Heterogeneity in Colicin E2 expression of Escherichia coli

细菌具有几种机制，使它们能够对变化和不利的环境条件作出反应，或胜过其他细菌。一种特殊的机制是产生和释放毒素，如细菌素。目前的研究重点是细菌对这些毒素的产生、释放和吸收。然而，很少有人知道的基本基因调控过程的定量方面。在第一个资助期，我们定量研究了大肠杆菌素E2操纵子在大肠杆菌中的表达动力学。大肠杆菌在单细胞水平上使用荧光延时显微镜。我们能够表明，异质定时是大肠杆菌素E2表达的特征在低浓度的外源性应激丝裂霉素C（MitC），而高浓度的外源性应激导致所有细胞的同步应激反应。在下一个资助期，我们的目标是了解SOS反应过程中大肠杆菌素基因表达的分子机制。扩展我们以前的分析，我们特别感兴趣的是基因表达如何响应随时间变化的环境条件。我们的方法将是模块化的：在研究cea和cel在一组广泛的突变株的表达，我们将解决的大肠杆菌素E2系统的各个模块的监管机制单独和组合。由于该系统的复杂性，它将是必要的，以解开的相对作用和转录和转录后水平的调控元件之间的相互作用，紧密结合的实验和理论分析。实验分析将为不同突变株的cea和cel基因的荧光报告提供大量的时间轨迹，其统计特性将与从相应数学模型的随机模拟获得的结果进行详细比较。这将使我们能够以自下而上的方式获得详细的分子理解。然后，这些分析将导致对调节大肠杆菌素E2表达的不同模块如何根据给定的环境条件被激活的分子理解。特别是，这应该让我们更深入地了解是什么原因导致在低应力水平下观察到的异质性时间，以及如何调节整个细胞群向同步反应的过渡。最后，我们想研究表型异质性如何在生长的细菌小菌落中传递。在这里，我们想了解是否大肠杆菌素依赖性反馈作用于邻近的大肠杆菌素产生细胞，或细胞密度（群体感应）影响表达大肠杆菌素E2的细胞的分数。这些实验将使我们能够更好地了解异质大肠杆菌素E2表达的生物学意义，以及它是如何在生长的小菌落内持续的。

Bacteria possess several mechanisms enabling them to respond to changing and unfavorable environmental conditions or to outcompete other bacteria. One particular mechanism is the production and the release of toxins such as bacteriocins. Current research focuses on production, release and uptake of these toxins by bacteria. However, little is known about the quantitative aspects of the underlying gene regulatory processes. In the first funding period, we quantitatively studied the expression dynamics of the Colicin E2 operon in E. coli on a single cell level using fluorescence time-lapse microscopy. We were able to show that heterogeneous timing is characteristic for Colicin E2 expression at low concentrations of the exogeneous stressor Mitomycin C (MitC), while high concentrations of the exogenous stressor lead to a synchronized stress response of all cells. In the next funding period, we aim to understand the molecular mechanisms underlying Colicin gene expression during SOS response. Extending our previous analysis we are in particular interested how gene expression responds to time-varying environmental conditions. Our approach will be modular: Upon studying cea and cel expression in a broad set of mutant strains we will address the regulatory mechanisms in the various modules of the Colicin E2 system separately and in combination. Due to the complexity of the system it will be essential to closely integrate experimental and theoretical analysis in order to disentangle the relative role of and the interplay between the regulatory elements at the transcriptional and the post-transcriptional level. The experimental analysis will provide large ensembles of time traces for fluorescence reporters of the cea and cel genes for the different mutant strains whose statistical properties will be compared in detail with the results obtained from stochastic simulations of corresponding mathematical models. This will allow us to arrive at a detailed molecular understanding in a bottom-up fashion. These analyses will then lead to a molecular understanding of how the different modules regulating Colicin E2 expression are activated in dependence on given environmental conditions. In particular, this should give us a deeper insight into what causes the observed heterogeneous timing at low stress levels, and how the transition towards a synchronized response of the whole cell population is regulated. Finally, we want to study how phenotypic heterogeneity is passed on within growing bacterial microcolonies. Here, we want to understand if either a Colicin-dependent feedback acting on neighboring Colicin producing cells, or cell density (quorum sensing) affect the fraction of cells expressing Colicin E2. These experiments shall enable us to better understand the biological significance of heterogeneous Colicin E2 expression and how it is sustained within growing microcolonies.