Genome-Wide Analysis of the Salmonella RpoN Regulon

智力优势。了解细菌如何调节其基因的表达对于操纵具有工业，环境或农业用途的细菌至关重要。基因调控的第一步是RNA聚合酶识别启动子，启动子是RNA合成的起始位点。在细菌中，这一初始步骤需要一个所谓的sigma因子，它与RNA聚合酶结合并将其引导到启动子。细菌使用主要的σ因子来转录其大多数基因，但通常具有表达特定基因所需的一种或多种替代σ因子。 RpoN是一种这样的替代σ因子，其是参与在农业、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的基因转录所需的。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核心和上下文的DNA序列，这是重要的识别RpoN-RNA聚合酶全酶和启动子活性的模式细菌沙门氏菌肠道血清型鼠伤寒沙门氏菌的关键信息。该项目还将通过研究基因内位点的作用（即，基因内位点）被RpoN-RNA聚合酶全酶识别。 具体而言，将检查这些基因内结合位点的潜力，以i）作为下游基因的内部启动子; ii）刺激附近RpoN依赖性启动子的活性;或iii）增强细菌内RpoN-RNA聚合酶全酶的水平。此外，该研究还将探索细胞内钾浓度（这对细菌应对周围环境的某些变化很重要）与特定RpoN依赖性基因调控之间的可能联系。由于S.肠道血清型鼠伤寒沙门氏菌参与氮吸收或特定糖的运输，这种联系可以提供一种调节氮和碳代谢的机制，以响应环境中的这些特定变化。本研究的结果为进一步了解RpoN-RNA聚合酶全酶的功能以及S.肠道血清型鼠伤寒杆菌将环境信号整合到控制其活性的调节网络中。这些新的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提供专业发展的培训和机会。此外，高中教师将在夏季参加研究。参与的高中教师将能够将他们学到的新的尖端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，除了接受研究指导外，他们还将通过校园研究生组织（如STEM学科多样性学者）获得社会支持和专业发展机会。参与该项目的研究生将帮助指导实验室的本科生，并通过进入指导计划接受正式培训。这种经验将促进研究生导师的专业发展，并提高本科生学生的研究经验的质量。 学生将在区域和国家会议上展示他们的研究结果，为他们提供网络机会，这将有助于他们的职业生涯。该项目还将通过为研究人员提供开发用于从基因组序列预测RpoN型启动子的软件来促进研究基础设施。

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.