Molecular mechanisms and benefits of phenotypic heterogeneity in Sinorhizobium meliloti populations

表型异质性在微生物界普遍存在，特别是在生物膜中。我们描述了一个案例，使用菌落的α -变形杆菌细菌Sinorhizobium meliloti。一个亚群体强烈表达外多糖（EPS）产生的特异性基因，而它们的遗传相同的邻居表现出很少或不表达。虽然控制EPS的遗传途径已经被很好地理解，比如群体感应，但关于EPS产生的多种表型是如何产生的，我们知之甚少。为了理解这一现象，我们使用了跨学科的方法，整合了数学建模、基因工程、活细胞成像和数据处理分析。该方法依赖于一个三重报告基因结构，它能够携带三个不同的启动子，每个启动子都融合到一个专用的荧光报告基因上。这提供了一种方法，通过这种方法可以监测单个细胞的菌落生长，并且可以根据其祖先谱系和贡献状态对每个个体进行系统分类。我们将每个人分为三类：无贡献者，弱贡献者和强贡献者。在此平台的基础上，我们概述了一个研究计划，以解决控制EPS产生的调控电路中如何出现异质性的问题。该计划侧重于双稳定必不可少的调节电路的几个方面。一种是能够产生异质信号的机制（例如，启动子区域的半甲基化和应激反应）的存在，另一种是能够产生异质信号放大的机制（例如，正反馈回路）。这两种特征都存在于S. meliloti控制EPS产生的调控电路中。这些将被整合到可在实验室测试的歧视性理论模型中。另一个重要的问题是表型异质性的生物学影响。为了回答这个问题，我们利用了我们最近的工作，将突变体（显示非贡献者表型）与野生型进行了比较。突变体生长更快，更灵活，而野生型更能在恶劣的物理条件下生存。在这个项目中，我们提出了一种方法，通过将控制EPS产生的启动子融合到阳性和阴性选择盒中来获得单个类别的种群。这将允许单类种群在没有进一步遗传扰动的情况下有条件地富集，并允许对贡献类和非贡献类进行表征。最后，我们提出了s.m iloti的合作/贡献表型如何在进化背景下得到青睐的问题，并建立了一个解决这个问题的模型。

Phenotypic heterogeneity is prevalent in the microbial world, especially in biofilms. We describe a case using colonies of the alpha-proteobacterium bacterium Sinorhizobium meliloti. A sub-population strongly expresses genes specific for exopolysaccharide (EPS) production, while their genetically identical neighbors exhibit either very little or no expression. Although the genetic pathways controlling EPS are relatively well understood, such as quorum sensing, little is known about how multiple phenotypes with respect to EPS production arise. To understand this phenomenon, we have used an interdisciplinary approach, integrating mathematical modeling, genetic engineering, live cell imaging, and data processing analyses. The approach relies upon a triple-reporter construct which is capable of carrying three different promoters, each fused to a dedicated fluorescent reporter gene. This provides a method whereby the growth of a colony from a single cell can be monitored, and each individual can be systematically classified with regards to its ancestral lineage and its contributor status. We classify each individual into one of three classes: non-contributor, weak contributor, and strong contributor.On the basis of this platform, we outline a research program to address the question of how heterogeneity arises in the regulatory circuitry controlling EPS production. The program focuses on several aspects of regulatory circuits essential for bi-stability. One is the presence of mechanisms capable of generating a heterogenic signal (e.g., hemi-methylation of promoter regions, and the stress response), and another is mechanisms capable of heterogenic signal amplification (e.g., positive feedback loops). Both features have been found in the regulatory circuitry controlling EPS production in S. meliloti. These will be integrated in discriminatory theoretical models which are testable in the laboratory. Another important question is the biological impact of phenotypic heterogeneity. To answer this, we make use of our recent work which compares a mutant (displaying a non-contributor phenotype) to the wild type. The mutant grew faster and was more motile, while the wild type was better at survival of harsh physical conditions. In this program, we propose a method to obtain populations that are selected for a single class by fusing the promoters controlling EPS production to positive and negative selection cassettes. This will allow the conditional enrichment of single-class populations without further genetic perturbation and allow characterization of the contributor and non-contributor classes. Finally, we ask the question of how the cooperative/contributor phenotypes of S. meliloti can be favored in the context of evolution and develop a model addressing this question.