# Insights on Osmotic Tolerance Mechanisms in _Escherichia coli_ Gained from an _rpoC_ Mutation
## 4 Conclusions  
In this work, we confirmed that the in-frame, 84 bp duplication in _rpoC_ (K370_A396 dup) does indeed contribute to osmotic tolerance of _E. coli_. The _rpoC_ K370_A396 dup mutation was  
Figure 11: Growth kinetics of MG1655, EYG1 and the knockout strains in MG1655 background. (**A**) M9 supplemented with 0.7 M NaCl with and without 50 \(\upmu\)g mL\({}^{-1}\) tryptophan (**B**) M9 (**C**) M9 supplemented with 0.7 M NaCl. Error bars are standard deviations. Figure 10: Growth kinetics of MG1655, EYG1, BW25113 and EJW3. (**A**) M9 (**B**) M9 supplemented with 0.6 M NaCl. Error bars are standard deviations. reconstructed in the BW25113 background (EJW3), and the _rpoC_ mutant performed much better than the wild-type strain under osmotic stress. From individual amino acid supplementation studies, we found several additional amino acids (e.g., tryptophan, phenylalanine, etc.) to play a role in improving osmotic tolerance in _E. coli_. Results from metabolite analysis revealed differences in intracellular and extracellular metabolites between the wild-type and _rpoC_ mutant, suggesting alterations in metabolism may be partially responsible for the enhanced hyperosmotic tolerance in the mutant. Compared with the wild-type strain, EJW3 produced and accumulated more known osmoprotectants (trehalose, proline, and glutamic acid) in response to hyperosmotic stress. In addition to known osmolytes, EJW3 also produce approximately 30% more acetic acid than BW25113. Subsequent supplementation studies confirmed that the addition of moderate concentration of acetic acid helps to improve the performance of BW25113 under hyperosmotic stress; which supported the theory that the higher production of acetic acid is likely related with the osmotic tolerance conferred by the _rpoC_ (K370_A396 dup) mutation. Membrane damage analysis using PI staining showed a lower effect on membrane integrity in the presence of hyperosmotic stress in _rpoC_ mutants that are actively growing (in exponential growth phase). Transcriptional analysis demonstrated that the _rpoC_ mutation indeed impacted relative transcript abundance of genes related with amino acids metabolism, including _metK_ and _mmuP_. When overexpressed in BW25113, _metK_ and _mmuP_ conferred enhanced osmotic tolerance, providing further validation that methionine metabolism is involved in enhanced hyperosmotic tolerance in the _rpoC_ mutant. Deletion of stress response genes _bolA_ and _hdeA_ also improved growth of BW25113 under osmotic stress, and the benefit of _bolA_ deletion on osmotic tolerance also applies to MG1655. Other genes perturbed by the _rpoC_ mutation in BW25113 and the synergistic effects of those genes remain to be investigated. In conclusion, the work demonstrated that the impacts of this particular _rpoC_ mutation on _E. coli_ metabolism and membrane integrity related with osmotic tolerance, and although the impacts of _rpoC_ mutation appeared to be strain dependent, some of the findings in BW25113 also apply to another K-12 strain. Results from this work can help to identify targets for metabolic engineering of _E. coli_ for enhanced tolerance to alternative feedstocks and water sources.