Article: article from journal or magazin.
Design of new promoters and of a dual-bioreporter based on cross-activation by the two regulatory proteins XylR and HbpR.
The HbpR protein is the sigma54-dependent transcription activator for 2-hydroxybiphenyl degradation in Pseudomonas azelaica. The ability of HbpR and XylR, which share 35% amino acid sequence identity, to cross-activate the PhbpC and Pu promoters was investigated by determining HbpR- or XylR-mediated luciferase expression and by DNA binding assays. XylR measurably activated the PhbpC promoter in the presence of the effector m-xylene, both in Escherichia coli and Pseudomonas putida. HbpR weakly stimulated the Pu promoter in E. coli but not in P. azelaica. Poor HbpR-dependent activation from Pu was caused by a weak binding to the operator region. To create promoters efficiently activated by both regulators, the HbpR binding sites on PhbpC were gradually changed into the XylR binding sites of Pu by site-directed mutagenesis. Inducible luciferase expression from mutated promoters was tested in E. coli on a two plasmid system, and from mono copy gene fusions in P. azelaica and P. putida. Some mutants were efficiently activated by both HbpR and XylR, showing that promoters can be created which are permissive for both regulators. Others achieved a higher XylR-dependent transcription than from Pu itself. Mutants were also obtained which displayed a tenfold lower uninduced expression level by HbpR than the wild-type PhbpC, while keeping the same maximal induction level. On the basis of these results, a dual-responsive bioreporter strain of P. azelaica was created, containing both XylR and HbpR, and activating luciferase expression from the same single promoter independently with m-xylene and 2-hydroxybiphenyl.
Bacterial Proteins/physiology, Base Sequence, Biphenyl Compounds/metabolism, DNA-Binding Proteins/physiology, Escherichia coli/genetics, Escherichia coli/metabolism, Gene Expression Regulation, Bacterial, Gene Order, Genes, Reporter, Luciferases/genetics, Luciferases/metabolism, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Promoter Regions, Genetic, Pseudomonas putida/genetics, Pseudomonas putida/metabolism, Trans-Activators/physiology, Transcription Factors/physiology, Xylenes/metabolism
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