A new gene expression system based on a fructose-dependent promoter from Rhodobacter capsulatus.
Translational lacZ fusions were constructed to analyse transcription of the fructose operon, encoding the fructose-specific phosphotransferase system of Rhodobacter capsulatus. It was demonstrated that transcription from the fru promoter (fruP) was negligible without fructose, and stimulated more than 100-fold by the presence of the inducer. A multiple cloning site, fruP, and a cassette conferring gentamycin resistance were assembled to form a cloning cartridge which is easily transferable to a broad-host-range vector. The sequence initiating the first gene of the fru operon was altered to introduce a NdeI site, allowing insertion of the 5' end of a gene at the correct distance from the ribosome-binding site. The system has been used to express the Escherichia coli lacZ gene in R. capsulatus. beta Gal activity was shown to be specifically and rapidly induced by fructose, at low concentrations. Vectors for fructose-dependent gene expression also proved to be useful in the complementation analysis of mutants. A fdxN mutant of R. capsulatus, markedly impaired in its ability to fix nitrogen due to the lack of a ferredoxin, could be fully complemented using a plasmid carrying a copy of fdxN behind fruP. Complementation, as well as the synthesis of the ferredoxin, were found to be strictly fructose dependent.
Duport
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C
Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
Meyer
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Naud
I
I
Jouanneau
Y
Y
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial; D005632:Fructose / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D011401:Promoter Regions, Genetic; D016354:Rhodobacter capsulatus / Q000235:genetics*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D005632:Fructose