Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida

Abstract

The 3-chlorocatechol operon ckcABD is central to the biodegradative pathway of 3-chlorobenzoate. The ckcR regulatory gene, which activates the ckABD operon, was cloned from the region immediately upstream of the operon and was shown to complement an insertion mutation for growth on 3-chlorobenzoate. ClcR activated the ckcA promoter, which controls expression of the clABD operon, in trans by 14-fold in an in vivo promoter probe assay in Pseudomonas putida when cells were incubated with 15 mM 3-chlorobenzoic acid. Specific binding of ClcR to the clcR-ckA intergenic promoter region was observed in a gel shift assay. Nucleotide sequence analysis of the ckcR gene predicts a polypeptide of 32.5 kDa, which was confirmed by using specific in vivo 35s labeling of the protein from a T7 promoter-controlled ATG fusion construct. ClcR shares high sequence identity with the LysR family of bacterial regulator proteins and has especially high homology to a subgroup of the family consisting of TcbR (57% amino acid sequence identity), TfdS, CatR, and CatM. ClcR was shown to autoregulate its own production in trans to 35% of unrepressed levels but partially relieved this autorepression under conditions that induced transcription at the ckcA promoter. Several considerations indicate that the ckcR-ckABD locus is most similar to the tcbR-tcbCDEF regulon.Modified ortho cleavage pathways are responsible for the biodegradation of a large array of chloroaromatic compounds and are widespread in soil bacterial communities. A well-characterized group of modified ortho cleavage pathway operons that encode functions for the dissimilation of catechol and chlorocatechols provides a model system for studying the regulation of evolutionarily related pathways. These include operons in degradative pathways for 3-chlorobenzoic acid (3Cba) from Pseudomonas putida, 1,2,4-trichlorobenzene from a Pseudomonas sp., 2,4-dichlorophenoxyacetic acid from Alcaligenes eutrophus, and benzoic acid from P. putida and from Acinetobacter calcoaceticus. In the above order, the relevant operons of the first three pathways include clcABD (15), tcbCDEF (50), and tfdCDEF (12), which convert 3-chlorocatechol, 3,4,6-trichlorocatechol, and 3,5-dichlorocatechol to maleylacetic acid, 5-chloromaleylacetic acid, and 2-chloromaleylacetic acid, respectively (12,14,44,45,52). The catechol-degradative genes in the benzoate pathways of P. putida andA. calcoaceticus are clustered in dissimilar operons. In P. putida (after the conversion of catechol to cis,cis-muconate by the catA gene product), the catBC operon encodes the conversion of the latter to 13-ketoadipate enol lactone, which is then transformed to 0-ketoadipate through the action of other loci (36).Catechol degradation in A. calcoaceticus is directed by the catA gene and the catBCEFD operon, which convert catechol through 13-ketoadipate to succinyl coenzyme A and acetyl coenzyme A (36). In nearly every case, the analogous reactions of the above five operons are catalyzed by homologous enzymes.Transcription of the catBCEFD, ...

Purification of the LysR family regulator, ClcR, and its interaction with the Pseudomonas putida clcABD chlorocatechol operon promoter

“…Consistent with this pattern, the expression of the biodegradation pathway for 3-chlorocatechol in Pseudomonas putida, which is encoded by the clcABD operon, has been shown to require the divergently transcribed lysR-type regulatory gene ckcR for activation (4). Cloning and sequencing of the clcR gene were reported in a previous study, along with data demonstrating that ClcR inducibly activates the clcABD operon and represses its own transcription (4). The extent of cross-binding and cross talk among LysR family members is as yet unclear.…”

“…4 are sites on the clcABD promoter showing sequence identity to known sites on the catBC promoter (11). ClcR and CatR are members of the same subgroup of the LysR family of transcriptional activators (4). The well-characterized sequences of the CatR binding site on the catBC promoter offer insights into the potential roles of similar sites within the clcABD promoter.…”

“…The promoter-binding properties of the ClcR protein were previously determined by gel shift assay (4). A more precise location of ClcR binding was determined by DNase I footprinting analysis (Fig.…”

Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

“…All identified LTTRs involved in aromatic degradation pathways act as transcriptional activators for their target metabolic operons in the presence of a chemical inducer, which is usually a pathway intermediate (33,128,212,215,227). In some cases, the effectors are (substituted) muconates which have lost their aromatic character, whereas in other systems aromatic compounds act as effectors (e.g., salicylate, catechol, and nitrotoluene [ Table 1]).…”

“…This diversity is also reflected in LTTRs associated with degradation pathways of aromatic compounds (Table 1) (44). A large group of LTTRs regulates a single target operon only, such as CatR controlling catBCA expression for catechol metabolism in Pseudomonas putida (215), ClcR controlling the clcABDE operon of plasmid pAC27 (33), TcbR controlling the tcbCDEF operon in plasmid pP51 of Pseudomonas sp. strain P51 (263), and CbnR controlling the cbnABCD operon for chlorocatechol metabolism in Ralstonia eutropha (163).…”