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

大多数细菌通过启动基因表达的全局变化来科普饥饿，称为严格反应。 这种严格的反应导致生长相关基因关闭，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人类病原体如衣原体中不存在，其仅在宿主细胞内生长。 该项目的目的是了解这些细菌病原体如何科普饥饿，因为它们缺乏严格的反应，但它们通常能够在氨基酸饥饿中存活，这是由人类宿主诱导的，以对抗感染。该项目将解决有关病原体进化的基本问题以及它们对饥饿的独特反应方式。 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，将采用若干战略来增加美洲印第安人参与大学一级的研究。其中一些学生将向他们的阿尔马母校高中展示他们的研究，以帮助鼓励学生攻读STEM的大学学位。此外，各种科学主题的活动将被用来提高美国印第安高中和大学学生在STEM的兴趣/意识/参与。本研究的目的是了解的适应和后果，在进化到专性细胞内生长。专性细胞内细菌衣原体通过消除严格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结合在核糖体的A位点时，严格反应被激活，导致（p）ppGpp合成，这是一种全局调节剂，部分再生氨基酸库。衣原体不产生色氨酸（trp）或（p）ppGpp，因此它如何在trp饥饿中存活还不清楚。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trp饥饿，同时在全球范围内减少其翻译。这一观察结果对于细菌来说是不寻常的，因此代表了一个独特的机会来推进知识。在饥饿期间，转录物的氨基酸含量如何影响基因表达尚未完全表征。该假说认为，在色氨酸限制期间，衣原体基因的表达是由编码蛋白质的色氨酸含量决定的。本项目的目标如下：1）确定开放阅读框的trp含量是否控制在trp饥饿期间的转录; 2）确定在trp饥饿期间是否优先翻译trp-free蛋白质; 3）比较和对比衣原体对trp饥饿的反应（1 - 2）与其他trp营养缺陷型，化脓链球菌（野生型和严格突变型）和柯克斯体。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。 在与本土研究计划相结合，PI还计划在美国印第安人世界观的背景下开发一门科学课程。

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.