# Insights on Osmotic Tolerance Mechanisms in _Escherichia coli_ Gained from an _rpoC_ Mutation
## 3 Results and Discussion  
### The rpoC K370_A396 dup Mutation Confer's Osmotic Tolerance in BW25113  
Our prior work investigating osmotic tolerance in _E. coli_ using adaptive laboratory evolution yielded several mutants with significantly increased tolerance to NaCl stress [24]. Two isolated osmotolerant mutants (G3 and G4) share an identical _rpoC_ K370_A396 dup mutation. These mutants were isolated from independent populations, which led us to hypothesize that this specific mutation is a causative mutation for the improved osmotic tolerance observed. Since both G3 and G4 also contain additional mutations, in order to study the specific function of the _rpoC_ K370_A396 dup mutation, we reconstructed this mutation in the BW25113 wild-type background to generate strain EJW3. In addition, since the parental strain from which G3 and G4 mutants were derived contains a _trpB_ deletion, we also generated strain EJW4 by deleting the _trpB_ gene from EJW3. The growth kinetics of strain BW25113, EJW3 (BW25113 _rpoC_ K370_A396 dup), JW1253 (BW25113 _rtrpB769:kan_), EJW4 (BW25113 _rpoC_ K370_A396 dup, _ArtrpB769:kan_), G3 (evolved osmotolerant mutant containing _rpoC_ K370_A396 dup mutation, BW25113 background), and Hfr-2\(\times\)SFX- (ancestor of G3, BW25113 background) [24] were compared in M9 supplemented with 0.6 M NaCl and 50 ug mL\({}^{-1}\) tryptophan (Figure 1). Since BW25113 and EJW3 are prototrophic for tryptophan, we also compared the growth of these two strains in the absence of tryptophan to assess the impact of the _rpoC_ K370_A396 dup mutation on osmotic tolerance in the absence of amino acid supplementation. The results showed that in the absence of tryptophan supplementation, the growth of BW25113 was drastically inhibited by 0.6 M NaCl 4 h after incubation with no significant growth observed within the next 20 h, while strain EJW3 continued to grow and reached significantly higher biomass than BW25113 after 24 h. The data showed that the addition of tryptophan significantly increased the growth of both BW25113 and the reconstructed _rpoC_ mutant EJW3 in the presence of 0.6 M NaCl (Figure 1A). Though the addition of tryptophan improved the performance of all strains tested in the presence of 0.6 M NaCl, strains with the _rpoC_ K370_A396 dup mutation (EJW3, EJW4, and G3) still outperformed their _rpoC_ wild-type counterparts (Figure 1A,B), strongly suggesting that the _rpoC_ K370_A396 dup mutation is a causative mutation for enhanced osmotic tolerance. Interestingly, the level of osmotic tolerance conferred by the _rpoC_ mutation alone was on the same level as that conferred by the addition of 50 ug mL\({}^{-1}\) tryptophan. Results in Figure 1 showed no significant difference in specific growth rates during the first few hoursof growth between BW25113 and EJW3; therefore, biomass concentrations after 24 and 48 h were used to assess osmotic tolerance for the majority of this work. To determine the impact of the _rpoC_ mutation on cell viability in hyperosmotic stress, BW25113 and EJW3 were grown in M9 supplemented with 0.6 M NaCl and the concentration of viable cells were quantified over time (Figure 2). The concentration of viable cells of both strains decreased within the first few hours, indicating the occurrence of cell death upon initial exposure to hyperosmotic stress. However, increases in the concentration of viable cells were observed in EJW3 sooner and at a faster rate than BW25113. The same trend was also observed using OD\({}_{600}\), indicating that OD\({}_{600}\) and the concentration of viable cells are correlated. The increase in growth (based on OD\({}_{600}\) measurements) observed in hyperosmotic conditions when tryptophan was supplemented also correlated with increased cell viability. Furthermore, there was no observable differences in cell size or shape between BW25113 and EJW3 in the presence or absence of 0.6 M NaCl at different growth phases (Figure S1). Thus, OD\({}_{600}\) was used as the measure of cell growth for the remainder of this study.