Transcriptional Activation of the Chlorocatechol Degradative Genes of <i>Ralstonia eutropha</i> NH9

<jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Ralstonia eutropha</jats:italic> (formerly <jats:italic>Alcaligenes eutrophus</jats:italic> ) NH9 degrades 3-chlorobenzoate via the modified <jats:italic>ortho</jats:italic> -cleavage pathway. A ca. 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the <jats:italic>cbnABCD</jats:italic> operon encoding the degradative enzymes (including <jats:italic>orfX</jats:italic> of unknown function) and the divergently transcribed <jats:italic>cbnR</jats:italic> gene encoding the LysR-type transcriptional regulator of the <jats:italic>cbn</jats:italic> operon. The <jats:italic>cbnRAB orfXCD</jats:italic> gene cluster is nearly identical to the chlorocatechol genes ( <jats:italic>tcbRCD orfXEF</jats:italic> ) of the 1,2,4-trichlorobenzene-degrading bacterium <jats:italic>Pseudomonas</jats:italic> sp. strain P51. Transcriptional fusion studies demonstrated that <jats:italic>cbnR</jats:italic> regulates the expression of <jats:italic>cbnABCD</jats:italic> positively in the presence of either 3-chlorobenzoate or benzoate, which are catabolized via 3-chlorocatechol and catechol, respectively. In vitro transcription assays confirmed that 2-chloro- <jats:italic>cis</jats:italic> , <jats:italic>cis</jats:italic> -muconate (2-CM) and <jats:italic>cis</jats:italic> , <jats:italic>cis</jats:italic> -muconate (CCM), intermediate products from 3-chlorocatechol and catechol, respectively, were inducers of this operon. This inducer-recognizing specificity is different from those of the homologous catechol ( <jats:italic>catBCA</jats:italic> ) and chlorocatechol ( <jats:italic>clcABD</jats:italic> ) operons of <jats:italic>Pseudomonas putida</jats:italic> , in which only the intermediates of the regulated pathway, CCM for <jats:italic>catBCA</jats:italic> and 2-CM for <jats:italic>clcABD</jats:italic> , act as significant inducers. Specific binding of CbnR protein to the <jats:italic>cbnA</jats:italic> promoter region was demonstrated by gel shift and DNase I footprinting analysis. In the absence of inducer, a region of ca. 60 bp from position −20 to position −80 upstream of the <jats:italic>cbnA</jats:italic> transcriptional start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position −50. Circular permutation gel shift assays demonstrated that CbnR bent the <jats:italic>cbnA</jats:italic> promoter region to an angle of 78° and that this angle was relaxed to 54° upon the addition of inducer. While a similar relaxation of bending angles upon the addition of inducer molecules observed with the <jats:italic>catBCA</jats:italic> and <jats:italic>clcABD</jats:italic> promoters may indicate a conserved transcriptional activation mechanism of <jats:italic>ortho</jats:italic> -cleavage pathway genes, CbnR is unique in having a different specificity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM. </jats:p>