Ghosh Sayantari, Sureka Kamakshi, Ghosh Bhaswar, Bose Indrani, Basu Joyoti, Kundu Manikuntala
Department of Physics, Bose Institute, Kolkata, India.
BMC Syst Biol. 2011 Jan 27;5:18. doi: 10.1186/1752-0509-5-18.
A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity.
In the present study, we characterise quantitatively the single cell promoter activity of the three key genes, namely, mprA, sigE and rel, in the stringent response pathway with gfp as the reporter. The origin of bimodality in the GFP distribution lies in two stable expression states, i.e., bistability. We develop a theoretical model to study the dynamics of the stringent response pathway. The model incorporates a recently proposed mechanism of bistability based on positive feedback and cell growth retardation due to protein synthesis. Based on flow cytometry data, we establish that the distribution of GFP levels in the mycobacterial population at any point of time is a linear superposition of two invariant distributions, one Gaussian and the other lognormal, with only the coefficients in the linear combination depending on time. This allows us to use a binning algorithm and determine the time variation of the mean protein level, the fraction of cells in a subpopulation and also the coefficient of variation, a measure of gene expression noise.
The results of the theoretical model along with a comprehensive analysis of the flow cytometry data provide definitive evidence for the coexistence of two subpopulations with overlapping protein distributions.
遭受应激的微生物的一种常见生存策略是在同基因微生物群体中产生表型异质性，使群体中的一部分能够在应激条件下存活。在最近的一项研究中，耻垢分枝杆菌的一个分枝杆菌群体在营养耗尽时表现出表型异质性。观察到的异质性表现为以绿色荧光蛋白（GFP）作为报告基因的表达水平的双峰分布形式，其中gfp与rel基因的启动子融合。严格反应途径在具有高rel活性的亚群体中启动。
在本研究中，我们以gfp为报告基因，定量表征了严格反应途径中三个关键基因，即mprA、sigE和rel的单细胞启动子活性。GFP分布双峰性的起源在于两种稳定的表达状态，即双稳态。我们开发了一个理论模型来研究严格反应途径的动力学。该模型纳入了最近提出的基于正反馈和蛋白质合成导致的细胞生长迟缓的双稳态机制。基于流式细胞术数据，我们确定在任何时间点分枝杆菌群体中GFP水平的分布是两个不变分布的线性叠加，一个是高斯分布，另一个是对数正态分布，只有线性组合中的系数随时间变化。这使我们能够使用分箱算法并确定平均蛋白质水平的时间变化、亚群体中细胞的比例以及变异系数（基因表达噪声的一种度量）。
理论模型的结果以及对流式细胞术数据的全面分析为具有重叠蛋白质分布的两个亚群体的共存提供了确凿证据。
