# Insights on Osmotic Tolerance Mechanisms in _Escherichia coli_ Gained from an _rpoC_ Mutation
## 3 Results and Discussion
### Effect of Acetic Acid on Osmotic Stress Tolerance  
To determine the role acetic acid plays on osmotic tolerance, we compared the growth kinetics of BW25113 and EJW3 in the presence of 10 mM acetic acid in M9 with or without the supplementation of 0.6 M NaCl. In the absence of osmotic stress, the addition of 10 mM acetic acid showed no significant impact on the growth of either strain (Figure 6A). However, the addition of acetic acid significantly improved the growth of BW25113 but only had a moderate impact on the growth of EJW3 in the presence of NaCl challenge (Figure 6B). Under higher osmotic stress with 0.7 M NaCl, acetic acid addition still had a benefit on the growth of BW25113, and showed a moderate benefit on the growth of EJW3 (Figure 6C). Prior work has shown that moderate concentration of NaCl can protect _E. coli_ from acetic acid toxicity [48]; here we show that the cross tolerance is reciprocal, that moderate concentrations of acetic acid can also protect _E. coli_ from NaCl stress. Interestingly, a statistically significant overlap (\(p<10^{-20}\), [PERSON]'s exact test) between metabolites level changes in organic acid and osmotolerant _E. coli_ mutants has been detected using an updated version of the Resistance combined with a recent  
\begin{table}
\begin{tabular}{c c c c c} \hline  & **Metabolite** & **BW25113** & **EJW3** & **_p-Value_ \\ \hline Intracellular & Trehalose (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 1190 \(\pm\) 190 & **1630 \(\pm\) 220** & 0.005 \({}^{*}\) \\  & Acetic acid (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 270 \(\pm\) 140 & 370 \(\pm\) 50 & 0.137 \\  & Glu (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 565,50 \(\pm\) 44.39 & **698.75 \(\pm\) 82.47** & 0.006 \({}^{*}\) \\  & Ala (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 38.49 \(\pm\) 9.03 & **71.58 \(\pm\) 13.51** & 0.001 \({}^{*}\) \\  & Arg (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 28.79 \(\pm\) 2.73 & **44.87 \(\pm\) 6.42** & 0.000 \({}^{*}\) \\  & Pro (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 94.31 \(\pm\) 6.14 & 107.31 \(\pm\) 18.37 & 0.131 \\ \hline Extracellular & Trehalose (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & - & - & - \\  & Acetic acid (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 260 \(\pm\) 20 & **360 \(\pm\) 20** & 0.000 \({}^{*}\) \\  & Glu (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 234.82 \(\pm\) 15.44 & **88.05 \(\pm\) 19.38** & 0.000 \({}^{*}\) \\  & Ala (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & - & - & - \\  & Arg (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & - & 7.90 \(\pm\) 12.34 & 0.148 \\  & Pro (\(\mathrm{\SIUnitSymbolMicro m}\) mL\({}^{-1}\) OD\({}_{600}\)\({}^{-1}\)) & 747.75 \(\pm\) 120.88 & 728.90 \(\pm\) 156.43 & 0.820 \\ \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 italicized 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 5: Quantification of intracellular and extracellular metabolites in M9 supplemented with 0.6 M NaCl.  
genome-wide survey of genotype-metabolite relationships [49; 50], providing additional evidence that the improved osmotic tolerance of EJW3 may also result from the higher production of acetic acid.