Intercellular Signalling in Vibrio harveyi

9506033 Bassler这项研究的主要目标是探索细菌用于细胞间通讯的分子机制。这项工作的重点是发光的细菌，哈维氏弧菌，与遗传和生化检查间和细胞内信号的途径的目标。定义的基因，蛋白质，相互作用，化学修饰，和信号参与细胞间的通信可能会导致一个分子的理解信号是如何检测到的，以及如何整合，处理和转导这些信息来控制发光基因（lux）和其他基因在同一个控制网络或调节子的表达。哈维氏弧菌中发光基因表达的调节是复杂的，并且似乎由调节编码发光酶的操纵子（luxCDABEGH）的转录的相互连接的信号转导途径组成。luxCDABEGH操纵子的表达受培养物密度的强烈影响。哈维氏弧菌分泌并响应细胞外信号分子，称为自诱导物，其在培养基中积累并诱导发光的表达。一个Lux信号响应系统由luxLMN基因座编码。 luxL和luxM基因是产生自诱导物（可能是（羟丁基高丝氨酸内酯））所需的，而luxN基因是对该自诱导物的应答所需的。 LuxL，M和N突变体的表型分析表明，一个额外的信号响应系统也控制密度传感。两个基因，luxP和luxQ，进行了鉴定，克隆和测序，并编码所需的第二个密度感应系统的功能。具有luxP和luxQ缺陷的突变体在对第二种自诱导物物质的反应中是有缺陷的。LuxQ和LuxN类似于双组分信号转导蛋白家族的成员，并且各自含有类似于组氨酸蛋白激酶和反应调节结构域的序列区域。突变体LuxN和LuxQ信号表型的分析表明，这两个信号响应途径收敛，以调节哈氏弧菌发光的表达。 另一个函数luxO是两个密度相关信号的积分所需的。 LuxO在氨基酸序列上与双组分信号转导蛋白家族的反应调节结构域相似，其对控制发光的表达起负作用。 LuxO在野生型中的抑制的缓解可能是由于与Lux信号系统中的其他组分的相互作用。由于调节过程很复杂，涉及细胞间和细胞内信号传递，因此可以揭示有趣的新机制。 然而，复杂性不应该成为障碍，因为感觉输入（化学信号）和输出（光发射）可以被定义，并且可以方便地控制和测量，并且遗传和生物化学方法学已经发展得很好。 这项研究包括探索勒克斯系统1信号继电器。 研究集中在系统1，因为自诱导物信号已被识别并可获得。将采用诱变程序来构建编码含有缺陷的蛋白质的lux基因，所述缺陷应导致转导序列中不同点处的Lux信号终止。通过反式分析鉴定突变基因并确定特定缺陷。将突变的lux调节基因座转移到哈维氏弧菌的基因组中，然后进行体内表型分析。 体外生物化学分析将用于研究野生型和突变型Lux信号转导子的组合，以确定哪些Lux系统1组分相互作用以及参与信号传递的事件序列。 将分析Lux调节蛋白的DNA结合。将采用凝胶迁移率变动试验和DNA酶I足迹分析来确定阳性和阴性调节蛋白LuxR和LuxO的DNA结合位点。 使用体外生物化学和体内遗传学相结合，应有助于发展一个全面的解释信号传导机制。 教育职责包括为普林斯顿大学的研究生设计和教授高级微生物遗传学课程和本科生微生物多样性课程。 首席研究员还将在1996-2000年在冷泉港教授高级细菌遗传学课程。 其他教育计划包括在我的实验室指导本科生和研究生，为少数民族暑期学生提供咨询，并参加高中教师的科学推广计划。 此外，首席研究员将是她所在系的本科生代表，在普林斯顿大学五所本科学院之一提供建议，并在课程审查委员会任职。 分析海洋细菌的发光基因将有助于了解这些细菌对环境的反应。 这反过来又可能带来实际应用。 光发射是基因表达的相对容易的量度，因此可用于在与生物技术相关的应用中改善细菌基因表达。 ***

9506033 Bassler The broad goal of this research is to explore the molecular mechanisms that bacteria use for intercellular communication. This work focuses on a luminous bacterium, Vibrio harveyi, with the objective of examining genetically and biochemically the pathways of inter- and intracellular signalling. Definition of the genes, proteins, interactions, chemical modifications, and signals involved in intercellular communication could lead to a molecular understanding of how signals are detected and how this information is integrated, processed, and transduced to control expression of luminescence genes (lux) and other genes in the same control network or regulon. Regulation of the expression of luminescence genes in V. harveyi is complex and appears to consist of interconnected pathways of signal transduction which modulate the transcription of the operon (luxCDABEGH) encoding the luminescence enzymes. The expression of the luxCDABEGH operon is strongly influenced by the density of the culture. V. harveyi secretes and responds to extracellular signal molecules, called autoinducers, which accumulate in the culture medium and induce the expression of luminescence. One Lux signal-response system is encoded by the luxLMN locus. The luxL and luxM genes are required for the production of an autoinducer (probably (hydroxybutryl homoserine lactone), and the luxN gene is required for the response to that autoinducer. Analysis of the phenotypes of LuxL, M and N mutants indicated that an additional signal-response system also controls density sensing. Two genes, luxP and luxQ, were identified, cloned and sequenced and encode functions required for this second density-sensing system. Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance. LuxQ and LuxN are similar to members of the family of two-component, signal transduction proteins and each contains regions of sequence resembling both the histidine protein kinase and the response regulator domains. Anal ysis of mutant LuxN and LuxQ signalling phenotypes indicated that these two signal-response pathways converge to regulate expression of luminescence in Vibrio harveyi. Another function, luxO, is required for the integration of the two density-dependent signals. LuxO, which is similar in amino acid sequence to the response regulator domain of the family of two-component, signal transduction proteins, acts negatively to control expression of luminescence. Relief of repression by LuxO in the wild-type could result from interactions with other components in the Lux signalling system. Since the regulatory process is complex and involves both intercellular and intracellular signal transmission interesting new mechanisms could be revealed. However, complexity should not be a barrier because the sensory input (chemical signal) and the output (light emission) can be defined and can be conveniently controlled and measured, and the genetic and biochemical methodology are well-developed. This research includes an exploration of the Lux system 1 signal relay. The studies are focused on system 1 because the autoinducer signal has been identified and is obtainable. Mutagenesis procedures will be employed to construct lux genes encoding proteins containing defects that should result in termination of the Lux signal at different points in the transduction sequence. The mutant genes will be identified by in trans analysis and the specific defects determined. The mutated lux regulatory loci will be transferred to the genome of V. harveyi followed by in vivo phenotype analysis. In vitro biochemical analyses will be used to study combinations of wildtype and mutant Lux signallers to determine which Lux system 1 components interact and the sequence of events involved in signal relay. DNA binding by Lux regulatory proteins will be analyzed. Gel mobility shift assays and DNase I footprint analyses will be employed to determine the DNA binding sites of the positive and negative regulatory proteins LuxR and LuxO. Using a combination of in vitro biochemistry and in vivo genetics should aid in development of a comprehensive explanation of the signalling mechanism. Educational responsibilities include both the design and instruction of an Advanced Microbial Genetics course for graduate students and a Microbial Diversity course for undergraduates at Princeton. The principal investigator will also teach the Advanced Bacterial Genetics course at Cold Spring Harbor from 1996-2000. Other educational plans include mentoring of both undergraduate and graduate students in my laboratory, counseling minority summer students, and participating in a science outreach program for high school teachers. Additionally, the principal investigator will be her department's undergraduate representative, advise at one of the five Princeton undergraduate colleges, and serve on the committee for reviewing curriculum. %%% Analyzing the light-producing genes of a marine bacterium will lead to an understalding of how these bacteria respond to their environment. This could, in turn, lead to practical applications. Light emission is a relatively easy measure of gene expression, and so can be used to improve bacterial gene expression in applications related to biotechnology. ***