Excellence in Research:Understanding environmental adaptation by elucidating the changes in molecular mechanisms associated with genetic selection in two-component response systems

细菌，就像所有的生物一样，经常发现自己处于恶劣的环境中，从极端的温度，到有毒的废物，甚至接触抗生素。为了生存，它们必须迅速做出反应。它们对生存的反应有两种方式：它们要么通过改变基因表达来适应环境，要么通过特异性地改变DNA来适应环境。双组分响应系统（TCRS）是细菌用于环境适应的研究最多的遗传因子之一。先前的研究表明，在极端条件下，TCRS基因会发生突变，从而使细菌得以存活，但这些突变对细菌生长和繁殖的确切影响尚不清楚。本研究旨在通过研究实验进化过程中获得的突变，以及这些突变如何影响细菌的整体适应性，了解TCRS在适应高水平银（一种已被充分研究的应激源）中的作用。该研究将有利于四个更广泛的影响领域：(1)更大的科学界，因为TCRS的合成工程已被提出用于从生物修复到抗菌药物设计的许多应用。(2)通过指导HBCU的本科生和博士生整合微生物学、分子生物学、进化、基因组学和计算生物学的方法，提高STEM领域代表性不足的少数族裔的保留率，使他们在专业学校和/或STEM职业中更具竞争力。(3)通过志愿参加当地的K-5课外活动，让当地社区接触微生物和进化的世界；(4)利用这些数据撰写教学案例研究，供世界各地的教育工作者使用。双组分反应系统（TCRS）是细菌环境适应中研究得最好的遗传因子之一，尽管对其在适应中的作用知之甚少。本研究的总体目标是表明进化适应通常是通过在长时间的驯化后获得TCRS基因突变而发生的。这就允许了差异繁殖的选择，导致适应细菌的生存，尽管可能对它们的适应性和功能产生负面影响。TCRS由两种蛋白质组成，一种是组氨酸激酶（HK），它能感知外部环境的变化，并通过自磷酸化激活。然后将磷酸盐转移到激活相关应答基因转录的应答调节因子（RR）。在这项研究中，研究人员将评估大肠杆菌通过实验进化适应银而获得的TCRS突变。在银适应菌株中，三个独立的研究发现了两种TCRS蛋白的突变：CusS，一种调节铜/银稳态的HK；OmpR，一种调节外膜孔蛋白表达的RR。本研究的动机是通过(1)将TCRS特定区域的突变与特定表型相关联，来显示与获得此类突变相关的细胞成本；(2)评估与TCRS生物学相关突变相关的适应度成本和逆转率；(3)识别单个TCRS突变影响的整体细胞生理变化。创新之处在于对突变的评估，这些突变是由生物体通过实验进化选择的，因此具有生物学相关性。该项目将进一步了解TCRS在适应中的作用以及与这些遗传适应相关的适应度成本，然后可以通过将这些表型与基因组决定因素联系起来，用于预测竞争和适应度的结果。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Bacteria, just as all living organisms, often find themselves in harsh environments ranging from extreme temperatures, to toxic waste, or even exposure to antibiotics. In order to survive, they must react quickly. Their response for survival comes in two ways: they either can acclimate through changes in gene expression, or they can adapt by specifically changing their DNA. Two-component response systems (TCRS) are one of the best studied genetic elements bacteria use for environmental acclimation. Previous work has shown that under extreme conditions, TCRS genes acquire mutations that allow the bacteria to survive, but the exact effect these mutations have on the growth and reproduction of bacteria are unknown. This research aims to understand the role that TCRS play in adaptation to high levels of silver (a well-studied stressor), by studying mutations acquired during experimental evolution, and how those mutations will affect the overall fitness of the bacteria. The research will benefit four broader impact areas: (1) The larger scientific community, as synthetic engineering of TCRS have been proposed for many applications ranging from bioremediation to antimicrobial drug design. (2) By improving underrepresented minority retention in STEM fields by mentoring undergraduates and a PhD student at an HBCU to integrate approaches in microbiology, molecular biology, evolution, genomics, and computational biology to make them more competitive for professional school and/or STEM careers. (3) By engaging and exposing local communities to the world of microbiology and evolution by volunteering in local K-5 afterschool programs; and (4) by using the data to write teaching case studies to be used by educators around the world.Two-component response systems (TCRS) are among the best studied genetic elements for environmental acclimation in bacteria, although very little is known about their role in adaptation. The overall goal of this research is to show that evolutionary adaptation commonly occurs through the acquisition of mutations in TCRS genes after prolonged acclimation. This then allows selection for differential reproduction leading to the survival of the adapted bacteria, although likely with negative consequences on their fitness and function. TCRS consist of two proteins, a histidine kinase (HK) that senses changes in the external environment and is activated via autophosphorylation. The phosphate is then transferred to a response regulator (RR) which activates transcription of relevant response genes. In this study, the investigators will assess TCRS mutations acquired through the adaptation of Escherichia coli to silver via experimental evolution. In silver adapted strains, three independent studies identified mutations in two TCRS proteins: CusS, a HK which regulates copper/silver homeostasis; and OmpR, a RR which regulates outer membrane porin expression. The motivation for this study is to show the cellular costs associated with the acquisition of such mutations by (1) associating mutations in specific domains of TCRS to a particular phenotype; (2) assessing the fitness cost and reversion rates associated with biologically relevant mutations in TCRS; and (3) identifying changes in global cellular physiology affected by mutations in a single TCRS. The innovation lies in the assessment of mutations that have been selected by the organism through experimental evolution and are therefore biologically relevant. The project will lead to further understanding of the role TCRS play in adaptation and the fitness cost associated with these genetic adaptations which may then be used to predict the outcome of competition and fitness by linking these phenotypes to genomic determinants.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.