Construction and testing of a bacterial luciferase reporter gene system forin Vivo measurement of nonsense suppression inStreptomyces

A reporter gene system, based on luciferase genes fromVibrio harvei, was constructed for measurement of translation nonsense suppression inStreptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of theluxB gene was replaced by all of the three stop codons individually. By cloning ofluxA andluxB genes under the control of strong constitutiveStreptomyces promoterermE* in plasmid pUWL201 we created Wlux1 with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively.Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal inStreptomyces, the effect was negligible.