Intercellular Signalling in Vibrio harveyi

9506033巴斯勒这项研究的广泛目标是探索细菌用于细胞间交流的分子机制。这项工作的重点是一种发光细菌，哈维氏弧菌，目的是从遗传和生物化学的角度研究细胞内和细胞内的信号传导途径。对参与细胞间通讯的基因、蛋白质、相互作用、化学修饰和信号的定义可能导致分子理解信号是如何被检测到的，以及这些信息是如何被整合、处理和转导以控制发光基因(Lux)和其他基因在同一控制网络或调节子中的表达。哈维氏弧菌中发光基因的表达调控是复杂的，似乎由相互连接的信号转导途径组成，这些信号转导途径调控编码发光酶的操纵子(LuxCDABEGH)的转录。豪华CDABEGH操纵子的表达受培养密度的强烈影响。哈维氏弧菌分泌细胞外信号分子并对其作出反应，这些分子被称为自身诱导剂，积聚在培养基中并诱导发光的表达。一个Lux信号-响应系统由LuxLMN轨迹编码。自体诱导物(可能是羟丁基高丝氨酸内酯)的产生需要Lux1和LuxM基因，而对该自体诱导物的反应需要LuxN基因。对Lux1、M和N突变体的表型分析表明，一个额外的信号反应系统也控制着密度感知。已鉴定、克隆、测序并编码第二密度敏感系统所需功能的两个基因：LuxP和LuxQ。具有LuxP和LuxQ缺陷的突变体对第二种自身诱导物质的反应是有缺陷的。LUXQ和LUXN类似于双组分信号转导蛋白家族的成员，每一个都包含类似组氨酸蛋白激酶和反应调节结构域的序列区域。对突变的LuxN和LuxQ信号表型的分析表明，这两条信号-反应通路在哈维氏弧菌中共同调节发光的表达。两个密度相关信号的积分需要另一个函数LUXO。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，对发光的表达起负性调控作用。野生型LUXO抑制的缓解可能是通过与Lux信号系统中的其他组件相互作用而产生的。由于调控过程是复杂的，涉及细胞间和细胞内的信号传递，因此可能会揭示有趣的新机制。然而，复杂性不应成为障碍，因为感觉输入(化学信号)和输出(光发射)是可以定义的，并且可以方便地控制和测量，而且遗传和生化方法发展得很好。本研究包括对Lux System 1信号继电器的探索。研究集中在系统1上，因为自动感应器信号已经被识别并可以获得。突变程序将被用来构建编码含有缺陷的蛋白质的Lux基因，这些缺陷应该导致Lux信号在转导序列的不同点处终止。突变基因将通过反式分析进行鉴定，并确定特定的缺陷。突变的lux基因将被转移到哈维氏弧菌的基因组中，然后进行体内表型分析。体外生化分析将被用来研究野生型和突变型Lux信号的组合，以确定哪些Lux系统1组件相互作用，以及信号传递中涉及的事件序列。将分析Lux调节蛋白与DNA的结合。凝胶迁移率改变分析和DNase I足迹分析将被用来确定正负调控蛋白LuxR和Luxo的DNA结合位点。使用体外生物化学和体内遗传学的组合应该有助于开发对信号机制的全面解释。教育职责包括为研究生设计和指导一门高级微生物遗传学课程，以及为普林斯顿大学本科生设计一门微生物多样性课程。首席研究员还将在1996-2000年间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，为少数族裔暑期学生提供咨询，以及参加一个面向高中教师的科学推广计划。此外，首席调查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中的一所提供建议，并在课程审查委员会任职。分析海洋细菌的发光基因将导致对这些细菌如何对环境做出反应的理解。这反过来可能导致实际应用。光发射是一种相对容易测量基因表达的方法，因此在生物技术相关应用中可以用来提高细菌的基因表达。***

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. ***