Cloning and characterization of the propanediol dehydratase genes in Salmonella typhimurium

The enteric bacterium Salmonella typhimurium utilizes 1,2-propanediol as both a carbon and energy source during aerobic growth but only as an energy source during anaerobic growth. The first enzyme in this pathway is adenosylcobalamin-dependent propanediol dehydratase. The polar effects of mini-tet insertions on pduiJac operon fusions suggest that the genes for propanediol utilization (pdu) are organized into a single operon. Using pduvAac operon fusions, it was determined that the pdu genes were induced by propanediol and that this induction was enhanced in the absence of oxygen. A mutant strain of S. typhimurium was constructed in which the transposable element Tn/Ocf-Kan was placed adjacent to the genes responsible for propanediol utilization. A cosmid library of genomic DNA from this strain was screened in Escherichia coli DH5a for resistance to kanamycin. All kanamycin-resistant clones were assayed for propanediol dehydratase enzyme activity. One kanamycin-resistant clone demonstrated enzyme activity. A 20.8-kbp subclone was trimmed with exonuclease HI and a 6.6-kbp //mdlll fragment was isolated which retained dehydratase activity. Four proteins visualized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, were expressed from this fragment, using the bacteriophage T7 RNA polymerase/promoter system.