Genetic Regulation of Bioluminescence in Marine Bacteria

本研究将从两个方面探讨哈维弧菌生物发光基因的调控：1)转录的密度依赖性调控；以及2)产生发光变体的机理。控制发光表达的调控基因与光产生的操纵子编码酶没有联系，也没有被克隆。然而，利用本地生物的转座子诱变，我们已经确定了编码调节功能的“缺失位点”。这个未连接的第二区域，现在被转座子插入标记，将被克隆并分析遗传电路：特定的lux基因和转录单位将被识别和测序；Lux功能将从突变表型分析中推断出来；并利用基因融合研究该区域基因的转录控制。众所周知，调节涉及一种叫做自诱导剂的信息素样物质，基因分析应该能深入了解细菌用来感知环境和对环境做出反应的机制。V. harveyi的发光变体在高频率下自发产生，并可以变回原始或新的发光形式。在发光变异DNA的Southern blot分析中，使用代表上述第二个位点部分的杂交探针获得了基因组组织变化与发光变异相关的迹象。我们将继续克隆涉及变异的DNA的整个区域，并精确地定义DNA结构的变化对发光强度的影响。发光变异可能是在海洋中遇到的各种变化的环境中生存所需的表型多样性产生的一般过程的一种表现，研究这一现象可以丰富我们对基因组可塑性如何产生多样性的理解。

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.