 10.1128/jb.176.10.3033-3039.1994

Enhancement of in vitro transcription by addition of cloned, overexpressed major sigma factor of Chlamydia psittaci 6BC.

A L Douglas 1, N K Saxena 1, T P Hatch 1
PMCID: PMC205461  PMID: 8188604

Abstract

Obligate parasitic bacteria of the genus Chlamydia possess a developmental cycle that takes place entirely within eucaryotic host cells. Because standard methods of genetic analysis are not available for chlamydiae, an in vitro transcription system has been developed to elucidate the mechanisms by which chlamydiae regulate gene expression. The in vitro system is specific for chlamydial promoters but is inefficient, presumably because the RNA polymerase is not saturated with sigma factor. Therefore, we prepared recombinant Chlamydia psittaci 6BC major sigma factor to enhance transcription in the in vitro system. The gene encoding the major sigma factor (sigA) was identified by using an rpoD box oligonucleotide and was subsequently cloned and sequenced. It was found to encode a potential 571-amino-acid protein (sigma 66) that is greater than 90% identical to the previously identified major sigma factors from the L2 and MoPn strains of Chlamydia trachomatis. sigA was recloned into a T7 RNA polymerase expression system to produce large quantities of sigma 66 in Escherichia coli. Overexpressed sigma 66 was identified by immunoblot by using monoclonal antibodies 2G10 (reactive) and 2F8 (nonreactive) generated against E. coli sigma 70. After purification by polyacrylamide gel electrophoresis, the recombinant protein was found to stimulate, by 10-fold or more, promoter-specific in vitro transcription by C. psittaci 6BC and C. trachomatis L2 RNA polymerases. Transcription was dependent on added chlamydial sigma 66, rather than on potentially contaminating E. coli sigma 70 or other fortuitous activators, since the monoclonal antibody 2G10, and not 2F8, inhibited transcription initiation. Recombinant omega(66) had no effect on transcription by E. coli core polymerase. The addition of recombinant omega(66) to the in vitro system should be useful for distinguishing omega(66)-dependent transcription of developmentally regulated chlamydial genes from omega(66)-independent transcription.

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Selected References

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

  1. Bavoil P., Ohlin A., Schachter J. Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infect Immun. 1984 May;44(2):479–485. doi: 10.1128/iai.44.2.479-485.1984. [DOI] [PMC free article] [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. 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]
  4. Engel J. N., Ganem D. Chlamydial rRNA operons: gene organization and identification of putative tandem promoters. J Bacteriol. 1987 Dec;169(12):5678–5685. doi: 10.1128/jb.169.12.5678-5685.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Erickson J. W., Gross C. A. Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression. Genes Dev. 1989 Sep;3(9):1462–1471. doi: 10.1101/gad.3.9.1462. [DOI] [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. Everett K. D., Hatch T. P. Sequence analysis and lipid modification of the cysteine-rich envelope proteins of Chlamydia psittaci 6BC. J Bacteriol. 1991 Jun;173(12):3821–3830. doi: 10.1128/jb.173.12.3821-3830.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fahr M. J., Sriprakash K. S., Hatch T. P. Convergent and overlapping transcripts of the Chlamydia trachomatis 7.5-kb plasmid. Plasmid. 1992 Nov;28(3):247–257. doi: 10.1016/0147-619x(92)90056-g. [DOI] [PubMed] [Google Scholar]
  9. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  10. Hatch T. P., Allan I., Pearce J. H. Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of Chlamydia spp. J Bacteriol. 1984 Jan;157(1):13–20. doi: 10.1128/jb.157.1.13-20.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hatch T. P., Vance D. W., Jr, Al-Hossainy E. Identification of a major envelope protein in Chlamydia spp. J Bacteriol. 1981 Apr;146(1):426–429. doi: 10.1128/jb.146.1.426-429.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  13. Igarashi K., Ishihama A. Bipartite functional map of the E. coli RNA polymerase alpha subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell. 1991 Jun 14;65(6):1015–1022. doi: 10.1016/0092-8674(91)90553-b. [DOI] [PubMed] [Google Scholar]
  14. Jovanovich S. B., Lesley S. A., Burgess R. R. In vitro use of monoclonal antibodies in Escherichia coli S-30 extracts to determine the RNA polymerase sigma subunit required by a promoter. J Biol Chem. 1989 Mar 5;264(7):3794–3798. [PubMed] [Google Scholar]
  15. 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]
  16. LaBell T. L., Trempy J. E., Haldenwang W. G. Sporulation-specific sigma factor sigma 29 of Bacillus subtilis is synthesized from a precursor protein, P31. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1784–1788. doi: 10.1073/pnas.84.7.1784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lambden P. R., Everson J. S., Ward M. E., Clarke I. N. Sulfur-rich proteins of Chlamydia trachomatis: developmentally regulated transcription of polycistronic mRNA from tandem promoters. Gene. 1990 Mar 1;87(1):105–112. doi: 10.1016/0378-1119(90)90500-q. [DOI] [PubMed] [Google Scholar]
  18. Lesley S. A., Burgess R. R. Characterization of the Escherichia coli transcription factor sigma 70: localization of a region involved in the interaction with core RNA polymerase. Biochemistry. 1989 Sep 19;28(19):7728–7734. doi: 10.1021/bi00445a031. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lu S., Halberg R., Kroos L. Processing of the mother-cell sigma factor, sigma K, may depend on events occurring in the forespore during Bacillus subtilis development. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9722–9726. doi: 10.1073/pnas.87.24.9722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Moulder J. W. Interaction of chlamydiae and host cells in vitro. Microbiol Rev. 1991 Mar;55(1):143–190. doi: 10.1128/mr.55.1.143-190.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Newhall W. J., Jones R. B. Disulfide-linked oligomers of the major outer membrane protein of chlamydiae. J Bacteriol. 1983 May;154(2):998–1001. doi: 10.1128/jb.154.2.998-1001.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Plaunt M. R., Hatch T. P. Protein synthesis early in the developmental cycle of Chlamydia psittaci. Infect Immun. 1988 Dec;56(12):3021–3025. doi: 10.1128/iai.56.12.3021-3025.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Raibaud O., Schwartz M. Positive control of transcription initiation in bacteria. Annu Rev Genet. 1984;18:173–206. doi: 10.1146/annurev.ge.18.120184.001133. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stephens R. S., Kuo C. C., Newport G., Agabian N. Molecular cloning and expression of Chlamydia trachomatis major outer membrane protein antigens in Escherichia coli. Infect Immun. 1985 Mar;47(3):713–718. doi: 10.1128/iai.47.3.713-718.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Stragier P., Losick R. Cascades of sigma factors revisited. Mol Microbiol. 1990 Nov;4(11):1801–1806. doi: 10.1111/j.1365-2958.1990.tb02028.x. [DOI] [PubMed] [Google Scholar]
  30. Strickland M. S., Thompson N. E., Burgess R. R. Structure and function of the sigma-70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription. Biochemistry. 1988 Jul 26;27(15):5755–5762. doi: 10.1021/bi00415a054. [DOI] [PubMed] [Google Scholar]
  31. Studier F. W. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol. 1991 May 5;219(1):37–44. doi: 10.1016/0022-2836(91)90855-z. [DOI] [PubMed] [Google Scholar]
  32. Su H., Watkins N. G., Zhang Y. X., Caldwell H. D. Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin. Infect Immun. 1990 Apr;58(4):1017–1025. doi: 10.1128/iai.58.4.1017-1025.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tanaka K., Shiina T., Takahashi H. Multiple principal sigma factor homologs in eubacteria: identification of the "rpoD box". Science. 1988 Nov 18;242(4881):1040–1042. doi: 10.1126/science.3194753. [DOI] [PubMed] [Google Scholar]
  35. Wichlan D. G., Hatch T. P. Identification of an early-stage gene of Chlamydia psittaci 6BC. J Bacteriol. 1993 May;175(10):2936–2942. doi: 10.1128/jb.175.10.2936-2942.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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