Collaborative Research: Iron limitation, carbon metabolism and siderophore production in marine bacteria - a systems biology approach

该奖项是根据2009年《美国复苏和再投资法》(公法111-5)资助的。异养细菌的铁限制具有重大的生物地球化学影响，包括同化效率降低和二氧化碳进入生物质的减少。海洋浮游细菌还通过产生铁载体对海水中铁的形态产生很大影响，铁载体是对铁有很高亲和力的配体。海水中99.9%以上的溶解铁与有机配体强烈结合，这些有机配体很可能包括铁载体，因为它们具有类似于现场检测到的螯合剂的铁的官能团和条件稳定常数。铁限制和铁载体合成是联系在一起的，因为这些配体是专门针对低细胞内铁浓度而产生的。在海洋细菌中，这两个过程都没有完全被理解。例如，当向HNLC区域添加铁时，海水中铁结合配体的产量出人意料地增加，而不是减少。一种可能的解释是碳的作用，它使铁限制和铁载体合成的研究变得复杂。低铁浓度降低浮游细菌生长效率或就地生产力的程度尚不能很好地量化，因为浮游植物的铁限制解除后，初级生产会迅速得到刺激。由此产生的有效碳的增加使得很难区分海洋细菌对铁(即碳)的直接限制和间接限制。碳源和可获得性也可能在调节铁载体的产生中扮演次要但重要的角色，而铁载体的产生既可以被葡萄糖刺激，也可以被抑制。该项目的目标是对控制伽马蛋白细菌中铁的获取和碳代谢的相互作用的基因调控网络进行建模，特别是费氏弧菌。铁和碳的调节途径在一个复杂的关系网络中紧密相连，该网络由全球转录调节因子(Fur和CRP)和小RNA RyhB介导。为了构建基因调控网络模型，PI将使用结合计算、基于生物信息学的研究和实验室实验的综合系统生物学方法。随着环境条件的变化，对基因表达、铁载体产量和铁的通量的预测将通过使用qRT-PCR和全球转录组分析来量化基因表达，以及确定铁配额和铁摄取率来验证。智力价值：这项研究将通过阐明铁与碳限制、碳源和铁载体生产之间的相互作用，有助于对上层海洋中铁的形态的总体理解。基因调控网络模型将能够识别对铁载体生产至关重要的环境变量，并评估铁限制的潜在生物标记物。该项目将有助于发现与铁代谢有关的新基因和控制机制，并揭示利用相同或相似的相互作用和转录因子的其他过程，如毒力和发光。最后，这种方法还提供了一个框架，用于综合遗传层面的信息，并利用这些信息预测具有生态重要性的铁载体生产等过程。更广泛的影响：该项目将通过指导年轻科学家并支持几个教育级别的外联计划和课程开发来加强科学研究和教育的基础设施。要求支持研究生参加拟议的研究，所有三个私人投资机构都将招募本科生参加暑期实习，目标往往是代表性不足的群体。为了让两名高中科学教育工作者参与我们在斯基德韦海洋研究所的研究，并设计了一项说明微生物如何对营养限制做出反应的实验室练习，我们要求教师补充研究经验。其目的是提供一种研究经验，加强地方和国家各级教育工作者之间的合作。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Iron limitation of heterotrophic bacteria has substantial biogeochemical implications, including lower assimilation efficiencies and reduced incorporation of CO2 into biomass. Marine bacterioplankton also have a large impact on iron speciation in seawater through their production of siderophores, ligands with a high affinity for iron. Over 99.9% of the dissolved iron in seawater is strongly bound to organic ligands, which are likely to include siderophores since they have functional groups and conditional stability constants for iron that are similar to chelators detected in situ. Iron limitation and siderophore synthesis are linked since these ligands are produced specifically in response to low intracellular iron concentrations. Neither process is completely understood in marine bacteria. For instance, the production of iron binding ligands in seawater unexpectedly increases, not decreases when iron is added to HNLC regions. One possible explanation is the role of carbon, which complicates studies of both iron limitation and siderophore synthesis. The extent to which low iron concentrations reduce bacterioplankton growth efficiencies or productivity in situ is not well-quantified because of the rapid stimulation of primary production that occurs after iron limitation of phytoplankton is relieved. The resulting increase in available carbon makes it is difficult to distinguish between direct and indirect iron (i.e. carbon) limitation of marine bacteria. Carbon source and availability may also play a secondary but important role in regulating siderophore production, which can be either stimulated or repressed by the addition of glucose. The objective of this project is to model the interacting gene regulatory networks that control iron acquisition and carbon metabolism in gamma -proteobacteria, specifically Vibrio fischeri. Iron and carbon regulatory pathways are tightly linked in a complex web of relationships mediated by global transcriptional regulators (Fur and CRP) and the small RNA RyhB. In order to construct a gene regulatory network model, the PIs will use an integrated systems biology approach that combines computational, bioinformatics based research and laboratory experimentation. Predictions of gene expression, siderophore production and the flux of iron with changing environmental conditions will be validated by quantifying gene expression using qRT-PCR and global transcriptome analyses, as well as determining iron quotas and iron uptake rates. Intellectual Merit: This study will contribute to the general understanding of iron speciation in the upper ocean by elucidating the interactions between iron and carbon limitation, carbon source and siderophore production. The gene regulatory network model will be capable of identifying environmental variables critical to siderophore production and evaluating potential biomarkers for iron limitation. This project will facilitate the discovery of new genes and control mechanisms involved in iron metabolism and shed light on other processes such as virulence and luminescence, which utilize the same or similar interactions and transcription factors. Finally, this approach also provides a framework for synthesizing information on the genetic level and using it to make predictions about processes such as siderophore production that are ecologically important. Broader Impacts: This project will enhance infrastructure for scientific research and education by mentoring young scientists and supporting both outreach programs and course development at several educational levels. Support is requested for graduate students to participate in the proposed research and all three PIs will recruit undergraduates for summer internships, often targeting underrepresented groups. Research Experiences for Teachers supplements are requested to allow two high school science educators to participate in our research at the Skidaway Institute of Oceanography and design a laboratory exercise that will illustrate how microbes respond to nutrient limitation. The aim is to provide a research experience that will strengthen collaborations among educators at both the local and national levels.