NahR: effects of replacements at Asn 169 and Arg 248 on promoter binding and inducer recognition.
NahR, a member of the LysR regulator family, is a positive transcriptional regulator for genes of the naphthalene degradation pathway in Pseudomonas sp. To study NahR binding properties, five single and six double mutants were made at residues 169 and/or 248, which are located in the central inducer recognition domain and the C-terminal multimerization domain of the protein, respectively. The effects of these mutations were examined by monitoring the expression of a firefly luciferase (luc) reporter gene under the control of NahR. We found that all mutants responded to induction by both salicylate and benzoate, whereas the wild-type NahR responded only to salicylate. Mutants N169E, N169E/R248C, and N169E/R248K showed low basal activities with high-level inducer responses, whereas mutant N169D/R248K showed high basal activity with inducer-independent responses. A gel retardation assay demonstrated that the different basal activities might be related to altered binding affinities of the NahR mutants to the Psal promoter. Together, these data suggest that NahR residues 169 and 248 might be involved in DNA binding as well as inducer recognition/binding.
Park
Hoo Hwi
HH
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea.
Lee
Hae Yong
HY
Lim
Woon Ki
WK
Shin
Hae Ja
HJ
D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001565:Benzoates / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D017930:Genes, Reporter; D007700:Kinetics; D049409:Luciferases, Firefly / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D001565:Benzoates; D004269:DNA, Bacterial; C063173:NahR protein, Bacteria; D011994:Recombinant Proteins; D014157:Transcription Factors; D049409:Luciferases, Firefly; D020156:Salicylic Acid