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

卓越的研究：通过阐明与二元反应系统中遗传选择相关的分子机制的变化来了解环境适应

• 批准号: 1900220
• 负责人: Misty Thomas
• 金额: $ 49.99万
• 依托单位: North Carolina Agricultural & Technical State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1900220&HistoricalAwards=false
• 关键词: Excellence; Research; Understanding; environmental; adaptation

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.

细菌，就像所有活着的有机体一样，经常发现自己处于从极端温度到有毒废物，甚至接触抗生素的恶劣环境中。为了生存，他们必须迅速做出反应。它们对生存的反应有两种方式：一种是通过基因表达的变化来适应，另一种是通过改变自己的DNA来适应。两组分反应系统(TCRS)是细菌用于环境适应的最好研究的遗传元件之一。以前的工作表明，在极端条件下，TCRS基因会获得允许细菌存活的突变，但这些突变对细菌生长和繁殖的确切影响尚不清楚。这项研究的目的是通过研究在实验进化过程中获得的突变，以及这些突变将如何影响细菌的整体适应性，来了解TCRS在适应高水平的银(一种研究得很好的压力源)方面所起的作用。这项研究将惠及四个更广泛的影响领域：(1)更大的科学界，因为已经提出了TCRS的合成工程，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、进化论、基因组学和计算生物学的方法，使他们在职业学校和/或STEM职业生涯中更具竞争力，从而改善STEM领域中代表性较低的少数群体的留住情况。(3)通过志愿参加当地的K-5课外项目，让当地社区接触微生物学和进化的世界；以及(4)使用这些数据编写教学案例研究，供世界各地的教育工作者使用。两组分反应系统(TCRS)是细菌环境适应的遗传因素研究最多的因素之一，尽管人们对它们在适应中的作用知之甚少。这项研究的总体目标是表明，进化适应通常是通过在长期驯化后获得TCRS基因突变而发生的。这就允许选择进行差异繁殖，导致适应细菌的生存，尽管可能会对它们的适应性和功能产生负面影响。TCR由两种蛋白质组成，一种是组氨酸激酶(HK)，它可以感知外部环境的变化，并通过自身磷酸化来激活。然后，磷酸盐被转移到反应调节器(RR)，后者激活相关反应基因的转录。在这项研究中，研究人员将评估通过实验进化使大肠杆菌适应银而获得的TCRS突变。在银适应菌株中，三项独立的研究发现了两种TCRS蛋白的突变：cuss，一种调节铜/银动态平衡的HK；以及OmpR，一种调节外膜孔蛋白表达的RR。这项研究的动机是通过(1)将TCRS的特定区域的突变与特定的表型相关联；(2)评估与生物相关的TCRS突变相关的适合度成本和返回率；以及(3)确定受单个TCRS突变影响的全球细胞生理学的变化，从而显示与获得此类突变相关的细胞成本。创新之处在于评估有机体通过实验进化选择的突变，因此它们在生物学上是相关的。该项目将导致进一步了解TCRS在适应中所起的作用，以及与这些遗传适应相关的适应成本，然后通过将这些表型与基因组决定因素联系起来，可以用来预测竞争和适应的结果。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Reporting off-target effects of recombinant engineering using the pORTMAGE system

使用 pORTMAGE 系统报告重组工程的脱靶效应

• DOI: 10.1016/j.mimet.2022.106627
• 发表时间: 2023
• 期刊: Journal of Microbiological Methods
• 影响因子: 2.2
• 作者: Sanders, Brittany R.;Townsend, Sydney E.;Ford, Maria L.;Graves, Joseph L.;Thomas, Misty D.
• 通讯作者: Thomas, Misty D.