Genetic Regulation of Bioluminescence in Marine Bacteria

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This investigation will focus on two aspects of the regulation of genes for bioluminescence (lux) of Vibrio harveyi: (1) The control of expression of lux genes by cell density and other environmental signals and (2) the mechanism which generates diversity of spontaneous variants with different intensities of luminescene. The genetic circuitry controlling expression of luminescence appears to be complex and to consist of interconnected pathways of signal transduction which modulate transcription of operon (luxC,D,A,B,E) encoding the luminescence enzymes. At least seven loci controlling expression of luminescence (luxJ,K,L,M,N,O,R) have now been identified by a variety of methods in this laboratory. These loci are not linked to the operon encoding the luminescence enzymes and have been cloned for further analysis. Density- dependent regulation is known to involve a pherome-like substance called autoinducer, and studies are planned to identify those genes responsible for synthesis and response to this signal molecule as well as those genes required for the responses to other signals such as iron availability. Specific experiments include: (1) transposon mutagenesis of cloned fragments to localize the lux regulatory genes; (2) gene replacement mutagenesis in the native organism to obtain mutants for characterization of the function of the regulatory genes; (3) subcloning of lux regulatory genes to reassemble control function in recombinant E.coli; (4) construction of indicator gene fusions and mRNA measurements to define regulatory interactions and hierarchical control relationships; (5) DNA sequencing to analyze the structure of the regulatory proteins and the cis-acting control regions; and (6) genome structure probing with cloned lux genes to detect and characterize DNA rearrangements thought to cause variation in luminescence phenotypes. Genetic analysis of luminescence regulation should yield insight into mechanisms bacteria use to perceive and react to their environment, and analysis of variation should reveal mechanisms for creating the genetic diversity needed for adaption to varied and changing circumstances.