Transcriptional activation of the chlorocatechol degradative genes of Ralstonia eutropha NH9

Ralstonia eutropha (formerly Alcaligenes euthrophus) NH9 degrades 3-chlorob enzoate via the modified ortho-cleavage pathway. A ca. 5.7-kb six-gene clus ter is responsible for chlorocatechol degradation: the cbnABCD operon encod ing the degradative enzymes (including orfX of unknown function) and the di vergently transcribed cbnR gene encoding the LysR-type transcriptional regu lator 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 stud ies demonstrated that cbnR regulates the expression of cbnABCD positively i n the presence of either 3-chlorobenzoate or benzoate, which are catabolize d via 3-chlorocatechol and catechol, respectively. In vitro transcription a ssays confirmed that 2-chloro-cis,cis-muconate (2-CM) and cis,cis-muconateR alstolria eutropha (formerly Alcaligenes eutrophus) NH9 degrades 3-chlorobe nzoate via the modified ortho-cleavage pathway. A ca. 5.7-kb six-gene clust er is responsible for chlorocatechol degradation: the cbnABCD operon encodi ng the degradative enzymes (including orfX of unknown function) and the div ergently transcribed cbnR gene encoding the LysR-type transcriptional regul ator of the cbn operon. The cbnRAB orfXCD gene cluster is nearly identical to the chlorocatechol genes (tcbRCD orfXEF) of the 1,2,4-trichlorobenzene-d egrading bacterium Pseudomonas sp. strain P51. Transcriptional fusion studi es 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 as says confirmed that 2-chloro-cis,cis-muconate (2-CM) and cis,cis-muconate ( CCM), intermediate products from 3-chlorocatechol and catechol, respectivel y, were inducers of this operon. This inducer-recognizing specificity is di fferent from those of the homologous catechol (catBCA) and chlorocatechol ( clcABD) operons of Pseudomonas putida, in which only the intermediates of t he regulated pathway, CCM for catBCA and 2-CM for clcABD, act as significan t inducers. Specific binding of CbnR protein to the cbnA promoter region wa s demonstrated by gel shift and DNase I footprinting analysis. In the absen ce of inducer, a region of ca. 60 bp from position -20 to position -80 upst ream of the cbnA transcriptional start point was protected from DNase I cle avage by CbnR, with a region of hypersensitivity to DNase I cleavage cluste red at position -50. Circular permutation gel shift assays demonstrated tha t CbnR bent the cbnA promoter region to an angle of 78 degrees and that thi s angle was relaxed to 54 degrees upon the addition of inducer. While a sim ilar relaxation of bending angles upon the addition of inducer molecules ob served with the catBCA and clcABD promoters may indicate a conserved transc riptional activation mechanism of ortho-cleavage pathway genes, CbnR is uni que in having a different specificity of inducer recognition and the extend ed footprint as opposed to the restricted footprint of CatR without CCM.hol and catechol, respectively, were inducers of this operon. This inducer-rec ognizing specificity is different from those of the homologous catechol (ca tBCA) and chlorocatechol (clcABD) operons of Pseudomonas putida, in which o nly the intermediates of the regulated pathway, CCM for catBCA and 2-CM for clcABD, act as significant inducers. Specific binding of CbnR protein to t he cbnA promoter region was demonstrated by gel shift and DNase I footprint ing analysis. In the absence of inducer, a region of ca. 60 bp from position -20 to position -80 upstream of the cbnA transcriptiona l start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position -50. Circular p ermutation gel shift assays demonstrated that CbnR bent the cbnA promoter r egion to an angle of 78 degrees and that this angle was relaxed to 54 degre es upon the addition of inducer. While a similar relaxation of bending angl es upon the addition of inducer molecules observed with the catBCA and clcA BD promoters may indicate a conserved transcriptional activation mechanism of ortho-cleavage pathway genes, CbnR is unique in having a different speci ficity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM.