Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli

Abstract

The DNA encoding the luciferase .alpha. and .beta. subunits in the luminous marine bacterium V. harveyi (strain 392) is contained within a 4.0-kilobase HindIII fragment. DNA from V. harveyi was digested with HindIII, and the resulting fragments were inserted into the HindIII site of plasmid pBR322. The recombinant plasmids were introduced by transformation into E. coli RR1. The colonies were supplied with n-decanal, the substrate for the bioluminescence reaction, and 12 colonies (of .apprx. 6000 total) were observed to luminesce brightly. One of the recombinant plasmids, pTB7, was studied in detail. The high level of expression of bioluminescence in pTB7 was the result not of native V. harveyi promoters but rather of a promoter in pBR322 which is within the tetracycline resistance gene but oriented in the direction opposite to the transcription of the tetracycline gene. Using antiluciferase antibody to probe proteins transferred from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose paper, it was shown that the E. coli transformants produce luciferase that cross-reacts with antiluciferase antibody and is the same MW as V. harveyi luciferase. No .alpha. subunit could be detected by using antiluciferase antibody in lysates of a subclone, pTB104, which is identical with pTB7 except for deletion of the .beta.-subunit gene. Thus, the .alpha. subunit may be unstable and be degraded unless it is associated with .beta.. The bioluminescence emission spectra of V. harveyi and of E. coli transformants carrying pTB7 are indistinguishable. On the basis of these observations, it is concluded that enzyme(s) within E. coli is (are) capable of supplying luciferase with FMNH2 and that no energy transfer system is required for bioluminescence.