A F Graham

Proximal and distal sites bind LuxR independently and activate expression of the Vibrio harveyi lux operon

The LuxR regulatory protein of Vibrio harveyi as well as the autoinducer molecule, N‐(3‐hydroxybutanoyl) homoserine lactone, are known to be required for expression of luminescence. Although LuxR has been implicated in the activation of the promoter of the lux operon of V. harveyi, and can bind to two distinct sites upstream of the transcription initiation start site, its mode of action is unknown, in the present experiments, mobility shift assays were used to demonstrate that LuxR bound to the distal and proximal sites in an independent rather than co‐operative interaction with a much tighter binding to the distal site. Deletion and mutation analyses of DNA upstream of the lux promoter followed by transconjugation Into V. harveyi in trans using the chloramphenicol acetyl‐transferase (cat) gene as a reporter demonstrated, however, that the proximal site for LuxR was absolutely critical for promoter activation while the distal LuxR site was only necessary for maximum activation. This result was confirmed by mutation of the proximal site which blocked activation of the lux promoter and binding of LuxR to this site, but did not prevent LuxR binding to the distal site.

LUX C, D AND E GENES OF THE Vibrio fischeri LUMINESCENCE OPERON CODE FOR THE REDUCTASE, TRANSFERASE, AND SYNTHETASE ENZYMES INVOLVED IN ALDEHYDE BIOSYNTHESIS

Abstract

Expression of bioluminescence by Escherichia coli containing recombinant Vibrio harveyi DNA.

When isogenic strains of Escherichia coli, RR1 (rec+) and HB101 (recA), were transformed with mapped recombinant plasmids known to contain Vibrio harveyi luciferase genes and large regions of DNA flanking on both sides, a small percentage (0.005%) of the colonies expressed high levels of luminescence (up to 10(12) quanta s-1 ml-1) in the absence of added aldehyde. The altered ability to express light was found to be due to a mutation in the host and not to an alteration in the recombinant DNA. When these bright colonies were cured of plasmid, they could be retransformed with cloned V. harveyi gene fragments in cis and in trans to yield luminescent colonies at 100% frequency. The maximum length of V. harveyi DNA required to produce light-emitting E. coli was shorter (6.3 kilobase pairs) than that required for expression of the V. fischeri system in E. coli. Cell extracts from bright clones contained wild-type levels of activity for the heteropolymeric (alpha beta) luciferase; fatty acid labeling revealed the presence of the three acylated polypeptides of the fatty acid reductase system which is involved in aldehyde biosynthesis for the luminescence reaction. The increased light emission in the mutant bacteria appeared to arise in part from production of higher levels of polycistronic mRNAs coding for luciferase.

[7] Cloning and expression of the genes from the bioluminescent system of marine bacteria

Publisher Summary Genes encoding at least five polypeptides are required to produce the enzymes necessary for bioluminescence in marine bacteria. These include genes coding for the two subunits of luciferase (α and β) and the three polypeptides of a fatty acid reductase complex, which supplies aldehyde for the bioluminescent reaction. Two regulatory polypeptides have been identified in the Vibriofischeri luminescence system. Transfer of the genes from luminescent bacteria into Escherichia coli and the study of their regulation can now readily be accomplished as very rapid and highly sensitive techniques are available for detection of the cloned genes and the analyses of the mRNAs and gene products. Selection of clones containing genes from the bioluminescence operons is generally based on the detection of light emission. Only the luciferase genes need be present as the aldehyde substrate can be supplied exogenously to E. coli and sufficient FMNH 2 and O 2 are present intracellularly to allow generation of light. Screening may be accomplished by the following approaches: visual scanning, exposure of colonies to film, scintillation counter readings, and hybridization probes.