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J. Bacteriol. doi:10.1128/JB.00717-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Investigation of transcription repression and small molecule responsiveness by TetR-like transcription factors using a heterologous E. coli-based assay

Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5; Department of Medical Genetics and Microbiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8

^* To whom correspondence should be addressed. Email: nodwellj{at}mcmaster.ca.

	Abstract

SCO7222 and ActR are two of 150 TetR-like transcription factors encoded in the Streptomyces coelicolor genome. Using bioluminescence as readout, we have developed E. coli-based biosensors that accurately report the regulatory activity of these proteins and used it to investigate their interactions with DNA and small molecule ligands. We found that SCO7222 and ActR repress the expression of their putative target genes, SCO7223 and actII-ORF2 (actA) by interacting with operator sequence in the promoters. The operators recognized by the two proteins are related such that O⁷²²³ (an operator for SCO7223) could be bound by both SCO7222 and ActR with similar affinities. In contrast, O^act (an operator for actII-ORF2) was bound tightly by ActR and more weakly by SCO7222. We demonstrated ligand specificity of these proteins by showing that while TetR (but not ActR or SCO7222) interacts with tetracyclines, ActR (but not TetR or SCO7222) interacts with actinorhodin and related molecules. Through operator-targeted mutagenesis, we found that at least two nucleotide changes in O⁷²²³ were required to disrupt its interaction with SCO7222, while ActR was more sensitive to changes on O^act. Most importantly, we found that the interaction of each protein with wild type and mutant operator sequences in vivo and in vitro correlated perfectly. Our data suggest that E. coli-based biosensors of this type should be broadly applicable to TetR-like transcription factors.