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Journal of Bacteriology, August 2006, p. 5524-5531, Vol. 188, No. 15
0021-9193/06/$08.00+0     doi:10.1128/JB.00480-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli {sigma}28 RNA Polymerase

Hilda Hiu Yin Yu,1 Elizabeth G. Di Russo,1 Megan A. Rounds,1 and Ming Tan1,2*

Department of Microbiology and Molecular Genetics,1 Department of Medicine, School of Medicine, University of California-Irvine, Irvine, California 92697-40252

Received 5 April 2006/ Accepted 15 May 2006

{sigma}28 RNA polymerase is an alternative RNA polymerase that has been postulated to have a role in developmental gene regulation in Chlamydia. Although a consensus bacterial {sigma}28 promoter sequence has been proposed, it is based on a relatively small number of defined promoters, and the promoter structure has not been systematically analyzed. To evaluate the sequence of the {sigma}28-dependent promoter, we performed a comprehensive mutational analysis of the Chlamydia trachomatis hctB promoter, testing the effect of point substitutions on promoter activity. We defined a –35 element recognized by chlamydial {sigma}28 RNA polymerase that resembles the consensus –35 sequence. Within the –10 element, however, chlamydial {sigma}28 RNA polymerase showed a striking preference for a CGA sequence at positions –12 to –10 rather than the longer consensus –10 sequence. We also observed a strong preference for this CGA sequence by Escherichia coli {sigma}28 RNA polymerase, suggesting that this previously unrecognized motif is the critical component of the –10 promoter element recognized by {sigma}28 RNA polymerase. Although the consensus spacer length is 11 nucleotides (nt), we found that {sigma}28 RNA polymerase from both Chlamydia and E. coli transcribed a promoter with either an 11- or 12-nt spacer equally well. Altogether, we found very similar results for {sigma}28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. The preferred {sigma}28 promoter that we defined in the context of the hctB promoter is TAAAGwwy-n11/12-ryCGAwrn, where w is A or T, r is a purine, y is a pyrimidine, n is any nucleotide, and n11/12 is a spacer of 11 or 12 nt.

* Corresponding author. Mailing Address: Department of Microbiology and Molecular Genetics, University of California-Irvine, B240 Med Sci I, Irvine, CA 92697-4025. Phone: (949) 824-3397. Fax: (949) 824-8598. E-mail: mingt{at}uci.edu.

Journal of Bacteriology, August 2006, p. 5524-5531, Vol. 188, No. 15
0021-9193/06/$08.00+0     doi:10.1128/JB.00480-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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