Single cell analysis of Escherichia coli outer membrane
porin composition in response to nutrient depletion
A. Delepierre
1, A. Brognaux
1, J. Bauwens
2, F. Francis
2, F. Delvigne
1.
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)
Context & Objectives
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)
several phenotypic traits
Ferenci T. (2005) Maintaining a healthy SPANC balance through regulatory and mutational adaptation. Mol. Microbiol. 2005;57:1-8
Bioprocess conditions: limitation of carbon substrate
Inflow stream
Outflow stream
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.
M
icrobial 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.M
icrobial 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.Methodology
Bacterial strains
Single-gene knockout deletion (KEIO)
ΔompC
Bioreactor Propidium iodide staining Flow cytometry analysis
laser
+
+
-FSC Fl 3. detector
Membrane intactness Permeabilized Cell
Substrate limitation
chemostat at D=0.1h-1 Cell sorting
Proteomic subpopulations
MS/MS spectrometry
LFQ Quantification (MaxQuant)
Results Discussion Conclusion
Deletion of gene encoding outer-menbrane porin OmpC
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+ +).
PI UPTAKE IS RELATED TO OUTER/ INNER MEMBRANE INTEGRITY
Control Osmotic shock Heat Shock
Outer membrane disruption
Subpopulation proteomic profile Subpopulation sorting
Lower outer membrane protein composition: OmpF
Substrate limitation Growth arrest
• Outer membrane integrity
• Inner membrane transporters (carbohydrates, amino acids,…) • Quality control: Chaperones and proteases (RpoH-response) • Carbon storage and energy-saving metabolic pathways
Cra cAMP-Crp RpoS/N … E. coli ΔompC Nutritional competence
Higher outer membrane protein composition: OmpF
