Characterization and analysis of bacterial sugar utilization strategies employing regulated phenotypic heterogeneity

本项目研究细菌对糖摄取系统的调节策略。在这些系统中，表型异质性不仅与给定时间点表型的统计分布有关，而且还与表型开关时间的显着细胞间变异性有关。我们的中心目标是(i)表征异质时序作为一种动态基因调控策略；（ii）分析竞争性糖利用系统的表型异质性。在SPP1617的第一个资助期，我们对大肠杆菌阿拉伯糖系统单细胞基因表达动力学中的异质时序现象进行了详细的实验和理论分析。我们发现这种糖利用系统的生理开关行为是不对称的，即关闭开关总是快速且均匀的，而打开开关在亚饱和诱导水平下是缓慢且不均匀的。我们在一个单一的定量模型中整合了这些和突变的行为。关于（ii），我们研究了两种相互竞争的PTS底物，山梨醇和n -乙酰氨基葡萄糖（Nag），在广泛的外部底物浓度下的细胞生长，同时定量了Nag和山梨醇利用系统在单细胞水平上的表达。我们的数据支持一个分层相互作用的模型，该模型在低浓度的优势糖（Nag）下分解，允许随机共利用。在SPP的第二个阶段，我们计划研究观察到的表型异质性在糖利用系统行为中的可能益处。我们的理论分析预测，在固定阶段，异质计时可以是一种有用的下注对冲策略：异质计时可以最大限度地降低在感知营养来源后诱导昂贵的基因表达的风险，这些营养来源可能是短暂的，在细胞从表达投资中受益之前就消失了。为了量化固定阶段基因表达的成本，我们将测量诱导启动子无偿表达基因对适应性指标（如活力、代谢活性、膜电位和膜完整性）的影响。我们还将寻求确定限制固定相细胞总基因表达能力的能量和/或资源池。获得的数据将整合到固定相基因表达的定量成本效益分析中，这将成为有益调控策略的实验基础理论分析的基础。本提案的另一项建模任务将是分析大肠杆菌中在过渡到固定阶段时被激活的营养传感信号网络。

This project studies bacterial regulation strategies for sugar uptake systems. In these systems, phenotypic heterogeneity is not only associated with a statistical distribution of phenotypes at a given point in time, but also with a significant cell-to-cell variability in the timing of phenotype switches. Our central goals are (i) to characterize heterogeneous timing as a dynamic gene regulation strategy and (ii) to analyze phenotypic heterogeneity in competing sugar utilization systems. In the first funding period of the SPP1617, we performed a detailed experimental and theoretical analysis of the phenomenon of heterogeneous timing in the single-cell gene expression dynamics of the arabinose system of E. coli. We found that the physiological switching behavior of this sugar utilization system is asymmetric, such that OFF-switching is always rapid and homogeneous, while ON-switching is slow and heterogeneously timed at sub-saturating inducer levels. We consolidated these and mutant behaviors within a single quantitative model. Regarding (ii), we studied cell growth on two competing PTS substrates, Sorbitol and N-acetylglucosamine (Nag), in a broad range of external substrate concentrations, while quantifying the expression of the Nag and Sorbitol utilization systems on the single-cell level. Our data supports a model of a hierarchical interaction that breaks down to allow stochastic co-utilization at low concentrations of the dominant sugar (Nag). For the second period of the SPP, we plan to study possible benefits of the observed phenotypic heterogeneity in the behavior of sugar utilization systems. Our theoretical analysis predicts that heterogeneous timing can be a useful bet-hedging strategy during stationary phase: Heterogeneous timing can minimize the risk of inducing costly gene expression after sensing nutrient sources that may be transient and disappear before a cell benefits from its expression investment. To quantify the cost of gene expression during stationary phase, we will measure the effects of gratuitous gene expression from inducible promoters on fitness indicators such as viability, metabolic activity, membrane potential, and membrane integrity. We will also seek to identify the pool of energy and/or resources that limits the total gene expression capacity of stationary phase cells. The obtained data will be integrated into a quantitative cost-benefit analysis of stationary phase gene expression, which will then be the basis for an experimentally well-founded theoretical analysis of beneficial regulation strategies. The other task for the modeling component of this proposal will be the analysis of a nutrient-sensing signaling network in E. coli that is activated at the transition to stationary phase.