Denitrification Linked to Methanol Oxidation in Methylotenera Mobilis: a New Paradigm of Methylotrophy

智力优点：Methylotenera mobilis是一种最近描述的细菌，是华盛顿湖沉积物中活跃的甲基营养菌群落的重要成员。最近的实验表明，M.在氧气存在下，运动细菌和相关细菌进行与甲醇氧化相关的反硝化作用，导致N2 O的排放。这是一个新的过程，可能是湖泊氮循环的重要组成部分。对M. mobilis鉴定了经典反硝化途径的几个传统组分，表明涉及替代酶。这种细菌也缺乏传统的甲醇脱氢酶。这一现象之所以有趣，有几个原因。首先，这是第一例由嗜甲基菌科的成员进行的反硝化作用;第二，依赖氧的反硝化作用在微生物世界中是不寻常的;第三，由Methylotenera和相关生物进行的反硝化作用可能代表了一个被忽视的重要的生物地球化学过程，占从C1代谢活跃的环境（如淡水湖泊）释放到大气中的N2 O的一部分。本项目的主要目标是了解Methylotenera反硝化的生物化学和生理学，并揭示反硝化与甲醇氧化耦合的特定性质。这项工作将提出甲基营养和反硝化的新范例，并将为全球碳和氮循环之间的联系提供新的见解。M. mobilis系统将是一个模型系统。具体目标是1）鉴定涉及脱氮和甲醇氧化的基因、酶和途径，2）通过突变测试预测涉及甲醇氧化和脱氮两者的基因的功能，和3）通过M. mobilis，主要候选者是一种新的周质硝酸还原酶。这一建议的智力价值在于获得与反硝化有关的甲基营养的生物化学见解，改变这两个过程的范式，并扩大微生物中能量产生途径的知识。更广泛的影响：该项目将涉及研究生、本科生和高中生，特别强调代表性不足的群体。与当地学校和学院建立的伙伴关系将提供公共宣传。这些数据将通过教育材料、会议介绍、科学和大众出版物以及专门网站广泛传播。社会影响将是提供生物化学和基因组学的见解，一个新的，以前被忽视的生物地球化学过程，可能有助于气候变化。

Intellectual Merit:Methylotenera mobilis is a recently described bacterium that is a prominent member of the active methylotroph community in Lake Washington sediment. More recent experiments suggested that M. mobilis and related bacteria carry out denitrification linked to methanol oxidation, in the presence of oxygen, resulting in emission of N2O. This is a novel process that may be an important part of the nitrogen cycle in lakes. Genomic analysis of M. mobilis identified few traditional components of the classic denitrification pathway, suggesting involvement of alternative enzymes. The bacterium also lacks a traditional methanol dehydrogenase. This phenomenon is interesting for a number of reasons. First, this is the first case of denitrification by a member of the Methylophilaceae family; second, oxygen-dependent denitrification is unusual in the microbial world; third, denitrification by Methylotenera and related organisms may represent an important biogeochemical process that has been overlooked, accounting for a portion of N2O released into the atmosphere from the environments active in C1 metabolism, such as freshwater lakes. The major goal of this project is to understand the biochemistry and the physiology of denitrification by Methylotenera and to uncover the specific nature of coupling of denitrification to methanol oxidation. This work will present new paradigms of both methylotrophy and denitrification and will provide new insights into the connection between the global cycles of carbon and nitrogen. M. mobilis will be the model system. The specific objectives are 1) identification of genes, enzymes and pathways involved in denitrification and methanol oxidation, 2) testing the functions of the genes predicted to be involved in both methanol oxidation and denitrification, via mutation, and 3) purification and characterization of one or more key enzymes involved in denitrification by M. mobilis, the primary candidate being a novel periplasmic nitrate reductase. The intellectual merit of this proposal is in obtaining biochemical insights into methylotrophy linked to denitrification, transforming the paradigms of both processes and broadening the knowledge on energy-generating pathways in microbes in general. Broader Impacts:This project will involve graduate, undergraduate and high school students, with special emphasis on the members of underrepresented groups. Established partnerships with local schools and colleges will provide the public outreach. The data will be broadly disseminated via educational materials, conference presentations, scientific and popular publications and the dedicated web site. The societal impact will be in providing biochemical and genomic insights into a novel, previously overlooked biogeochemical process that potentially contributes to climate change.