# Insights on Osmotic Tolerance Mechanisms in _Escherichia coli_ Gained from an _rpoC_ Mutation
## 3 Results and Discussion
### Effects of Amino Acid Supplementation  
Since we observed an improvement in growth performance under osmotic stress with tryptophan supplementation, and prior work have reported the impact of some amino acids (e.g., glutamic acid, proline) on osmotic stress response [15; 38], we hypothesized that there are additional amino acids that also play a role in osmotic tolerance in _E. coli_. Thus, we assessed the impact of individual amino acid supplementation on osmotic tolerance of BW25113 and EJW3. Preliminary data with tryptophan supplementation showed a more observable benefit in the presence of higher NaCl concentration, therefore a higher osmotic stress with 0.65 M NaCl was used. The summary of amino acid supplementation on optical cell density (OD\({}_{600}\)) is listed in Table S6. With a few exceptions (valine, methionine, proline, alanine), the addition of 10 ug mL\({}^{-1}\) or 100 ug mL\({}^{-1}\) of most amino acids tested significantly improved the growth of both BW25113 and EJW3 under osmotic stress. This may be partially due to the decreased ATP requirements for amino acid biosynthesis when these amino acids are supplemented [39], which also is the likely reason that higher osmotic tolerance is observed when _E. coli_ is grown in rich media rather than in minimal media without amino acids. However, higher concentrations of some amino acids (e.g., serine, cysteine, threonine, isoleucine, and leucine) reduced growth, likely due to feedback inhibition. Interestingly, the addition of 100 ug mL\({}^{-1}\) methionine appears to result in a faster accumulation of biomass (based on the higher biomass concentration at 24 h), but a 39% lower final biomass (\(p\)-value \(<\) 0.001) after 48 h in strain EJW3. However, the addition of 100 ug mL\({}^{-1}\) methionine led to a 72% lower final biomass (\(p\)-value \(<\) 0.001) in BW25113 after 48 h, which suggests a higher feedback inhibition of methionine in strain BW25113 compared with EJW3 (Figure 3). The supplementation of methionine has been reported to improve \begin{table}
\begin{tabular}{c c c c c} \hline
**Strains** & **0 h** & **24 h** & **48 h** & **72 h** \\ \hline BW25113 & – & + & + & + \\ EJW3 & – & + & +++ & ++++ \\ JW1253 * & – & + & +++ & ++++ \\ EJW4 * & – & + & +++ & ++++ \\ Hfr-2\(\times\)SFX- * & – & + & + & + \\ G3 * & – & +++ & ++++ & ++++ \\ \hline \end{tabular}
\end{table} 
Table 2: Growth in micro-aerobic condition in M9 supplemented with 0.8 M NaCl. \begin{table}
\begin{tabular}{c c c c c} \hline
**Strains** & **0 h** & **24 h** & **48 h** & **72 h** \\ \hline BW25113 & – & + & + & + \\ EJW3 & – & + & +++ & ++++ \\ JW1253 * & – & + & +++ & ++++ \\ EJW4 * & – & + & +++ & ++++ \\ EJW4 * & – & + & +++ & ++++ \\ Hfr-2\(\times\)SFX- * & – & + & + & + \\ EJW4 * & – & + & + & + \\ EJW4 * & – & + & + & + \\ G3 * & – & +++ & ++++ & +++ \\ \hline \end{tabular}
\end{table} 
Table 3: Growth in aerobic condition in M9 supplemented with 0.75 M NaCl. the tolerance to NaCl in _Saccharomyces cerevisiae_[40], but has not been reported in _E. coli_. Our results suggest potential synergy between the _rpoC_ mutation and methionine supplementation on osmotic tolerance in _E. coli_. The supplementation with 100 \(\upmu\)g mL\({}^{-1}\) and 1000 \(\upmu\)g mL\({}^{-1}\) of proline (Figure 4) or with 1000 \(\upmu\)g mL\({}^{-1}\) of alanine (Figure 5) resulted in significant growth inhibition in EJW3, but were beneficial to BW25113, in the presence of osmotic stress. This led us to hypothesize that EJW3 may produce more proline and alanine, making it more sensitive to additional supplementation of these two amino acids, compared with BW25113. Proline has been reported as an osmoprotectant in _E. coli_[38; 41] and other microorganisms [42]. Thus, the overproduction of proline may be one of the causes that contribute to the higher osmotic tolerance in EJW3. Figure 4: Cell density in M9 supplemented with 0.65 M NaCl and proline. (**A**) Cell density after 24 h. (**B**) Cell density after 48 h. Error bars are standard deviations.