Interspecific luciferase β subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi

Abstract

Bacterial luciferase (EC 1.14.14.3) is a heterodimer composed of α- and β-chains encoded by luxA and luxB, respectively. Although some interspecific combinations of these subunits lead to active enzyme, others do not. The β subunits of Vibrio fischeri and Photobacterium leiognathi form active enzyme with the α subunits of V. fischeri, P. leiognathi and Vibrio harveyi, while the β subunit from V. harveyi only complements the α subunit of V. harveyi. Inactivity is caused by a lack of dimerization of the β subunit of V. harveyi with the α subunits of V. fischeri and P. leiognathi. These observations served as the basis for a search to discover which segment of the β polypeptide confers the ability to dimerize with the α subunits of V. fischeri and P. leiognathi. Intragenic β subunit hybrids were made between V. harveyi, V. fischeri and P. leiognathi. Unique restriction sites were introduced into the respective luxB genes to divide them into four roughly equal segments. In all, 78 hybrids were constructed by in vitro techniques. The N-terminal segment of the peptide contains the signals that differentiate between the β subunits of V. fischeri and P. leiognathi and the β subunit of V. harveyi, and allow the former to dimerize with their α subunits. The second segment has no major effect on enzyme activity but does exhibit some context effects. Important interactions were found between the third and fourth segments of the polypeptide with respect to enzymatic activity.