Results Discussion Conclusion Single cell analysis of Escherichia coli outer membrane porin composition in response to nutrient depletion A. Delepierre1, A. Brognaux1, J. Bauwens2, F. Francis2, F. Delvigne1. Context & Objectives Microbial phenotypic heterogeneity characterizes the simultanous presence of several phenotypic traits, among an isoclonal cellular population. Noise, defined as stochastic fluctuations in the biochemical reactions, cell age, as well as mutations are at the bottom of the phenomenon and can be reinforced by variations in the microenvironment, mostly under bioprocessing conditions. Indeed, spatial heterogeneity , due to the decrease of mixing efficiency at large scale, leads to gradients of dissolved oxygen, pH and substrate concentration. Bacterial cells such as Escherichia coli counteract nutrient limitation by several cellular adaptations. Among them, outer cellular membrane permeabilization, by induction of large porin such as OmpF and LamB , optimizes nutrient uptake. The drawback of higher membrane permeability is a low resistance in extracytoplasmic stresses. At the population level, both paradoxical strategies, self-preservation and nutrient competence, are developed. This phenotypic diversification illustrates Bet-hedging strategy, with the aim to increase fitness in temporally variable conditions. To track this phenomenon at the single-cell level and define molecular markers underlining the SPANC, specific tools adapted to the bioprocesses have been developed: Propidium iodide staining combined to FACS as well as MS/MS spectrometry. Microbial phenotypic heterogeneity Isogenic microbial population Noise: extrinsec and intrinsec Cell age: asymetric repartition of proteins during cell division Mutations Bioprocess conditions: spatial heterogeneity (external noise) Inflow stream Outflow stream several phenotypic traits Bioprocess conditions: limitation of carbon substrate Modulation of outer–membrane porin composition: SPANC Self-Preservation and Nutritional Competence Shimizu K(2013) Regulation systems of bacteria such as escherichia coli in response to nutrient limitation and environmental stresses. Metabolites 2013, 4(1):1-35. Ferenci T. (2005) Maintaining a healthy SPANC balance through regulatory and mutational adaptation. Mol. Microbiol. 2005;57:1-8 Methodology Bacterial strains Flow cytometry analysis Bioreactor Propidium iodide staining Proteomic subpopulations Single-gene knockout deletion (KEIO) ΔompC FSC laser Fl 3. detector Membrane intactness Permeabilized Cell - + MS/MS spectrometry LFQ Quantification (MaxQuant) Deletion of gene encoding outer-menbrane porin OmpC Substrate limitation chemostat at D=0.1h-1 Cell sorting Results Discussion Conclusion PI uptake is related to outer/ inner membrane integrity Phenotypic heterogeneity according to outer membrane porin composition Subpopulation sorting Subpopulation proteomic profile Control Osmotic shock Heat Shock Higher outer membrane protein composition: OmpF Nutritional competence Outer membrane disruption Lower outer membrane protein composition: OmpF Substrate limitation Growth arrest E. coli ΔompC Cra cAMP-Crp RpoS/N … Under growth optimal conditions, membrane integrity of wild-type E. coli cells limits PI uptake. Density plot of PI fluorescence intensity (FL3A) vs. Forward Scatter (FSC) shows a predominant subpopulation, characterized by the emission of weak red fluorescence intensity (Subpopulation PI-). Outer membrane permeabilization induced by osmotic shock is responsible for PI uptake and its periplasmic localization (Subpopulation PI+). In the case of heat treatment, membranes are irreversibly damaged; PI covalently interacts with DNA. Consequently, cells emit red fluorescence at higher intensity (Subpopulation PI++). Outer membrane integrity Inner membrane transporters (carbohydrates, amino acids,…) Quality control: Chaperones and proteases (RpoH-response) Carbon storage and energy-saving metabolic pathways 1Univ. Liege- Gembloux Agro-Bio Tech. MiPI Unit. Passage des Déportés, 2. B-5030 Gembloux (Belgium) 2Univ. Liege-Gembloux Agro-BioTech. Functional and Evolutive Entomolgy Unit. Passage des Déportés, 2. B-5030 Gembloux (Belgium)