Molecular mechanisms and benefits of phenotypic heterogeneity in Sinorhizobium meliloti populations

表型异质性在微生物世界中普遍存在，尤其是在生物膜中。我们描述了一个使用阿尔法蛋白杆菌菌落的案例。一个亚群强烈表达胞外多糖(EPS)生产所特有的基因，而它们遗传上相同的邻居表现出很少或没有表达。虽然控制EPS的遗传途径相对较好，如群体感应，但关于EPS产生的多种表型是如何产生的，人们知之甚少。为了理解这一现象，我们使用了一种跨学科的方法，整合了数学建模、基因工程、活细胞成像和数据处理分析。这种方法依赖于能够携带三种不同启动子的三重报告结构，每个启动子都融合到一个专门的荧光报告基因上。这提供了一种方法，借此可以监测单个细胞的群体生长情况，并根据其祖先谱系和贡献者地位对每个个体进行系统分类。我们将每个个体分为三类：非贡献者、弱贡献者和强贡献者。在这个平台的基础上，我们概述了一个研究计划，以解决控制EPS生产的调控电路如何产生异质性的问题。该计划侧重于对双稳定至关重要的调节电路的几个方面。一种是存在能够产生异源信号的机制(例如，启动子区域的半甲基化和胁迫反应)，另一种是能够产生异源信号放大的机制(例如，正反馈环)。这两个特征已经在控制苜蓿EPS产生的调节电路中被发现。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(显示非贡献者表型)与野生型进行了比较。突变体生长更快，能动性更强，而野生型更善于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生产的启动子融合到正选择盒和负选择盒来获得为单个类别选择的群体。这将允许在没有进一步遗传扰动的情况下有条件地丰富单一类别的种群，并允许确定贡献者和非贡献者类别的特征。最后，我们提出了一个问题，即如何在进化的背景下支持紫花苜蓿的合作/贡献者表型，并开发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.