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Design of new promoters and of a dual‐bioreporter based on cross‐activation by the two regulatory proteins XylR and HbpR
Author(s) -
Tropel David,
Bähler Alexandra,
Globig Kathrin,
Van Der Meer Jan Roelof
Publication year - 2004
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2004.00645.x
Subject(s) - pseudomonas putida , promoter , biology , luciferase , repressor , bioreporter , mutant , activator (genetics) , reporter gene , transcription (linguistics) , transcription factor , bacterial transcription , microbiology and biotechnology , gene , genetics , gene expression , transfection , linguistics , philosophy
Summary The HbpR protein is the σ 54 ‐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 P hbpC and P u promoters was investigated by determining HbpR‐ or XylR‐mediated luciferase expression and by DNA binding assays. XylR measurably activated the P hbpC promoter in the presence of the effector m‐xylene, both in Escherichia coli and Pseudomonas putida . HbpR weakly stimulated the P u promoter in E. coli but not in P. azelaica . Poor HbpR‐dependent activation from P u was caused by a weak binding to the operator region. To create promoters efficiently activated by both regulators, the HbpR binding sites on P hbpC were gradually changed into the XylR binding sites of P u 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 P u itself. Mutants were also obtained which displayed a tenfold lower uninduced expression level by HbpR than the wild‐type P hbpC , 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.