Collaborative Research: Expanding the diversity of iron oxidation mechanisms via genetics, microscopy and 'omics in Leptothrix

铁在地球上几乎无处不在，使其成为生命化学以及土壤、沉积物和地下水化学的重要组成部分。铁与氧化剂如氧气接触可导致铁氧化。当铁氧化发生在土壤中时，它会导致营养物质和金属的螯合;因此，了解铁氧化的机制和控制至关重要。微生物可以催化铁氧化，但微生物对氧化的贡献程度仍然相对未知。该项目评估微生物铁氧化机制（基因/蛋白质），以帮助确定何时以及在环境中发生微生物铁氧化的程度。本项目专门利用铁氧化细菌Leptothrix cholodnii SP-6来探索新的铁氧化机制。先进的知识可以应用于确定对铁氧化细菌活性的控制，以及如何利用它进行生物修复，水处理，资源回收和其他应用。该项目培养了两名研究生和许多本科生。除了公开讲座，动手活动，和实验室图尔斯之旅，一个主要的外展活动包括“微生物在野外”为中学女生和第一代大学生的研讨会，谁了解微生物的力量，以应对环境挑战。培养、遗传学和组学研究任务正作为多周体验式学习单元纳入大学课堂。异养铁氧化细菌Leptothrix cholodnii SP-6中的新遗传系统为揭示新的铁氧化机制提供了前所未有的机会。L. cholodnii SP-6易于培养，其基因组具有许多预测的金属氧化基因，包括多血红素细胞色素和多铜氧化酶的基因，其与其它铁氧化剂中的同源物。然而，基因组缺少众所周知的铁氧化酶基因，使L。cholodnii SP-6是研究铁氧化新机制的良好模式生物。该项目使用培养，比较基因组学，转录组学，蛋白质组学，遗传学和显微镜的组合来确定Leptothrix中的铁氧化机制，特别是SP-6。本研究的主要目的包括：（1）寻找L.通过转座子测序（TnSeq）、转录组学和蛋白质组学对cholodnii SP-6进行了研究。正在开发用于培养和测定铁氧化的高通量管道，以便可以筛选尽可能多的基因。(2)通过对新的纤发菌的分离和基因组特征分析，探索纤发菌铁氧化基因的环境多样性和频率。其次是比较基因组学研究。(3)铁氧化相关基因。cholodnii SP-6的基因敲除和互补。(4)通过标记和定位关键的铁氧化蛋白，并通过共聚焦和电子显微镜对细胞和生物矿物进行成像，确定铁氧化蛋白在生物矿化中的作用。总之，这些方法将建立一个更大的铁氧化基因/蛋白质的集合，同时了解这些如何催化形成对环境重要的羟基氧化铁biominerals.This奖项反映了NSF的法定使命，并已被认为是值得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的支持。

Iron is practically ubiquitous across Earth, making it an important component in the chemistry of life and in the chemistry of soils, sediments, and groundwater. Iron in contact with an oxidant such as oxygen can result in iron oxidation. When iron oxidation occurs in soils, it can lead to the sequestration of nutrients and metals; thus, understanding the mechanisms and controls on iron oxidation is critical. Microorganisms can catalyze iron oxidation, yet the extent of microorganism contribution to oxidation remains relatively unknown. This project evaluates microbial iron oxidation mechanisms (genes/proteins) to help identify when, and how much, microbial iron oxidation is occurring in the environment. This project specifically uses the iron-oxidizing bacterium Leptothrix cholodnii SP-6 to explore new iron-oxidizing mechanisms. The advanced knowledge can be applied to determine controls on iron-oxidizing bacterial activity and how to harness it for bioremediation, water treatment, resource recovery, and other applications. This project trains two graduate students and numerous undergraduates. In addition to public lectures, hands-on activities, and lab tours, a major outreach activity includes "Microbes in the Wild" workshops for middle school girls and first-generation college-bound students, who learn about the power of microbes to address environmental challenges. Culturing, genetics, and 'omics research tasks are being incorporated into college classes as multi-week experiential learning units.A new genetic system in the heterotrophic iron-oxidizing bacterium Leptothrix cholodnii SP-6 provides an unprecedented opportunity to reveal new iron oxidation mechanisms. L. cholodnii SP-6 is easily culturable and its genome has numerous predicted metal oxidation genes including genes for multiheme cytochromes and multicopper oxidases with homologs in other iron oxidizers. Yet, the genome is missing well-known iron oxidase genes, making L. cholodnii SP-6 a good model organism to investigate novel iron oxidation mechanisms. This project is using a combination of culturing, comparative genomics, transcriptomics, proteomics, genetics, and microscopy to determine the iron oxidation mechanisms in Leptothrix, notably SP-6. The research aims include (1) discover candidate iron oxidation genes in L. cholodnii SP-6 via transposon sequencing (TnSeq), transcriptomics, and proteomics. A high throughput pipeline for culturing and assaying iron oxidation is being developed so that as many genes as possible can be screened. (2) Explore the environmental diversity and frequency of Leptothrix iron oxidation genes via isolation and genomic characterization of new Leptothrix spp. followed by comparative genomics study. (3) Validate iron oxidation genes in L. cholodnii SP-6 via genetic methods of knockout and complementation. (4) Determine the role of iron oxidation proteins in biomineralization by labeling and localizing key iron oxidation proteins and imaging cells and biominerals by confocal and electron microscopy. Together, these approaches will establish a larger set of iron oxidation genes/proteins while understanding how these catalyze formation of environmentally important iron oxyhydroxide biominerals.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.