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中的重要组成部分。铁与氧等氧化剂接触会导致铁氧化。当土壤中发生铁氧化时，它会导致养分和金属的封存；因此，了解铁氧化的机制和控制是至关重要的。微生物可以催化铁氧化，但微生物对铁氧化的贡献程度尚不清楚。该项目评估微生物铁氧化机制(基因/蛋白质)，以帮助确定环境中微生物铁氧化发生的时间和程度。本项目专门使用铁氧化菌Leptothrix cholodnii SP-6来探索新的铁氧化机制。先进的知识可用于确定对氧化铁细菌活性的控制，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众多的本科生。除了公开讲座、动手活动和实验室参观外，一项重要的外展活动还包括为中学女生和第一代大学学生举办的“野外微生物”讲习班，他们了解微生物应对环境挑战的力量。培养、遗传学和组学研究任务作为为期数周的体验学习单元被纳入大学课程。异养型铁氧化细菌Leptothrix cholodnii SP-6中的一个新的遗传系统提供了一个前所未有的机会来揭示新的铁氧化机制。霍乱弧菌SP-6易于培养，其基因组中有许多预测的金属氧化基因，包括多血红素细胞色素基因和多铜氧化酶基因，这些基因与其他铁氧化剂中的同源物有关。然而，基因组中缺少众所周知的铁氧化酶基因，这使得霍乱弧菌SP-6成为研究新的铁氧化机制的很好的模式生物。该项目结合了培养、比较基因组学、转录组学、蛋白质组学、遗传学和显微技术来确定细毛虫的铁氧化机制，特别是SP-6。本研究的目的包括：(1)通过转座子测序、转录组学和蛋白质组学的方法发现霍乱弧菌SP-6的铁氧化候选基因。正在开发一种高通量的培养和检测铁氧化的流水线，以便尽可能多地筛选基因。(2)通过对新的细毛藻的分离和基因组特征的研究，探索细毛藻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)通过基因敲除和互补的方法验证霍乱弧菌SP-6中的铁氧化基因。(4)通过共聚焦显微镜和电子显微镜对关键的铁氧化蛋白、成像细胞和生物矿物进行标记和定位，以确定铁氧化蛋白在生物矿化中的作用。总而言之，这些方法将建立一个更大的铁氧化基因/蛋白质集，同时了解这些基因/蛋白质如何催化形成对环境重要的氢氧化铁生物矿物。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.