INTERSPECIFIC LUCIFERASE BETA-SUBUNIT HYBRIDS BETWEEN VIBRIO-HARVEYI,VIBRIO-FISCHERI AND PHOTOBACTERIUM-LEIOGNATHI

Bacterial luciferase (EC 1.14.14.3) is a heterodimer composed of alpha - and beta-chains encoded by luxA and luxB, respectively. Although som e interspecific combinations of these subunits lead to active enzyme, others do not. The beta subunits of Vibrio fischeri and Photobacterium leiognathi form active enzyme with the alpha subunits of V.fischeri, P.leiognathi and Vibrio harveyi, while the beta subunit from V.harveyi only complements the alpha subunit of V.harveyi. Inactivity is caused by a lack of dimerization of the beta subunit of V.harveyi with the a lpha subunits of V.fischeri and P.leiognathi. These observations serve d as the basis for a search to discover which segment of the beta poly peptide confers the ability to dimerize with the alpha subunits of V.f ischeri and P.leiognathi. Intragenic beta subunit hybrids were made be tween V.harveyi, V.fischeri and P.leiognathi. Unique restriction sites were introduced into the respective luxB genes to divide them into fo ur roughly equal segments. In all, 78 hybrids were constructed by in v itro techniques. The N-terminal segment of the peptide contains the si gnals that differentiate between the beta subunits of V.fischeri and P .leiognathi and the beta subunit of V.harveyi, and allow the former to dimerize with their alpha subunits. The second segment has no major e ffect on enzyme activity but does exhibit some context effects. Import ant interactions were found between the third and fourth segments of t he polypeptide with respect to enzymatic activity.