A NEW GENE-EXPRESSION SYSTEM BASED ON A FRUCTOSE-DEPENDENT PROMOTER FROM RHODOBACTER-CAPSULATUS

Translational lacZ fusions were constructed to analyse transcription o f the fructose operon, encoding the fructose-specific phosphotransfera se system of Rhodobacter capsulatus. It was demonstrated that transcri ption from the fru promoter (fruP) was negligible without fructose, an d stimulated more than 100-fold by the presence of the inducer. A mult iple cloning site, fruP, and a cassette conferring gentamycin resistan ce were assembled to form a cloning cartridge which is easily transfer able to a broad-host-range vector. The sequence initiating the first g ene of the fru operon was altered to introduce a NdeI site, allowing i nsertion of the 5' end of a gene at the correct distance from the ribo some-binding site. The system has been used to express the Escherichia coli lacZ gene in R. capsulatus. beta Gal activity was shown to be sp ecifically and rapidly induced by fructose, at low concentrations. Vec tors for fructose-dependent gene expression also proved to be useful i n the complementation analysis of mutants. A fdxN mutant of R. capsula tus, 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 fdxN behind fruP. Complementation, as well as the synthesis of the ferredoxin, were found to be strictly fructose dependent.