Evolution of Function of Bacterial PecS Family Transcription Factors

该项目关注细菌性植物病原体对宿主环境的适应有助于控制基因表达的机制。中心假设是细菌病原体在宿主感染时遇到的压力或营养限制导致代谢变化，进而导致疾病进展所需的基因表达。了解导致毒力基因表达的步骤对于最终控制经济上重要的植物的疾病进展是重要的。该计划还侧重于整合研究和教学，为研究生和本科生提供机会，以获得研究经验和熟练掌握与细菌遗传学和转录调控定量分析相关的技术。研究生将是开发这些研究项目不可或缺的。本科生也将有助于这一项目，特别强调增加代表性不足的群体成员的研究机会。对本科生的培训不仅为他们的深造做好准备，而且有助于培养一支有竞争力的STEM劳动力队伍。细菌性植物病原体在宿主组织中定殖时会遇到胁迫或营养限制。一种“严格反应”，其中基因活性发生全局变化，有利于有助于生存的基因的表达（对于植物病原体，这包括毒力基因）。严格的反应取决于“alarmones”的产生，例如磷酸化的鸟苷核苷酸通常被标记为（p）ppGpp。这个项目解决了嘌呤补救途径在严格反应过程中被动员的假设。酶黄嘌呤脱氢酶（Xdh）是嘌呤补救的关键，它偏向于鸟嘌呤核苷酸的形成途径。该项目的第一个目的是解决假设（p）ppGpp是一个转录因子的配体，抑制土壤细菌天蓝色链霉菌中编码Xdh的基因。使用S。coelicolor和植物病原体根癌农杆菌中，将编码（p）ppGpp合成酶的基因失活以研究（p）ppGpp在xdh基因活性中的作用。还将测定纯化的转录因子的DNA结合和（p）ppGpp减弱DNA结合的能力。Xdh也负责产生尿酸盐，该项目的另一个目的是解决以下假设：在严格反应期间产生的尿酸盐反过来作为可扩散的信使，在转录因子佩奇的控制下诱导基因的表达，该转录因子最初被鉴定为毒力基因表达的主调节因子。将在Xdh酶活性失活后以及在缺失xdh基因的菌株中检查pecS和其他确认的佩奇靶基因的表达，以确定尿酸盐作为体内配体的作用。还将对缺失佩奇基因的细菌菌株进行基因表达分析，以比较A. tumefaciens和S.天蓝色。因此，一个基本的假设是要测试的严格的反应过程中产生的尿酸盐作为一个可扩散的信使和佩奇的配体的功能，建立严格的反应和植物病原体的毒力基因的调节之间的新的连接。

This project concerns the mechanisms by which adaptation of bacterial plant pathogens to the host environment contributes to control of gene expression. The central hypothesis is that stress or nutrient limitation encountered by the bacterial pathogen upon host infection results in metabolic changes that in turn lead to expression of genes required for disease progression. Understanding the steps that lead to virulence gene expression is important for eventual control of disease progression in economically important plants. This program also focuses on integrating research and teaching by offering the opportunity for both graduate and undergraduate students to acquire research experience and proficiency in techniques associated with bacterial genetics and quantitative analysis of transcriptional regulation. Graduate students will be integral to developing these research projects. Undergraduate students will also contribute to this project, with special emphasis on increasing research access to members of underrepresented groups. The training of undergraduate students will not only prepare them for advanced studies, but contribute to the development of a competitive STEM workforce. Bacterial plant pathogens encounter stress or nutrient limitation when they colonize host tissues. A "stringent response" ensues in which global changes in gene activity occur, favoring expression of genes that contribute to survival (for plant pathogens, this includes virulence genes). The stringent response depends on production of "alarmones", such as the phosphorylated guanosine nucleotides commonly referred-to as (p)ppGpp. This project addresses the hypothesis that purine salvage pathways are mobilized during the stringent response. The enzyme xanthine dehydrogenase (Xdh) is critical for purine salvage, and it biases the pathway towards formation of guanine nucleotides. The first aim of this project is to address the hypothesis that (p)ppGpp is a ligand for a transcription factor that represses the gene encoding Xdh in the soil bacterium Streptomyces coelicolor. Using both S. coelicolor and the plant pathogen Agrobacterium tumefaciens, the genes encoding (p)ppGpp synthetases will be inactivated to address the role of (p)ppGpp in xdh gene activity. DNA binding by purified transcription factors and the ability of (p)ppGpp to attenuate DNA binding will also be determined. Xdh is also responsible for producing urate, and another aim of this project is to address the hypothesis that urate produced during the stringent response in turn functions as a diffusible messenger to induce expression of genes under control of the transcription factor PecS, which was originally identified as a master regulator of virulence gene expression. Expression of pecS and other confirmed PecS target genes will be examined upon inactivation of Xdh enzyme activity as well as in strains deleted for the xdh gene to determine the role of urate as a ligand in vivo. Gene expression analyses in bacterial strains deleted for the pecS gene will also be implemented to compare the PecS regulons in A. tumefaciens and S. coelicolor. Thus, a fundamental hypothesis to be tested is that urate produced during the stringent response functions as a diffusible messenger and as a ligand for PecS, establishing a novel connection between the stringent response and the regulation of virulence genes by plant pathogens.