CAREER: Effects of Reductive Evolution on Amino Acid Stress Responses in Bacteria

大多数细菌通过启动被称为严格反应的全球基因表达变化来应对饥饿。这种严格的反应导致生长相关基因被关闭，而在营养限制条件下生存所需的基因被激活。这种反应在专性细胞内的人类病原体中是不存在的，例如衣原体，它们只在宿主细胞内生长。这个项目的目的是了解这些细菌病原体如何应对饥饿，因为它们缺乏严格的反应，但它们往往能够在氨基酸饥饿中生存下来，氨基酸饥饿是由人类宿主诱导的，以对抗感染。这个项目将解决有关病原体的进化以及它们对饥饿做出反应的独特方式的基本问题。更实际的是，它将导致确定潜在的药物和诊断目标，以治疗和识别由衣原体等病原体引起的疾病。为了扩大参与，将使用几种策略来增加美国印第安人对大学水平研究的参与。其中一些学生将把他们的研究成果提交给他们的母校高中，以帮助鼓励学生攻读STEM专业的大学学位。此外，将利用各种以科学为主题的活动，提高美国印第安人高中生和大学生对STEM的兴趣/认识/参与。这项研究的目的是了解适应及其后果的进化，以专职细胞内生长。专性细胞内细菌衣原体通过消除严格的反应和氨基酸生物合成途径使其基因组最小化。当不带电荷的tRNA与核糖体的A位结合时，就会激活严格的反应，导致(P)ppGpp合成，这是一种全球调节因子，部分地再生氨基酸库。衣原体不产生色氨酸(Trp)或(P)ppGpp，因此它如何在Trp饥饿中生存尚不清楚。PI先前的工作表明，衣原体在全球范围内增加其转录，以响应色氨酸饥饿，而全球减少其翻译。这种观察对细菌来说是不寻常的，因此，代表着一个独特的机会来促进知识的发展。转录本的氨基酸含量如何影响饥饿期间的基因表达还没有完全的特征。假设在色氨酸限制期间，衣原体基因的表达由编码蛋白的色氨酸含量决定。本项目的目标如下：1)确定Trp饥饿期间开放阅读框中Trp的含量是否控制转录；2)确定Trp饥饿过程中无Trp的蛋白是否优先翻译；3)比较和对比衣原体对Trp饥饿的反应(1和2)与其他Trp营养缺陷菌、化脓性链球菌(野生型和严格突变体)和Coxiella的反应。该项目更广泛影响的主要目标是增加美洲印第安人对STEM研究的参与。与土著研究计划相结合，该协会还计划在美洲印第安人世界观的背景下开发一门关于科学的课程。

Most bacteria cope with starvation by launching a global change in gene expression known as the stringent response. The stringent response causes growth-related genes to be turned off and genes that are needed for survival under nutrient-limiting conditions to be activated. This response is absent in obligate intracellular human pathogens such as Chlamydia, which only grow inside a host cell. The aim of this project to understand how these bacterial pathogens cope with starvation, since they lack the stringent response and yet they are often able to survive amino acid starvation, which is induced by the human host to combat the infection. This project will address fundamental questions about the evolution of pathogens and the unique manner in which they respond to starvation. More practically, it will result in the identification of potential drug and diagnostic targets to treat and identify diseases caused by pathogens such as Chlamydia. In an effort to broaden participation, several strategies will be used to increase participation of American Indians in college-level research. Some of these students will present their research to their alma mater high schools to help encourage the students to pursue college degrees in STEM. In addition, various science-themed activities will be used to increase interest/awareness/participation of American Indian high school and college students in STEM. This study aims to understand the adaptations and their consequences in evolving to obligate intracellular growth. The obligate intracellular bacterium Chlamydia has minimized its genome by eliminating the stringent response and amino acid biosynthesis pathways. The stringent response is activated when an uncharged tRNA binds in the A site of the ribosome, leading to (p)ppGpp synthesis, a global regulator that, in part, regenerates amino acid pools. Chlamydia does not make tryptophan (trp) or (p)ppGpp thus how it survives trp starvation is not well understood. Prior work by the PI has shown that Chlamydia globally increases its transcription in response to trp starvation while globally decreasing its translation. This observation is unusual for a bacterium, and therefore, represents a unique opportunity to advance knowledge. How the amino acid content of a transcript affects gene expression during starvation has not been fully characterized. The hypothesis is that, during trp limitation, chlamydial gene expression is determined by the trp content of the encoded protein. The goals of this project are the following: 1) to determine if the trp content of open reading frames controls transcription during trp starvation; 2) to determine if trp-free proteins are preferentially translated during trp starvation; 3) to compare and contrast the chlamydial response to trp starvation (1&2) with that of other trp auxotrophs, Streptococcus pyogenes (wild-type and stringent mutant) and Coxiella. The major goal of the broader impacts of this project is to increase participation of American Indians in STEM research. In conjunction with the Native Studies Program, the PI also plans to develop a course on science in the context of the American Indian worldview.