Population-level analyses are rapidly becoming inadequate to answer many of biomedical science and microbial ecology's most pressing questions. The role of microbial populations within ecosystems and the evolutionary selective pressure on individuals depend fundamentally on the metabolic activity of single cells. Yet, many existing single-cell technologies provide only indirect evidence of metabolic specialization because they rely on correlations between transcription and phenotype established at the level of the population to infer activity. In this study, we take a top-down approach using isotope labels and secondary ion mass spectrometry to track the uptake of carbon and nitrogen atoms from different sources into biomass and directly observe dynamic changes in anabolic specialization at the level of single cells. We investigate the classic microbiological phenomenon of diauxic growth at the single-cell level in the model methylotroph Methylobacterium extorquens In nature, this organism inhabits the phyllosphere, where it experiences diurnal changes in the available carbon substrates, necessitating an overhaul of central carbon metabolism. We show that the population exhibits a unimodal response to the changing availability of viable substrates, a conclusion that supports the canonical model but has thus far been supported by only indirect evidence. We anticipate that the ability to monitor the dynamics of anabolism in individual cells directly will have important applications across the fields of ecology, medicine, and biogeochemistry, especially where regulation downstream of transcription has the potential to manifest as heterogeneity that would be undetectable with other existing single-cell approaches.IMPORTANCE Understanding how genetic information is realized as the behavior of individual cells is a long-term goal of biology but represents a significant technological challenge. In clonal microbial populations, variation in gene regulation is often interpreted as metabolic heterogeneity. This follows the central dogma of biology, in which information flows from DNA to RNA to protein and ultimately manifests as activity. At present, DNA and RNA can be characterized in single cells, but the abundance and activity of proteins cannot. Inferences about metabolic activity usually therefore rely on the assumption that transcription reflects activity. By tracking the atoms from which they build their biomass, we make direct observations of growth rate and substrate specialization in individual cells throughout a period of growth in a changing environment. This approach allows the flow of information from DNA to be constrained from the distal end of the regulatory cascade and will become an essential tool in the rapidly advancing field of single-cell metabolism.

中文翻译：

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直接观察微生物双生生长过程中单细胞代谢活性的动态。

人口水平分析正迅速不足以回答许多生物医学和微生物生态学最紧迫的问题。生态系统中微生物种群的作用以及个体的进化选择压力从根本上取决于单细胞的代谢活性。但是，许多现有的单细胞技术仅提供代谢专一化的间接证据，因为它们依赖于在人群水平上建立的转录和表型之间的相关性来推断活性。在这项研究中，我们采用自上而下的方法，使用同位素标记和二次离子质谱技术来跟踪来自不同来源的碳和氮原子对生物质的吸收，并直接观察单细胞水平合成代谢专业化的动态变化。我们在模型甲基营养型甲基芽孢杆菌中研究了单细胞水平上双生生长的经典微生物现象。甲基芽孢杆菌在自然界中，这种生物栖息在叶球体中，在那里它经历了可用碳底物的昼夜变化，因此需要对中央碳代谢进行全面检查。我们表明，人口对可行的底物的可用性变化表现出单峰响应，这一结论支持经典模型，但迄今为止仅得到间接证据的支持。我们预计直接监测单个细胞合成代谢动力学的能力将在生态学，医学和生物地球化学领域中具有重要的应用，特别是在转录下游调控可能表现为异质性的情况下，这可能是其他现有单细胞方法无法检测到的。重要信息了解如何将遗传信息作为单个细胞的行为实现是生物学的长期目标，但它代表着重要的意义。技术挑战。在克隆微生物种群中，基因调控的变异通常被解释为代谢异质性。这遵循生物学的核心教条，即信息从DNA到RNA再到蛋白质，并最终表现为活性。目前，DNA和RNA可以在单个细胞中表征，但是蛋白质的丰度和活性却无法。因此，关于代谢活性的推论通常依赖于转录反映活性的假设。通过跟踪它们从中构建生物质的原子，我们可以在不断变化的环境中的整个生长期间直接观察单个细胞的生长速率和底物专一性。这种方法允许从DNA的信息流从调节级联的远端受到限制，并且将成为快速发展的单细胞代谢领域中必不可少的工具。