Present and Potential Functions of Bacterial Transporters

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。知识价值。该项目将测试这样一种假设，即营养转运体具有功能的多样性和遗传的延展性，使它们能够在细菌的进化分化中充当楔子。单个转运蛋白基因将在细菌中进行研究，其中所有具有重叠或潜在重叠活性的转运蛋白都已被删除。然后确定每个转运体赋予的属性。将选择导致生长表型改变的突变基因，并对这些基因进行测序，以确定导致转运蛋白活性变化的氨基酸序列替换。这项工作将与贝氏不动杆菌一起进行，这种细菌具有独特的优势，因为它具有丰富的营养和极高的自然转化能力。将来，baylyi的遗传结构将允许该程序适用于确定来自其他细菌的转运蛋白的功能，包括那些具有复杂基因组或难以生长的细菌。细菌学的一个核心问题是，是什么让一个有机体与众不同？差异的明显来源是在基因组中，基因重排标志着细胞系进化分化的不连续性。不太明显，但可能同样重要的是活动上的差异，比如跨膜运输，它决定了细胞如何适应经常变化的环境。跨膜转运蛋白非常适合适应进化分化，因为它们与环境直接接触，数量丰富（通常由占基因组10%或更多的基因编码），并且它们具有冗余性，可以允许一个转运蛋白保持活性，而另一个具有重叠功能的转运蛋白适应新的挑战。要了解转运蛋白在多大程度上促进了进化分化，取决于对它们的功能和基因简单性的了解，它们可以从一种活动转移到另一种活动。目前的信息，基于大量的基因组序列数据，只提供了必要知识的一瞥，因为序列相似性是转运蛋白功能相似性的不良预测指标。该项目旨在开发一个系统，以了解baylyi转运蛋白的主要子集如何促进生理和潜在的遗传适应。该系统的一个优点是，它将为研究从其他细菌中容易克隆的基因提供基础。更广泛的影响。该项目将对科学培训和研究产生广泛影响。耶鲁大学的研究生和本科生将轮流参与这项研究。baylyi的遗传系统是世界各地教学实验室的基础，因为自然转化提供了一个基因是什么以及它做什么的快速演示。对这一研究项目的支持将有助于继续为大学入门教学提供细菌菌株，并创建有关运输遗传分析的高级教学实验室。作为耶鲁大学外展项目的一部分，主要研究成果将被纳入当地高中的课堂。这笔资金将允许进一步参与耶鲁大学SURF暑期研究项目，该项目向少数族裔本科生介绍研究性科学。研究结果将通过在国家会议上的发言和科学文献传播。细菌菌株将有助于其他实验室克隆和鉴定细菌转运体的其他基因，从而为科学界的进一步研究提供有用的资源。该项目的结果可能不仅会深入了解转运体进化的基本问题，即底物特异性如何演变，而且还会提供有助于生物修复和生物技术应用的知识库，这两者都依赖于代谢物的运输和加工。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual merit. The project will test the hypothesis that nutrient transporters possess functional versatility and genetic malleability that allow them to serve as wedges in the evolutionary divergence of bacteria. Individual transporter genes will be studied in bacteria in which all transporters with overlapping or potentially overlapping activities have been deleted. The properties conferred by each transporter will then be determined. Mutant genes that confer altered growth phenotypes will be selected, and these genes will be sequenced to determine amino acid sequence substitutions that caused the change in transporter activity. This work will be conducted with Acinetobacter baylyi, a bacterium that offers singular advantages for genetic investigation because of its nutritional breadth and its extraordinarily high competence for natural transformation. In the future, genetic constructs in A. baylyi will allow the procedures to be adapted to determine the functions of transporters from other bacteria, including those with complex genomes or those that are difficult to grow.A central question in bacteriology is what makes an organism stand apart? Obvious sources of differences are in genomes where gene rearrangements mark discontinuities in the evolutionary divergence of cell lines. Less obvious, but perhaps as significant, are differences in activities, such as transmembrane transport, that determine how a cell adapts to an often shifting environment. Transmembrane transporters are well suited for accommodating evolutionary divergence because they are poised to have direct contact with the environment, they are abundant (frequently encoded by genes accounting for 10% or more of a genome), and they possess a redundancy that may allow one transporter to maintain an activity while another transporter with overlapping function adapts to a new challenge. To understand the extent to which transporters contribute to evolutionary divergence depends upon knowledge about both their functions and the genetic simplicity with which they can shift from one activity to another. Present information, based upon an overwhelming amount of genome sequence data, provides only a glimpse of the necessary knowledge because sequence similarities are poor predictors of similarities in transporter function. This project is designed to develop a system for learning how a major subset of transporters contributes to physiology and potential genetic adaptation in A. baylyi. A merit of the system is that it will provide a foundation for study of genes easily cloned from other bacteria.Broader impacts. The project will have a broad impact on scientific training and research. Yale graduate students on rotation through the lab and undergraduates will participate in the research. The A. baylyi genetic system is the basis for teaching laboratories around the world because natural transformation provides a swift demonstration of what a gene is and what it does. Support for this research program will facilitate the continued supply of bacterial strains for introductory college teaching and to create upper level teaching laboratories on the genetic analysis of transport. Key research findings will be integrated into classes at local high schools as part of the Yale Outreach Program. Funding will allow further participation in the Yale SURF summer research program, which introduces minority undergraduate students to investigative science. Results of the research will be disseminated through presentations at national meetings and the scientific literature. Bacterial strains will assist other laboratories in cloning and characterizing other genes for bacterial transporters and will thereby provide a useful resource for further research in the scientific community. Results from the project are likely not only to give insight into a basic question of transporter evolution, that is how substrate specificity evolves, but also to provide a knowledge base that will contribute to bioremediation and biotechnological applications, both of which depend upon transport and processing of metabolites.