The β subunit polypeptide of Vibrio harveyi luciferase determines light emission at 42° C

Summary

The nucleotide sequence of the 1uxA and luxB genes encoding the αβ heterodimeric luciferase from thermotolerant Vibrio harveyi CTP5 was determined. The DNA sequence of the CTP5 luxA and luxB genes is identical to the DNA sequence of the luxA and luxB genes from mesophilic V. harveyi MAV (B 392), with minor exceptions. The sequence differences result in 5 amino acid substitutions in the α subunit polypeptide and 7 amino acid substitutions in the β subunit polypeptide. Escherichia coli cells grown on solid medium and expressing CTP5 or MAV luxAB genes emit similar amounts of light at 37° C, while at 42° C cells containing CTP5 luxAB genes show more than tenfold increased bioluminescence compared to cells with MAV luxAB genes. When grown in liquid medium E. coli cells with CTP5 or MAV luxAB genes emit equivalent amounts of light at 37° C; however, in liquid medium at 42° C cells containing CTP5 luxAB genes show only three times higher bioluminescence than cells with MAV luxAB genes. Expression of T7 promoter-linked hybrid luxAB transcriptional units luxA _CTP5-luxB _MAV and luxA _MAV-luxB _CTP5 in E. coli reveals that (i) the MAV luxB gene product is responsible for the decreased activity of MAV luciferase at 42° C; (ii) the CTP5 luxB gene encodes the information required for most of the increased activity of CTP5 luciferase relative to MAV luciferase at 42° C; and (iii) E. coli cells containing MAV luxB gene show an increase in bioluminescence when grown in liquid medium at 42° C, which coincides with elevated GroEL chaperonin levels. The MAV-CTP5 hybrid luciferases, with amino acid changes at known positions, provide a sensitive and simple system to study temperature-dependent assembly of multi-subunit enzymes in vivo.