Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD chlorocatechol operon in Pseudomonas putida.
The 3-chlorocatechol operon clcABD is central to the biodegradative pathway of 3-chlorobenzoate. The clcR regulatory gene, which activates the clcABD 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 clcA promoter, which controls expression of the clcABD 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-clcA intergenic promoter region was observed in a gel shift assay. Nucleotide sequence analysis of the clcR 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 clcA promoter. Several considerations indicate that the clcR-clcABD locus is most similar to the tcbR-tcbCDEF regulon.
Coco
W M
WM
Department of Microbiology and Immunology, University of Illinois, Chicago 60612.
Rothmel
R K
RK
Henikoff
S
S
Chakrabarty
A M
AM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002396:Catechols / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D009876:Operon; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002396:Catechols; D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C031137:3-chlorocatechol; C073470:LysR protein, Bacteria; D001616:beta-Galactosidase