Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology.IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis, very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.

中文翻译：

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枯草芽孢杆菌生物膜发育过程中的代谢重塑。

生物膜是由紧密结合的细胞组成的结构化群落，它们构成了自然和人造环境中细菌生长的主要状态。尽管控制模型细菌（如枯草芽孢杆菌）中生物膜形成的核心遗传回路已得到很好的表征，但人们对新陈代谢在这一复杂发育过程中所起的作用知之甚少。在这里，我们通过结合代谢组学、转录组学和蛋白质组学分析，对枯草芽孢杆菌中与表膜生物膜形成和发育相关的代谢变化进行了时间分辨分析。我们报告了影响中心碳代谢、初级生物合成途径、发酵途径和次级代谢的代谢的广泛而动态的重塑。迄今为止，这些代谢改变中的大多数未被识别为与生物膜相关。例如，我们观察到在早期生物膜生长过程中三羧酸 (TCA) 循环的活性增加、从脂肪酸生物合成到脂肪酸降解的转变、铁代谢和运输的重组以及从醋酸盐发酵到乙偶姻发酵的转变。代谢组学、转录组学和蛋白质组学测量之间的密切一致性表明，生物膜发育过程中的代谢重塑很大程度上控制在转录水平。我们的结果还提供了对参与这种复杂代谢重塑的转录因子和调控网络的见解。根据这些结果，我们证明了通过乙酰乳酸合酶产生乙偶姻对于生物膜的强劲生长至关重要，并且具有保护氧化还原平衡和维持细胞外 pH 值的双重作用。本报告代表了对枯草芽孢杆菌生物膜发育过程中发生的代谢重塑的全面系统级调查，这将作为未来生物膜生理学研究的有用路线图。重要性细菌生物膜在自然环境中无处不在，并在许多生物膜中发挥重要作用临床、工业和生态环境。尽管对控制模型细菌（如枯草芽孢杆菌）中生物膜形成的转录调控网络了解很多，但对代谢在这一复杂发育过程中的作用知之甚少。为了解决这一重要的知识鸿沟，我们通过结合代谢组学、转录组学和蛋白质组学分析，对枯草芽孢杆菌中与细菌生物膜发育相关的代谢变化进行了时间分辨分析。在这里，我们报告了影响中心碳代谢、初级生物合成途径、发酵途径和次级代谢的代谢的广泛和动态重塑。该报告可作为未来细菌生物膜生理学研究的独特假设生成资源。在生物膜研究领域之外，这项工作也应该与任何对微生物生理学和代谢感兴趣的研究人员相关。我们报告了影响中心碳代谢、初级生物合成途径、发酵途径和次级代谢的代谢的广泛和动态重塑。该报告可作为未来细菌生物膜生理学研究的独特假设生成资源。在生物膜研究领域之外，这项工作也应该与任何对微生物生理学和代谢感兴趣的研究人员相关。我们报告了影响中心碳代谢、初级生物合成途径、发酵途径和次级代谢的代谢的广泛和动态重塑。该报告可作为未来细菌生物膜生理学研究的独特假设生成资源。在生物膜研究领域之外，这项工作也应该与任何对微生物生理学和代谢感兴趣的研究人员相关。