Genome-Wide Analysis of the Salmonella RpoN Regulon

智力上的功绩。了解细菌是如何调节其基因表达的，对于操纵具有工业、环境或农业用途的细菌至关重要。基因调控的第一步是让RNA聚合酶识别启动子，即RNA合成的起始位置。在细菌中，这一初始步骤需要一种所谓的西格玛因子，该因子与RNA聚合酶结合，并将其导向启动子。细菌使用主要的西格玛因子来转录他们的大多数基因，但通常拥有表达特定基因所需的一个或多个替代西格玛因子。RpoN是一种在农业、生物能源生产、生物修复和宿主-微生物相互作用中重要的各种微生物过程中涉及的基因转录所需的一种替代西格玛因子。从基因组序列中准确预测RpoN-RNA聚合酶全酶识别的启动子的能力对于剖析控制这些重要微生物过程的调控网络至关重要。这项研究计划将提供关于核心和上下文DNA序列的关键信息，这些序列对于模式菌鼠伤寒沙门氏菌的RpoN-RNA聚合酶全酶和启动子活性的识别非常重要。该项目还将通过研究RpoN-RNA聚合酶全酶识别的基因内位点(即基因内位点)的作用，以新的方式扩大对RpoN功能的理解。具体地说，将检查这些基因内结合位点的潜力：1)作为下游基因的内部启动子；2)刺激附近依赖RpoN的启动子的活性；或3)提高细菌内RpoN-RNA聚合酶全酶的水平。此外，这项研究将探索细胞内钾浓度(这对细菌对周围环境的某些变化做出反应非常重要)与特定RpoN依赖基因的调节之间可能存在的联系。由于鼠伤寒沙门氏菌中许多依赖于RpoN的基因参与了氮的吸收或特定糖的运输，这种联系可以提供一种机制来调节氮和碳代谢，以响应环境中的这些特定变化。从研究中获得的信息将为RpoN-RNA聚合酶全酶的功能以及肠杆菌血清型鼠伤寒沙门氏菌如何将环境信号整合到调控其活动的调控网络中提供新的线索。这些新信息可以应用于其他具有工业、环境或农业意义的细菌。该项目将通过让本科生和研究生参与研究来整合研究和教育，并将为参与该项目的学生的专业发展提供培训和机会。此外，高中教师将在暑期参与研究。参与的高中教师将能够将他们学习的新的、尖端的方法整合到他们教授的实验室课程中，这将有助于激励他们的学生考虑从事生物研究。来自代表性不足群体的学生将参与该项目，除了接受研究方面的指导外，他们还将通过STEM学科多样性学者等校园研究生组织获得社会支持和专业发展机会。参与该项目的研究生将在实验室帮助指导本科生，并通过入门指导计划接受这方面的正式培训。这一经历将促进研究生导师的专业发展，并提高本科生门生S的研究经验的质量。学生将在地区和国家会议上展示他们的发现，为他们提供网络机会，这将有助于他们的职业生涯。该项目还将为研究基础设施做出贡献，使研究人员能够访问为根据基因组序列预测RpoN型启动子而开发的软件。

Intellectual Merit. Understanding how bacteria regulate the expression of their genes is critical for the manipulation of bacteria that have industrial, environmental or agricultural uses. The first step in the regulation of a gene is for RNA polymerase to recognize the promoter, the site of initiation of RNA synthesis. In bacteria, this initial step requires a so-called sigma factor that binds to RNA polymerase and directs it to the promoter. Bacteria use a primary sigma factor for transcription of most their genes, but generally possess one or more alternative sigma factors required for expression of specific genes. RpoN is one such alternative sigma factor that is required for the transcription of genes involved in a variety of microbial processes that are important in agriculture, bioenergy production, bioremediation, and host-microbe interactions. The ability to accurately predict promoters recognized by RpoN-RNA polymerase holoenzyme from genome sequences is critical for dissecting the regulatory networks that control these important microbial processes. This research program will provide crucial information on core and contextual DNA sequences that are important for recognition by RpoN-RNA polymerase holoenzyme and promoter activity in the model bacterium Salmonella enterica serovar Typhimurium. The project will also expand understanding of RpoN function in new ways by examining the roles of sites within genes (i.e., intragenic sites) that are recognized by RpoN-RNA polymerase holoenzyme. Specifically, these intragenic binding sites will be examined for their potential to i) function as internal promoters for downstream genes; ii) stimulate the activity of nearby RpoN-dependent promoters; or iii) enhance the levels of RpoN-RNA polymerase holoenzyme inside the bacterium. In addition, the research will explore a possible link between cellular concentrations of potassium (which are important for the bacterium to respond to certain changes in the surrounding environment) and the regulation of specific RpoN-dependent genes. Since many of the RpoN-dependent genes in S. enterica serovar Typhimurium are involved either in nitrogen uptake or the transport of specific sugars, such a link could provide a mechanism for regulating nitrogen and carbon metabolism in response to those specific changes in the environment. The information gained from the research will shed new light on the function of RpoN-RNA polymerase holoenzyme and how S. enterica serovar Typhimurium integrates environmental signals into regulatory networks that govern its activity. Such new information can be applied to other bacteria that have industrial, environmental or agricultural significance.Broader Impacts. The project will integrate research and education by involving undergraduate and graduate students in the research, and it will provide training and opportunities for the professional development of the students involved in the project. In addition, high school teachers will participate in the research during the summer. Participating high school teachers will be able to integrate the new, cutting-edge methods they learn into the laboratory classes they teach, which will help inspire their students to consider careers in biological research. Students from underrepresented groups will be involved with the project and in addition to receiving mentoring in research, they will also receive social support and opportunities for professional development through campus graduate student organizations such as the Scholars for Diversity in STEM Disciplines. Graduate students involved with the project will help mentor undergraduates in the laboratory and receive formal training for this through an Entering Mentoring program. This experience will foster the professional development of the graduate mentors and enhance the quality of the research experience for the undergraduate protégés. Students will present their findings at regional and national conferences, providing them with networking opportunities, which will assist them in their careers. The project will also contribute to research infrastructure by providing researchers access to software developed to predict RpoN-type promoters from genome sequences.