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Journal of Bacteriology, November 2009, p. 6654-6664, Vol. 191, No. 21
0021-9193/09/$08.00+0     doi:10.1128/JB.00902-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mechanism of Transcriptional Activation by Pseudomonas aeruginosa ExsA{triangledown} ,{dagger}

Christopher A. Vakulskas, Keith M. Brady,{ddagger} and Timothy L. Yahr*

Department of Microbiology, University of Iowa, Iowa City, Iowa

Received 10 July 2009/ Accepted 17 August 2009

ExsA is a transcriptional activator of the Pseudomonas aeruginosa type III secretion system (T3SS). The T3SS consists of >40 genes organized within 10 transcriptional units, each of which is controlled by the transcriptional activator ExsA. ExsA-dependent promoters contain two adjacent ExsA binding sites that when occupied protect the –30 to –70 region from DNase I cleavage. The promoters also possess regions bearing strong resemblance to the consensus –10 and –35 regions of {sigma}70-dependent promoters. The spacing distance between the putative –10 and –35 regions of ExsA-dependent promoters, however, is increased by 4 to 5 bp compared to that in typical {sigma}70-dependent promoters. In the present study, we demonstrate that ExsA-dependent transcriptional activation requires a 21- or 22-bp spacer length between the –10 and –35 regions. Despite the atypical spacing in this region, in vitro transcription assays using {sigma}70-saturated RNA polymerase holoenzyme (RNAP-{sigma}70) confirm that ExsA-dependent promoters are indeed {sigma}70 dependent. Potassium permanganate footprinting experiments indicate that ExsA facilitates an early step in transcriptional initiation. Although RNAP-{sigma}70 binds to the promoters with low affinity in the absence of ExsA, the activator stimulates transcription by enhancing recruitment of RNAP-{sigma}70 to the PexsC and PexsD promoters. Abortive initiation assays confirm that ExsA enhances the equilibrium binding constant for RNAP while having only a modest effect on the isomerization rate constant.

* Corresponding author. Mailing address: University of Iowa, 540B Eckstein Medical Research Building, Iowa City, IA 52242-1101. Phone: (319) 335-9688. Fax: (319) 335-8228. E-mail: tim-yahr{at}uiowa.edu

{triangledown} Published ahead of print on 28 August 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Present address: JMI Laboratories, 345 Beaver Kreek Centre, Suite A, North Liberty, IA 52317.

Journal of Bacteriology, November 2009, p. 6654-6664, Vol. 191, No. 21
0021-9193/09/$08.00+0     doi:10.1128/JB.00902-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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