Starter grant: Investigation of the role of alternative oxidase (AOX) in Vibrio fischeri respiration

替代氧化酶（AOX）是一种不依赖血红素的末端氧化酶，存在于所有植物以及某些真菌、原生生物、动物和细菌的呼吸链中。与其他呼吸道氧化酶不同，AOX不直接促进质子动力的产生。在产热植物中，AOX被用来在开花期间产生热量，但它的功能尚不确定，尽管在真核生物中，它可能有助于代谢稳态，保护免受氧化应激或毒性。替代氧化酶在细菌生理中的作用尚不清楚。最近对序列数据库的调查显示，许多细菌和海洋宏基因组样本的基因组编码一种aox样蛋白。研究AOX在细菌生理中的作用的一个模式生物是费氏弧菌，一种与夏威夷短尾乌贼形成共生关系的生物发光海洋细菌。在费氏弧菌中，微阵列和报告基因分析结果已经确定了aox基因表达水平与一氧化氮（NO）之间的联系。本研究的总体目标是探讨AOX和NO之间的生理联系。首先，我们将利用定量PCR技术评估NO诱导的aox转录物水平的变化；其次，我们将研究NO对费氏弧菌末端呼吸氧化酶功能的影响。这项工作将提供有关AOX在细菌生理学中的功能的新信息，这是一个很大程度上未被探索的领域，但对理解细菌对海洋生态系统的贡献具有重要意义。这些研究将为进一步探索AOX在不同细菌中的作用奠定基础，并最终更好地理解细菌呼吸与生态系统功能之间的联系。此外，将为一名研究生和两名本科生提供基本微生物技术、分子生物学、细菌生理学和相关仪器技术的培训机会。

Alternative oxidase (AOX) is a heme-independent terminal oxidase present in the respiratory chains of all plants, as well as in certain fungi, protists, animals, and bacteria. Unlike other respiratory oxidases, AOX does not directly contribute to the generation of a proton motive force. With the exception of thermogenic plants, where AOX is used to generate heat during flowering, its function is uncertain, although in eukaryotes it may contribute to metabolic homeostasis, protection from oxidative stress, or virulence. The role of alternative oxidase in bacterial physiology is unclear. A recent survey of the sequence databases revealed numerous bacteria and marine metagenomic samples whose genomes encode an AOX-like protein. A model organism for studying the role of AOX in bacterial physiology is Vibrio fischeri, a bioluminescent marine bacterium that forms a symbiotic relationship with the Hawaiian bobtail squid. In V. fischeri, microarray and reporter assay results have identified a link between aox gene expression levels and nitric oxide (NO). The overall goal of the research is to investigate the physiological link between AOX and NO. First, quantitative PCR will be used to assess NO-induced changes in aox transcript levels, and second, the effect of NO on the function of terminal respiratory oxidases in V. fischeri will be investigated. This work will provide novel information related to the function of AOX in bacterial physiology, an area that is largely unexplored, but has implications for understanding bacterial contribution to marine ecosystems. The studies will provide a foundation for future exploration of the role of AOX in diverse bacteria and ultimately a better understanding of the link between bacterial respiration and ecosystem function. In addition, training opportunities will be provided for one graduate and two undergraduate students in basic microbiological techniques, molecular biology, bacterial physiology, and related instrumentation techniques.