Heterogeneous cell wall homeostasis as a survival strategy in stationary phase: balancing cell growth, antibiotic production, and envelope stress response in Bacillus subtilis

细胞被膜是一种重要的但高度动态的结构，它保护细菌细胞免受环境的影响。在整个生长周期中保持其完整性-即使面对抗生素的威胁-对生存绝对至关重要。虽然近年来在了解平衡生长期间的包膜稳态方面取得了重大进展，但对稳定期的调节和生理调节知之甚少。当面临不利的生长条件时，革兰氏阳性模式生物枯草芽孢杆菌开始进行复杂的分化程序，这导致形成不同的亚群，例如变成感受态、形成生物膜或分化成休眠的内生孢子。这种多样化归因于赌注对冲策略，旨在通过为许多不同的命运做好准备来最大限度地提高人口的生存率。但到目前为止，人们对其他稳定期生存策略的生理相关性和相关性知之甚少，包括靶向细胞壁生物合成的抗菌肽（AMP）的产生，所产生的细胞包膜应激反应（CESR）以及剩余营养细胞中的细胞生长。这些将是本提案的重点，该提案基于以下基石：（i）单个细胞的生长速率是广泛异质的，导致营养细胞、分化细胞和裂解细胞的广泛混合群体。(ii)AMP生产也是一种异质和短暂的策略。(iii)由于AMP靶向细胞壁生物合成，它们的抑制作用与活跃生长的细胞偶联，因此也应该是异质的。这表示在（iv）观察到的损伤敏感Lia系统，响应于细胞壁生物合成的脂质II周期的扰动双峰诱导。基于此，我们提出，在稳定期的Lia响应代表了一个“生理三重与门”，需要AMP的生产，细胞生长，和不足的内在保护。为了解决这一假设，并最终解释AMP生产，稳定期生长和诱导CESR的异质性之间的相关性，我们追求四个主要目标。(i)我们的目标是提取一个定量的，基于单细胞的图片之间的相关性和表观遗传细胞生长，AMP的生产，和CESR。(ii)我们希望开发一个定量和动态的脂质II循环的数学模型，以模拟AMP的生产和由此产生的诱导CESR之间的相关性。(iii)在结合上述两个目标，我们的目标是确定随机/调节过程负责决定细胞的命运决定有关孢子形成，AMP生产和诱导的CESR。(iv)最后，我们想解开的生理相关性的表型异质性在细胞包膜稳态和利亚介导的CESR稳定期生存。

The cell envelope is an essential yet highly dynamic structure that protects the bacterial cell from its environment. Maintaining its integrity throughout the growth cycle - even in the face of antibiotic threat - is absolutely essential for survival. While significant progress has been made in recent years on understanding envelope homeostasis during balanced growth, little is known on the regulatory and physiological adjustments in stationary phase. When faced with unfavorable growth conditions, the Gram-positive model organism Bacillus subtilis embarks on a complex differentiation program, which results in the formation of different subpopulations that e.g. become competent, form biofilms or differentiate into dormant endospores. This diversification is attributed to bet-hedging strategies that aim at maximizing the survival of the population by 'being prepared' for a number of different fates. But so far little is known about the physiological relevance and correlation of other stationary phase survival strategies, including the production of antimicrobial peptides (AMPs) that target cell wall biosynthesis, the resulting cell envelope stress responses (CESR), and the cell growth in the remaining vegetative cells. These will be the focus of this proposal, which rests on the following cornerstones: (i) The growth rate of individual cells is broadly heterogeneous resulting in widely mixed populations of vegetative, differentiating and lysing cells. (ii) AMP production is also a heterogeneous and transient strategy. (iii) Since AMPs target cell wall biosynthesis, their inhibitory action is coupled to actively growing cells and should therefore also be heterogeneous. This is expressed in (iv) the bimodal induction observed for the damage-sensing Lia system, which responds to perturbations of the Lipid II cycle of cell wall biosynthesis. Based on this, we propose that the Lia response in stationary phase represents a 'physiological triple AND-gate' that requires AMP production, cell growth, and insufficient intrinsic protection. To address this hypothesis and ultimately explain the correlation between the heterogeneity in AMP production, stationary phase growth and induction of CESR, we pursue four major goals. (i) We aim at extracting a quantitative, single-cell based picture of the correlation and epigenetic inheritance between cell growth, AMP production, and CESR. (ii) We want to develop a quantitative and dynamic mathematical model of the Lipid II cycle, in order to simulate the correlation between AMP production and the resulting induction of the CESR. (iii) In combining the above two goals, we aim at Identifying the stochastic/regulatory processes responsible for determining the cell fate decision regarding sporulation, AMP production and induction of the CESR. (iv) Ultimately, we would like to unravel the physiological relevance of phenotypic heterogeneity in cell envelope homeostasis and Lia-mediated CESR for stationary phase survival.