1. 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)