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 Vibrio 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 Escherichia coli RR1. The colonies were supplied with n-decanal, the substrate for the bioluminescence reaction, and 12 colonies (of ca. 6000 total) were observed to luminesce brightly. One of the recombinant plasmids, pTB7, has been 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 nitro-cellulose paper, we have shown that the E. coli transformants produce luciferase that cross-reacts with antiluciferase antibody and is the same molecular weight 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphenyl-inducible Promoters in a Polychlorinated Biphenyl-degrading Bacterium,Rhodococcussp. RHA1

“…Solid compounds were supplied as powder at a final concentration of 0.2%, and volatile compounds were supplied in vapor. The cell suspension was then diluted 1:10 in lux buffer, 14) and a 100-l aliquot was mixed with 390 l of lux buffer. After adding 10 l of 0.1% (v/v) 1-decanal in lux buffer to the resulting 490 l of diluted cell suspension, luciferase activity was measured using a luminometer (Lumitester K-100, Kikkoman, Noda, Japan).…”

Structure of the β2 homodimer of bacterial luciferase from vibrio harveyi: X‐ray analysis of a kinetic protein folding trap

“…Plasmid pbT7, encoding the luciferase p subunit from c! harveyi under control of the T7 promoter of pT7-6 was constructed by inserting the Pstl-Sac1 fragment carrying a fragment of l u d , all of luxB, and a fragment of luxE from pTB7 (Baldwin et al, 1984) into pT7-6 that had been opened by Pst 1 and Sac I . The medium used was LB supplemented with ampicillin (100 pg/mL).…”

ORGANIZATION and REGULATION OF BACTERIAL LUMINESCENCE GENES

“…harveyi, several mRNA species are produced by this DNA (Miyamoto et al, 1985), and the expression of luminescence by E. coli containing fragments of V . harveyi 1uxAB DNA up to 18 kb long (thereby encompassing luxCDABEGI-2) is constitutive, and light levels are low except when the cloned insert is under the control of a strong promoter and aldehyde is added (Belas et al, 1982;Cohn et al, 1983;Baldwin et al, 1984;Gupta et al, 1986), or when the DNA is present in certain E. coli mutants (Miyamoto et al, 1987), o r when expression is under the control of T7 RNA polymerase (Miyamoto et al, 1988b). The results of the latter studies suggest that low expression of luminescence by E. coli containing V .…”