# Insights on Osmotic Tolerance Mechanisms in _Escherichia coli_ Gained from an _rpoC_ Mutation
## 3 Results and Discussion
### Metabolite Analysis
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **Metabolite** & **BW25113** & **EJW3** & \(p\)**-Value** \\ \hline Intracellular & Trehalose (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & - & - & - \\  & Acetic acid (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 650 \(\pm\) 90 & **390 \(\pm\) 80** & 0.000 \({}^{*}\) \\  & Glu (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 187.72 \(\pm\) 35.94 & 212.37 \(\pm\) 27.2 & 0.210 \\  & Ala (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 81.18 \(\pm\) 16.55 & 83.71 \(\pm\) 3.15 & 0.721 \\  & Arg (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & - & 4.78 \(\pm\) 7.41 & 0.145 \\  & Pro (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 61.64 \(\pm\) 6.12 & **75.06 \(\pm\) 1.95** & 0.000 \({}^{*}\) \\ \hline Extracellular & Trehalose (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & - & - & - \\  & Acetic acid (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 280 \(\pm\) 20 & **370 \(\pm\) 10** & 0.000 \({}^{*}\) \\  & Glu (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & - & 6.42 \(\pm\) 12.41 & 0.234 \\  & Ala (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 10.86 \(\pm\) 6.33 & **1.93 \(\pm\) 4.72** & 0.020 \({}^{*}\) \\  & Arg (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 2.95 \(\pm\) 7.21 & - & 0.341 \\  & Pro (ug mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 516.13 \(\pm\) 35.09 & **776.02 \(\pm\) 117.02** & 0.000 \({}^{*}\) \\ \hline \hline \end{tabular} - Not detectible. \({}^{*}\) Statistically significantly different between BW25113 and EJW3 (\(p\)-values \(<\) 0.05). Values bolded are significantly higher in EJW3. Values bolded and italiczed are significantly lower in EJW3. \(p\)-values are calculated by using a two-tailed student’s \(t\)-test with six biological replicates.  
\end{table} 
Table 4: Quantification of intracellular and extracellular metabolites in M9. Trehalose is one of the most well-known osmoprotectants. Prior reports showed increased production of trehalose in response to osmotic stress to prevent water loss and to maintain the intracellular pressure of the cells [15; 18]. We found that the intracellular concentration of trehalose under osmotic stress to be \(\sim\)37% higher in EJW3 than BW25113 (\(p\)-value = 0.005), which may be another reason for the higher osmotic tolerance conferred by the _rpoC_ mutation. In addition to known osmoprotectants and amino acids, we also compared the intracellular and extracellular levels of organic acids between the two strains. The results showed a 30%-40% higher extracellular acetic acid concentration in EJW3 cultures (\(p\)-value < 0.001) with or without osmotic stress challenge. However, EJW3 showed a \(\sim\)40% lower intracellular acetic acid concentration in the absence of (\(p\)-value < 0.001), and a similar level of intracellular acetic acid concentration in the presence of (\(p\)-value = 0.137) hyperosmotic stress. Taking into account the relative total volume of extracellular versus intracellular metabolites collected (a ratio of 100:1.5), the total amount of acetic acid produced by EJW3 (370 \(\pm\) 20 \(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) was about 30% higher (\(p\)-value < 0.001) than that of BW25113 (290 \(\pm\) 20 \(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)). During growth on glucose as carbon source, the acetate secretion of _E. coli_ has been shown to depend on growth rate [46; 47], thus the higher level of extracellular acetic acid in strain EJW3 may not be a direct consequence of the _rpoC_ mutation but a side effect of the higher growth rate of the _rpoC_ mutant under osmotic stress. However, it is possible that the higher acetic acid production may also contribute to the higher osmotic tolerance of EJW3.