Comparative Cross-Strain Analysis of Stress Resistance Mechanisms Revealed By Transposon Insertion Sequencing

Escherichia coli is exposed to a number of stress conditions during high-cell density fed batch fermentation due to non-ideal mixing and pH maintenance.  Two of the most prominent stresses are accumulation of acetate under sub-optimal glucose feeding regimes, and high osmolarity resulting from pH neutralization and high substrate, product, and byproduct concentrations.  Different laboratory strains of E. coli exhibit remarkable variation in their growth phenotypes under these applied stresses, however virtually nothing is known to explain the source of this variation and to enable rational engineering to impart stress tolerance.  We have constructed diverse transposon insertion libraries (>50000 mutants) in 4 strains of E. coli: K-12 MG1655, BL21(DE3), W, and Crooks.  Short-term growth selections were performed in media supplemented with high acetate concentrations, high NaCl concentrations, and both high acetate and high NaCl concentrations.  Insertions frequencies in each gene were determined by massively parallel sequencing of transposon-chromosome junctions, allowing an analysis of both conditionally essential and conditionally detrimental genes.  Many differences and similarities in resistance mechanisms at the genetic level could be revealed across strains, allowing correlations to be made with growth phenotypes.  Cross-strain comparisons of conditionally essential genes and their relative essentiality also suggest a large degree of variation in metabolic flux distributions and regulation of gene expression between strains.  A number of direct targets for metabolic engineering of stress resistance via loss-of-function mutations were also discovered,  and we show that deletion of a selection of these genes results in increased growth rates and/or decreased lag times under the original selection condition.