CAREER: The Mechanistic Interplay Between Biofilm Metabolism and Morphogenesis

细菌可以以多细胞群落的形式存在，这种群落由一种名为生物膜的粘性基质结合在一起。与其他多细胞生物体一样，这些结构中的细胞受到独特条件的挑战，但技术限制阻碍了对生物膜微环境和细胞的化学和生理描述。本项目将阐明铜绿假单胞菌生物膜特定区域对自身产生的氧气限制和形态变化的代谢和信号机制。采用电化学和样品处理技术的跨学科方法将使代谢物和基因表达的直接测绘和测量成为可能。该项目将利用生物膜的各种图案和强烈的色彩，通过展览和讲习班，激发当地学生和公众对多细胞和新陈代谢概念的兴趣，这些概念与医学和工业有关。PI将扩展他的实验室课程，包括设计和维护一个免费网站，该网站托管分析软件和用于编目生物膜图案的交互数据库。技术描述：尽管多细胞生命展示了看似无穷无尽的形状和图案，但它们在形态上受到驻留细胞新陈代谢需求的限制。新陈代谢的灵活性可以在这种结构内形成的化学梯度中产生能量；或者，细胞群落可以改变它们的形态，以改善对资源的获取。为了解决新陈代谢和形态发生之间的相互作用，这个项目将探索在铜绿假单胞菌生物膜发育过程中形成的自我生成的氧梯度如何改变生物膜的形态。铜绿假单胞菌产生氧化还原活性吩嗪，当氧气有限时，吩嗪可以支持能量的产生，在氧气不足的情况下，突变株形成复杂的皱折结构，增加生物膜表面积，并且缺乏缺氧亚区。这项研究将询问一个模型，在该模型中，缺氧区的细胞进行吩嗪依赖的新陈代谢，传感器调节蛋白对氧化还原信号做出反应，调节菌落皱纹。该项目将：i)测试呼吸酶是否在缺氧区催化吩嗪还原，并需要特定的呼吸酶；ii)测试生物膜细胞是否将吩嗪还原与碳源氧化或发酵结合在一起，使用菌落薄片的色谱/质谱分析；iii)确定控制皱纹形成的蛋白质的微环境效应，并确定建立皱纹菌落所需的基质蛋白质。

Bacteria can reside as multicellular communities held together by a sticky matrix called biofilms. Cells within these structures, like other multicellular organisms, are challenged by unique conditions but technical limitations have hindered developing chemical and physiological descriptions of biofilm microenvironments and cells. This project will elucidate metabolic and signaling mechanisms in specific regions of Pseudomonas aeruginosa biofilms in response to self-generated oxygen limitations and morphological changes. An interdisciplinary approach employing electrochemical and sample processing techniques will enable direct mapping and measurement of metabolites and gene expression. This project will capitalize on the varied patterns and intense coloration of biofilms to pique the interest of local students and the public in the concepts of multicellularity and metabolism, concepts relevant to medicine and industry, through exhibits and workshops. The PI will expand his laboratory course to include design and maintenance of a free website that hosts analytical software and an interactive database for cataloguing biofilm patterns.Technical description: Although multicellular life exhibits a seemingly endless array of shapes and patterns, they are morphologically constrained by the metabolic needs of resident cells. Metabolic flexibility can enable energy generation in the chemical gradients that form within such structures; alternatively, cellular communities can change their morphologies to improve access to resources. To address the interplay between metabolism and morphogenesis, this project will explore how self-generated oxygen gradients that form during Pseudomonas aeruginosa biofilm development alter biofilm morphology. Pseudomonas aeruginosa produces redox-active phenazines that can support energy generation when oxygen is limiting and in their absence mutants form intricate wrinkled structures that increase biofilm surface area, and lack an anoxic subzone. This research will interrogate a model in which cells in the anoxic zone carry out phenazine-dependent metabolism and sensor-regulator proteins modulate colony wrinkling in response to redox cues. This project will: i) test whether respiratory enzymes catalyze phenazine reduction in the anoxic zone and require specific respiratory enzymes; ii) test whether biofilm cells couple phenazine reduction to carbon source oxidation or fermentation using chromatography/mass spectrometry of colony thin sections; and iii) define the microenvironmental effects of proteins that control wrinkle formation and identify the matrix protein required to build wrinkled colonies.