Bioluminescence in soybean root nodules: Demonstration of a general approach to assay gene expression in vivo by using bacterial luciferase

Abstract

Two plasmid vectors pFIT001 and pPALE001, containing luxAB genes encoding bacterial luciferase [alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] from Vibrio harveyi, have been constructed. Escherichia coli carrying derivatives of pFIT001 with DNA inserts in the unique EcoRI site located in luxB form "dark" colonies that can be readily distinguished from the bioluminescent or "bright" colonies. In contrast, promoterless pPALE001 is used as a promoter-search vector based on bioluminescence. The control and regulation of gene expression can be analyzed in vivo using promoter-luxAB fusions by a variety of simple methods, including a technique called "luxdot." As an example, we have introduced nitrogenase nifD and nifH promoter-luxAB fusions into the Bradyrhizobium japonicum chromosome and shown symbiotically regulated bioluminescence in soybean root nodules. B. japonicum transconjugants containing a single copy per genome of the nif promoter-controlled luciferase structural genes did not produce light in free-living cultures, but the same transconjugants did express bioluminescence in root nodules that was strong enough to be detected by the naked eye.