Characterization of MobR, the 3-hydroxybenzoate-responsive transcriptional regulator for the 3-hydroxybenzoate hydroxylase gene of Comamonas testosteroni KH122-3s.
Comamonas testosteroni KH122-3s is an aerobic soil bacterium that utilizes 3-hydroxybenzoate as a sole carbon and energy source. In this strain, 3-hydroxybenzoate hydroxylase (MobA) acts on the initial step of the degradation to produce 3,4-dihydroxybenzoate, which is subsequently subjected to the meta-cleavage pathway leading to tricarboxylic acid cycle intermediates. Gene walking analysis of the upstream region of mobA revealed an open reading frame (mobR) that encodes a transcriptional regulator of the MarR family. Here, we report that MobR negatively regulates the expression of mobA, and that the repression is relieved by binding of 3-hydroxybenzoate, the substrate for MobA. A primer extension experiment was performed to determine the transcription start site for mobA and identified it at 83 bp upstream of the mobA start codon, accompanied by a typical sigma70-type promoter. The mobR gene was expressed in Escherichia coli cells and the recombinant product was purified to homogeneity. Gel mobility-shift assays and DNase I footprinting analyses indicated that MobR binds as a homodimer to an imperfect inverted repeat within the mobA-mobR intergenic region, with an apparent dissociation constant of 11.5(+/- 0.5) nM. The operator site is located between the start codon and the promoter region for mobA, suggesting that MobR functions as a transcriptional repressor for mobA expression. The results of effector-binding assays indicated that MobR, but not its isomers 4-hydroxybenzoate and salicylate, is released from the operator site by the addition of 3-hydroxybenzoate. This dissociation process is highly cooperative, with a Hill coefficient of approximately 2. In addition, CD spectroscopic studies demonstrated that MobR adopts two conformational states corresponding to the effector-bound and unbound forms. These results suggest that the MobR dimer possesses at least two effector-binding sites, and that the effector binding to MobR induces an allosteric conformational change required for dissociation of the protein-DNA complex.
Hiromoto
Takeshi
T
Department of Chemistry, Nanobiothechnology Research Center, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
Matsue
Hanako
H
Yoshida
Mariko
M
Tanaka
Takeshi
T
Higashibata
Hiroki
H
Hosokawa
Keiichi
K
Yamaguchi
Hiroshi
H
Fujiwara
Shinsuke
S
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000235:genetics / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D020604:Comamonas testosteroni / Q000201:enzymology / Q000235:genetics*; D018983:DNA Footprinting; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D062385:Hydroxybenzoates / Q000494:pharmacology*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010226:Parabens / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins; D020156:Salicylic Acid / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D062385:Hydroxybenzoates; D010226:Parabens; D011994:Recombinant Proteins; D015534:Trans-Activators; C032948:3-hydroxybenzoic acid; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase; C021385:3-hydroxybenzoate-4-monooxygenase; C038193:4-hydroxybenzoic acid; D020156:Salicylic Acid