Intercellular Signalling in Vibrio harveyi

[506033] Bassler本研究的主要目标是探索细菌用于细胞间通讯的分子机制。这项工作的重点是一种发光细菌，哈维弧菌，目的是研究细胞间和细胞内信号传导的遗传和生化途径。细胞间通讯中涉及的基因、蛋白质、相互作用、化学修饰和信号的定义可能导致对信号如何被检测以及这些信息如何被整合、处理和转导以控制发光基因（lux）和相同控制网络或调节中的其他基因表达的分子理解。发光基因的表达调控是复杂的，似乎由相互关联的信号转导通路组成，这些信号转导通路调节编码发光酶的操纵子（luxCDABEGH）的转录。luxCDABEGH操纵子的表达受培养密度的强烈影响。V. harveyi分泌并响应细胞外信号分子，称为自诱导剂，在培养基中积累并诱导发光表达。luxLMN基因座编码了一个Lux信号响应系统。luxL和luxM基因是产生一种自体诱导剂（可能是羟基丁基同丝氨酸内酯）所必需的，而luxN基因是产生对该自体诱导剂的反应所必需的。LuxL、M和N突变体的表型分析表明，另外一个信号响应系统也控制着密度感应。对两个基因luxP和luxQ进行了鉴定、克隆和测序，并编码了第二个密度传感系统所需的功能。在luxP和luxQ中有缺陷的突变体对第二种自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，每个都包含类似于组氨酸蛋白激酶和反应调节结构域的序列区域。对突变体LuxN和LuxQ信号表型的分析表明，这两种信号响应途径会聚在一起调节哈维弧菌的发光表达。另一个函数，luxO，是两个密度相关信号的积分所必需的。LuxO在氨基酸序列上与双组分信号转导蛋白家族的响应调节结构域相似，对发光的表达起负调控作用。在野生型中，LuxO抑制的解除可能是由于与Lux信号系统中其他成分的相互作用。由于调控过程复杂，涉及细胞间和细胞内的信号传递，可能会揭示有趣的新机制。然而，复杂性不应该成为障碍，因为感官输入（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，遗传和生化方法也很发达。本研究包括对Lux系统1信号继电器的探索。研究的重点是系统1，因为自感应信号已经被识别并可获得。诱变程序将被用来构建lux基因，编码含有缺陷的蛋白质，这些缺陷会导致lux信号在转导序列的不同点终止。突变基因将通过反式分析确定，并确定具体的缺陷。突变的勒克斯调节位点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。体外生化分析将用于研究野生型和突变型Lux信号传导者的组合，以确定哪些Lux系统1成分相互作用以及信号传递中涉及的事件顺序。将分析Lux调节蛋白的DNA结合。凝胶迁移位移测定和DNA酶I足迹分析将用于确定正调控蛋白LuxR和负调控蛋白LuxO的DNA结合位点。利用体外生物化学和体内遗传学的结合应该有助于对信号传导机制的全面解释。教育职责包括设计和指导研究生的高级微生物遗传学课程和普林斯顿大学本科生的微生物多样性课程。首席研究员还将在1996-2000年期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，为少数民族暑期学生提供咨询，并参与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，在普林斯顿五所本科学院之一提供建议，并在课程审查委员会中任职。分析海洋细菌的发光基因将有助于了解这些细菌如何对环境作出反应。反过来，这可能会导致实际应用。光发射是一种相对容易的基因表达测量方法，因此可以用于改善与生物技术相关的细菌基因表达。* * *

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. ***