P111: Genetic determinants for n-butanol tolerance in evolved E. coli mutants. Cross-adaptiation and antagonistic pleiotrophy between n-butanol and other stressor

One of the main challenges in biocatalyst-based production of different bioproducts is the toxicity associated of such products to the microbial system.  Since most tolerance phenotypes are complex, with largely unknown genetic determinants and mechanisms, we used the adaptive laboratory evolution method VERT (Visualizing Evolution in Real-Time) to help map-out part of the adaptive landscape of tolerance to n-butanol in Escherichia coli.  Since the use of VERT allows the tracking of independent lineages (via the use of either GFP or YFP marked cells) within the population, we were able to isolate mutants from independent lineages for further characterization.

Analysis of the evolutionary dynamics, coupled with phenotypic, transcriptomic, and genotypic analyses, helped us to identify diverse and divergent mechanisms of adaptation between different subpopulations during laboratory evolution.  Isolated mutants from the GFP-marked subpopulation exhibited cross tolerance between n-butanol and the cationic antimicrobial peptide polymyxin B, while the YFP-marked mutants did not.  Analysis of the isolated mutants from the two colored subpopulations revealed differential cross-tolerance and antagonistic pleiotropy between n-butanol and different stressors.  On the other hand, the YFP-marked mutants exhibited cross-adaptation between n-butanol and hyperosmotic stresses, which was not observed in the GFP-labeled mutants.  These results suggest different routes of adaptation between the GFP and YFP-labeled adaptive mutants.  Further analyses in the presence of different stressors demonstrate cross-adaptation and antagonistic pleiotrophy of isolated mutants from distinctive subpopulations.  Our results suggest that the genotypic space of the evolved E. coli population enables a high phenotypic plasticity during adaptation in stressful environments.