Expression of bacterial luciferase genes from Vibrio harveyi in Bacillus subtilis and in Escherichia coli

Abstract

For gene expression and cell physiology studies of Gram-positive Bacillus subtilis, novel shuttle vectors which cause the host organism to produce light have been constructed. These vectors carry genes encoding luciferase from Vibrio harveyi, a selectable kanamycin marker and an origin of replication for Gram-negative and -positive bacteria. The effect of DNA-insert size on light production in Escherichia coli and in B. subtilis was studied by also cloning into the shuttle vector a gene whose product participates in fatty acid metabolism. B. subtilis containing lux genes was found to differ from its Gram-negative counterpart in light emission characteristics. After addition of the substrate, light emission by B. subtilis was rapid but it decayed quickly, showing biphasic kinetics. In E. coli light emission is continuous, reflecting constant regeneration of substrates for the luciferase reaction. A promoter cloning vehicle, pCSS117, was constructed by inserting a transcriptional termination loop in the upstream sequences of luciferase genes. Using this vector, the mode of action of promoters of interest can be studied in E. coli and in B. subtilis.