Genetic Regulation of Bioluminescence in Marine Bacteria

本次调查将集中在两个方面的监管， 哈氏弧菌生物发光（lux）基因：（1）对照 lux基因的表达受细胞密度和其他环境因素的影响 信号和（2）产生多样性的机制 具有不同发光强度的自发变体。 控制发光表达的遗传电路 似乎是复杂的，并包括相互连接的途径， 调控操纵子转录信号转导 (luxC、D、A、B、E）编码发光酶。 至少七 控制发光表达的基因座（luxJ、K、L、M、N、O、R）具有 现在在这个实验室里用各种方法鉴定出来了。 这些基因座不与编码发光的操纵子相连 酶，并已被克隆用于进一步分析。 密度- 已知依赖性调节涉及信息素样物质 称为自诱导物，研究计划确定这些基因， 负责合成和响应这种信号分子， 以及对其他信号做出反应所需的基因 例如铁可用性。 具体实验包括：（1） 克隆片段的转座子诱变以定位lux 调控基因;（2）天然基因中的基因置换诱变 生物体获得突变体的功能表征 调控基因;（3）亚克隆lux调控基因， 在重组大肠杆菌中重组控制功能;（4）构建 的指示基因融合和mRNA测量，以确定 监管互动和层级控制关系;（5） DNA测序分析调控蛋白的结构 和顺式作用控制区;和（6）基因组结构 用克隆的lux基因探测以检测和表征DNA 被认为引起发光变化的重排 表型 发光调节的遗传分析应 深入了解细菌用来感知和反应的机制 他们的环境，和变异分析应该揭示 创造适应所需遗传多样性的机制 适应各种变化的环境

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.