Regulation of Type II Restriction-Modification Systems

智慧：生物圈被超过10 e30个细菌和古生菌所控制，这个数字大约相当于“大爆炸”以来的秒数。由于细菌是无性繁殖的，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，但控制细菌之间基因流动速率的因素却知之甚少。但似乎很明显，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（例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免受核酸酶侵害的甲基转移酶。关于这些系统的运作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，以及从植物疾病到地球化学和全球变暖的所有影响，解决我们理解中的这一缺陷至关重要。该项目的目的是阐明C蛋白控制的模型RM系统的调节设计，以及它如何在活细菌细胞中发挥作用。C蛋白激活其自身基因和下游核酸酶基因的转录，结果核酸酶表达被延迟，直到C蛋白积累。第一个目的是确定配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/核酸酶的比例过低，细胞可能会因DNA损伤而死亡。如果这个比例太高，整个种群可能会被逃脱核酸酶并甲基化的病毒（噬菌体）杀死。第二个目的是确定在包括应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发生的变化。第三个目的是表征控制核酸酶产生的时间敏感控制系统。数学建模提供了预测，将进行测试，看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科学家的教育和研究培训，从高中科学教师到博士后研究员。教师和本科生将各自研究许多已经克隆但未表征的C蛋白直向同源物之一。高中科学教师和本科科学专业的学生将被招募进行夏季研究实习。实验室成员将继续为高中学生举办科学博览会项目，并将在高中进行志愿生物/职业教学。此外，至少有一名研究生和一名博士后研究员将被鼓励获得生物信息学证书作为他们培训的一部分。这些人将为细菌基因组学的新兴领域做好充分的准备。该项目也将丰富研究生阶段的教学，并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.