Transcriptional Activation of the Chlorocatechol Degradative Genes of Ralstonia eutropha NH9

Abstract

ABSTRACT Ralstonia eutropha (formerly Alcaligenes eutrophus ) NH9 degrades 3-chlorobenzoate via the modified ortho -cleavage pathway. A ca. 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the cbnABCD operon encoding the degradative enzymes (including orfX of unknown function) and the divergently transcribed cbnR gene encoding the LysR-type transcriptional regulator of the cbn operon. The cbnRAB orfXCD gene cluster is nearly identical to the chlorocatechol genes ( tcbRCD orfXEF ) of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51. Transcriptional fusion studies demonstrated that cbnR regulates the expression of cbnABCD 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- cis , cis -muconate (2-CM) and cis , cis -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 ( catBCA ) and chlorocatechol ( clcABD ) operons of Pseudomonas putida , in which only the intermediates of the regulated pathway, CCM for catBCA and 2-CM for clcABD , act as significant inducers. Specific binding of CbnR protein to the cbnA 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 cbnA 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 cbnA 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 catBCA and clcABD promoters may indicate a conserved transcriptional activation mechanism of ortho -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.