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Journal of Bacteriology, March 2004, p. 1493-1502, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1493-1502.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Residues Required for Bacillus subtilis PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and Helix 3

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607¹

Received 22 September 2003/ Accepted 18 November 2003

Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP loop and helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP_I206A, PhoP_R210A, PhoP_L209A, and PhoP_H208A) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP_H205A and PhoP_V204A) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP_V202A and PhoP_D203A, had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP_V190A, PhoP_W191A, PhoP_Y193A, PhoP_F195A, PhoP_G197A, PhoP_T199A, and PhoP_R200A) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP_W191A and PhoP_R200A could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short helix 3, more similar to OmpR than to PhoB of Escherichia coli.

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