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Journal of Bacteriology, September 2009, p. 5458-5470, Vol. 191, No. 17
0021-9193/09/$08.00+0     doi:10.1128/JB.00355-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

CtrA, a Global Response Regulator, Uses a Distinct Second Category of Weak DNA Binding Sites for Cell Cycle Transcription Control in Caulobacter crescentus{triangledown}

William Spencer,{dagger} Rania Siam,{ddagger} Marie-Claude Ouimet, D. Patrick Bastedo, and Gregory T. Marczynski*

Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada

Received 13 March 2009/ Accepted 15 June 2009

CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA~P exhibits high affinity (dissociation constant [Kd], <10 nM) for consensus TTAA-N7-TTAA binding sites with "typical" (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (Kd, >500 nM) CtrA binding sites with "atypical" (N != 7) spacing. Footprints demonstrated that phosphorylated CtrA~P does not exhibit increased affinity for "atypical" sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 µM) accumulate, which can drive CtrA binding to "atypical" sites. In vivo cross-linking showed that when the stable CtrA{Delta}3 protein persists during the cell cycle, the "atypical" sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrA{Delta}3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA~P phosphorylation that provides the dominant "activation" signal. Protein dimerization is one potential means of "activation." The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the "atypical" sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how "atypical" sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada. Phone: (514) 398-3917. Fax: (514) 398-7052. E-mail: gregory.marczynski{at}mcgill.ca

{triangledown} Published ahead of print on 19 June 2009.

{dagger} Present address: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.

{ddagger} Present address: Biology Department and the YJ Science and Technology Research Center, The American University in Cairo, P.O. Box 2511, 113 Sharia Kasr El Aini, Cairo, Egypt.

Journal of Bacteriology, September 2009, p. 5458-5470, Vol. 191, No. 17
0021-9193/09/$08.00+0     doi:10.1128/JB.00355-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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