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

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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.