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. ***
