Abstract
We have constructed a shuttle plasmid for Bacillus megaterium and Escherichia coli that contains the promoter and repressor gene of the B. megaterium-borne operon for xylose utilization. A polylinker downstream of the promoter allows versatile cloning of genes under its transcriptional control. We have placed gdhA (encoding glucose dehydrogenase) from B. megaterium, lacZ (encoding beta-galactosidase) from E. coli, mro (encoding mutarotase) from Acinetobacter calcoaceticus, and human puk (encoding single-chain urokinase-like plasminogen activator, rscuPA) under xylose control in this vector. All four genes were between 130-fold and 350-fold inducible by 0.5% xylose in the growth medium in B. megaterium. Enzymatically active glucose dehydrogenase and mutarotase accumulated to 20% and 30% of the total soluble protein, respectively. beta-Galactosidase and rscuPA were also expressed at a high level. A gel analysis of the products demonstrated their proteolytic stability in the cytoplasm, even up to 5 h after induction. The expression properties of this new host-vector system are discussed in comparison to the ones available for B. subtilis and E. coli.
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