# Insights on Osmotic Tolerance Mechanisms in _Escherichia coli_ Gained from an _rpoC_ Mutation
## 3 Results and Discussion
### Metabolite Analysis  
In order to test our hypothesis that _rpoC_ K370_A396 dup mutation led to an overproduction of some amino acids such as proline and alanine, and to identify other potential effects of the mutation on the production of other metabolites, extracellular and intracellular metabolites of BW25113 and EJW3 during late exponential phase (OD\({}_{600}\) about 0.7-1.0) in the presence and absence of osmotic stress were analyzed. The results are shown in Table 4, Table 5, Tables S7 and S8. The results showed that strain EJW3 produced \(\sim\)22% more intracellular (\(p\)-value \(<\) 0.001) and \(\sim\)50% more extracellular (\(p\)-value \(<\) 0.001) proline than BW25113 in the absence of hyperosmotic stress, while the exposure to 0.6 M NaCl led to increased intracellular amount of proline in both strains and extracellular amount of proline produced by BW25113. Prior study has shown the overproduction of proline to confer enhanced osmotic stress tolerance [(43)]. Since the inoculum used in our study were prepared without the addition of excess NaCl, the EJW3 culture likely contained higher initial intracellular proline than BW25113, which may result in early protection to EJW3 from the inhibition of 0.6 M NaCl. Glutamic acid has been found to accumulate in osmotically stressed cells and serves as an osmoprotectant [(15)]. Consistent with prior work, results from our experiment also showed an increase in glutamic acid production under osmotic stress in both BW25113 and EJW3 strains. However, EJW3 appears to accumulate more glutamic acid intracellular, as the intracellular level of glutamic acid was \(\sim\)24% higher (\(p\)-value = 0.006), while the extracellular concentration was \(\sim\)62% lower (\(p\)-value \(<\) 0.001) in EJW3 compared with BW25113. This suggests that EJW3 may be better at accumulating glutamic acid intracellular under osmotic stress due to reduced export of the amino acid into the bulk medium. A similar trend was observed with arginine levels, with both strains producing more arginine in the presence of osmotic stress, and EJW3 maintaining \(\sim\)56% higher intracellular arginine concentration (\(p\)-value \(<\) 0.001) compared with the wild-type (Table 5). It has also been reported that cold osmotic shock reduced the ability of _E. coli_ to accumulate arginine [(44)]. The ability of EJW3 to produce and accumulate more arginine than BW25113 may also contribute to its higher osmotic tolerance. Alanine is known to be an important osmolyte in many organisms [(45)], but thus far has not been reported to serve as an osmolyte in _E. coli_. Our results showed that the production of alanine decreased under osmotic stress in both strains, but the intracellular concentration was \(\sim\)86% higher in EJW3 than in BW25113 (\(p\)-value = 0.001). Though the production of alanine did not increase in response to osmotic stress, the data suggests that biosynthesis of this amino acid was not significantly inhibited in EJW3 in the presence of osmotic stress compared with BW25113. In addition to known amino acids that are perturbed by osmotic stress in bacteria, our results showed that the production of methionine, tyrosine, tryptophan, phenylalanine and isoleucine were also perturbed by osmotic stress and the _rpoC_ mutation (Tables S7 and S8).