Regulation of Type II Restriction-Modification Systems

智力优势：生物圈由超过1030种细菌和古细菌控制，这个数字大致相当于“大爆炸”以来的秒数。由于细菌是无性的，它们快速获得新基因（如抗生素抗性或降解特定化学物质的能力）取决于物种之间大量的基因交换。尽管它至关重要，但控制细菌之间这种基因流动速率的因素却知之甚少。但很明显，限制修改（RM）系统起着关键作用。RM系统产生两种酶，一种是切割未受保护的DNA（如从另一个细胞或细菌病毒进入的DNA）的核酸酶，另一种是保护细菌自身DNA不受核酸酶破坏的甲基转移酶。关于这些系统的功能和作用的许多基本问题仍然存在。考虑到RM系统对于理解正在进行的细菌进化的重要性，它对从植物疾病到生物地球化学和全球变暖的所有事情都有影响，解决我们理解中的这一缺陷至关重要。本课题的目的是阐明C蛋白控制的模型RM系统的调控设计，以及它在活细菌细胞中的作用。C蛋白激活自身基因和下游核酸酶基因的转录，因此核酸酶的表达被延迟，直到C蛋白积累。第一个目的是确定成对核酸酶和甲基转移酶的可接受相对水平的范围。如果甲基转移酶/核酸酶比例过低，细胞可能因DNA损伤而死亡。如果这个比例太高，整个群体可能会被病毒（噬菌体）杀死，这些病毒（噬菌体）逃脱了核酸酶并被甲基化。第二个目标确定在包括压力在内的一系列生长条件下相对核酸酶和甲基转移酶水平的自然变化。第三个目的是表征控制核酸酶生产的时间敏感控制系统。数学模型提供了将被测试的预测，以查看系统设计是否被准确理解。更广泛的影响：该项目的一个组成部分是促进从高中科学教师到博士后的科学家的教育和研究培训。教师和本科生将分别研究众多已经克隆但尚未鉴定的C蛋白同源物中的一个。将招募高中科学教师和本科科学专业学生进行暑期研究实习。实验室成员将继续接待高中生参加科学展览项目，并将在高中做生物学/职业教学志愿者。此外，至少一名研究生和一名博士后将被鼓励获得生物信息学证书，作为他们培训的一部分。这些人将做好充分的准备，为细菌基因组学的新兴领域做出贡献。该项目还将丰富研究生水平的教学，进一步促进数学建模在细菌研究中的应用。

Intellectual Merit:The biosphere is dominated by over 10e30 bacteria and archaea, a number roughly equivalent to the number of seconds since the "Big Bang". Since bacteria are asexual, their rapid acquisition of new genes (for things like antibiotic resistance or ability to degrade specific chemicals) depends on the huge amount of gene exchange between species. Despite its critical importance, what controls the rate of this gene flow between bacteria is poorly understood. But it does seem clear that restriction-modification (RM) systems play a key role. RM systems produce two enzymes, a nuclease that cuts unprotected DNA (such as what enters from another cell or a bacterial virus), and a methyltransferase that protects the bacterium's own DNA from the nuclease. Many basic questions about the functioning and roles of these systems remain. Given the great importance of RM systems for understanding ongoing bacterial evolution, with implications for everything from plant diseases through biogeochemistry and global warming, it is critical that this deficiency in our understanding be addressed. The purpose of this project is to elucidate the regulatory design of a C protein-controlled model RM system, and how it acts in a living bacterial cell. The C protein activates transcription of its own gene, and of the downstream nuclease gene, and as a result nuclease expression is delayed until C protein accumulates. The first aim is to determine the range of acceptable relative levels of a paired nuclease and methyltransferase. If the methyltransferase/nuclease ratio is too low, cells may die from DNA damage. If this ratio is too high, the entire population may be killed by virus (phage) that escaped the nuclease and became methylated. The second aim determines naturally occurring changes in relative nuclease and methyltransferase levels, under a range of growth conditions including stresses. The third aim is to characterize the time sensitive control system that controls production of the nuclease. Mathematical modeling has provided predictions that will be tested, to see if the system design is accurately understood. Broader Impacts:An integral aspect of the project is to promote education and research training of scientists, ranging from high school science teachers through postdoctoral fellows. The teachers and undergraduates will each study one of the many already cloned but uncharacterized C protein orthologs. High school science teachers and undergraduate science majors will be recruited for summer research internships. The laboratory members will continue to host high school students for science fair projects and will do volunteer biology/career teaching at high schools. Additionally, at least one graduate student and one postdoctoral fellow will be encouraged to earn certificates in bioinformatics as part of their training. These individuals will be well prepared to contribute to the burgeoning field of bacterial genomics. The project will also enrich graduate level teaching and further promote the application to bacterial studies of mathematical modeling.