This investigation will focus on two aspects of the regulation of genes for bioluminescence (lux) from Vibrio harveyi: 1) the density-dependent control of transcription; and 2) the mechanism which generates luminous variants. The regulatory genes controlling expression of luminescence are not linked to the operon encoding enzymes for light production and have not been cloned. However, using transposon mutagenesis of the native organism, we have identified the "lost locus" encoding regulatory function(s). This unlinked second region, now marked by transposon insertion, will be cloned and the genetic circuitry analyzed: the specific lux genes and transcriptional units will be identified and sequenced; the Lux functions will be inferred from analysis of mutant phenotypes; and transcriptional control of genes in this region will be examined by using gene fusions. Regulation is known to involve a pheromone-like substance called autoinducer, and genetic analysis should yield insight into mechanisms bacteria use to perceive and react to their environment. Luminous variants of V. harveyi arise spontaneously at high frequencies and can change back to the original or new luminous forms. Indication that changes in genome organization correlate with luminescence variation has been obtained by using a hybridization probe representing part of the second locus mentioned above in Southern blot analysis of DNA from luminous variants. We will proceed to clone the entire region of DNA involved in variation and define precisely the changes in DNA structure which affect the intensity of light emission. Luminous variation could be one manifestation of a general process for creating the diversity of phenotypes needed for survival in the varied and changing circumstances encountered in the ocean, and studying this phenomenon could enrich our understanding of how genome plasticity generates diversity.
