Transcriptional regulation of the benabcde operon of acinetobacter sp. Strain adp1: benm-mediated synergistic induction in response to benzoate and cis,cis-muconate
Bundy, Becky Marie
MetadataShow full item record
BenM, a LysR-type transcriptional regulator of Acinetobacter sp. strain ADP1, controls expression of benABCDE, an operon involved in benzoate degradation via the ?- ketoadipate pathway. In vivo studies suggested BenM responds to either benzoate or cis,cis-muconate as inducer, and both compounds together have a synergistic effect on transcription. To investigate whether the effects of these inducers result from direct interactions with BenM, we utilized a run-off in vitro transcription assay and DNase I footprinting. These experiments demonstrated the synergistic effects of benzoate and cis,cis-muconate require BenM and no additional Acinetobacter proteins. A C-terminally his-tagged BenM was purified by metal affinity chromatography and shown to be a stable tetramer. His-tagged BenM was as active as native BenM in the transcription assay. This assay indicated basal transcription in the absence of BenM, and a 2-fold repression of transcription in the presence of BenM without inducer. The presence of either inducer alone with BenM yielded activation. cis,cis-Muconate elicited a response 2-fold greater than benzoate as the sole inducer. In concert, the two inducers had a greater effect on transcription than the sum of their individual contributions. Based on DNaseI protection patterns, a model is presented in which there are three sites in the benA operator-promoter which BenM may bind. These sites are centered at positions -64, -43, and -12 relative to the transcriptional start site, the A residue 216 nucleotides upstream of the translational start. Without inducer, BenM binds the -64 and -12 sites, bending the intervening DNA and causing several hypersensitive sites. In the presence of either inducer, BenM no longer protects the -12 site. When both inducers are present, BenM protects both the -64 and -43 sites. The pattern of hypersensitivity with both inducers differs from that produced with a sole inducer. Mutational analysis of the benABCDE promoter regulatory region suggested that Site 1 is required for cooperative binding of BenM to Sites 2 and 3, and that Site 2 is required activation of ben gene expression by BenM in response to either inducer alone as well as both inducers together. Therefore, both compounds in concert appear to induce conformational changes in the BenM-DNA complex that effect full induction of benA transcriptional activation.