# Insights on Osmotic Tolerance Mechanisms in _Escherichia coli_ Gained from an _rpoC_ Mutation
## 3 Results and Discussion
### Transcriptional Profile Analysis  
RpoC is the \(\beta\)' subunit of the RNA polymerase complex, and plays roles in promoter recognition, sigma factor binding and ion chelation [53; 54; 55]. The _rpoC_ K370_A396 dup mutation is located in domain 2 (amino acid residues 344-486) of the RpoC subunit [56], close to the clamp (amino acid residues 14-342) and the NADFDGD (amino acid residues 458-464) motif which is involved in Mg2+ binding [57; 55]; thus, this mutation in _rpoC_ may impact transcriptional initiation and elongation [58; 59]. Furthermore, the 3-D structure of the RNA polymerase places the K370_A396 dup mutation in close proximity to the proposed binding site of guanosine tetraphosphate (ppGpp) (amino acid residues 362, 417) [60; 61; 62], which is a global regulator involved in stringent response [63]. Thus, the _rpoC_ K370_A396 dup mutation is expected to have global impact on the transcriptional regulation of the cell; therefore, the transcriptional differences in BW25113 and EJW3 can potentially be used to identify new molecular mechanisms of osmotolerance in _E. coli_. Thus, we compared the transcriptional profiles between EJW3 and BW25113 in M9 with or without 0.6 M NaCl challenge to identify any transcriptional regulatory differences that may lead to new findings on molecular mechanisms for osmotic tolerance. The transcriptional data showed that most genes upregulated in strain EJW3 under osmotic stress are related with amino acids metabolism, which corroborated with our data from amino acid supplementation and metabolite analysis; and the genes downregulated are related with membrane composition and transporters. Several genes upregulated in strain EJW3 under osmotic stress were chosen for further validation for their role in osmotic tolerance in the _rpoC_ K370_A396 dup background. The selected genes (Table S9) are involved in amino acids metabolism and were chosen based on results from the amino acid supplementation and metabolite analysis. These genes were overexpressed in BW25113 by using clones from the ASKA collection [35], and the growth kinetics of the overexpression strains were compared against the control expressing the empty plasmid in M9 with or without NaCl supplementation. To avoid potential toxicity associated with high level gene expression, basal level expression (without Isopropyl \(\beta\)-D-1-thiogalactopyranoside (IPTG) induction) and a lower osmotic stress (0.55 M NaCl) were used. Among the genes tested, only the overexpression of _metK_ and _mmuP_ significantly improved the performance of BW25113 under osmotic stress. The overexpression of _metK_ and _mmuP_ did not impact the growth of BW25113 in the absence of hyperosmotic stress, but rather conferred a benefit in the presence of 0.55 M NaCl (Figure 8). This suggests that the osmotic tolerance conferred by the _rpoC_ mutation in BW25113 is partially due to the upregulation of _metK_ and _mmuP_. _MetK_ encodes the methionine adenosyltransferase that catalyzes the formation of the S-adenosylmethionine (AdoMet) [64], which is involved in many biological reactions. It plays important roles as donors of methyl, sulfur, and aminopropyl groups, thus is frequently involved in the regulation of gene expression, including genes involved in methionine metabolism [65; 66; 67; 68], but was not known to be related to osmotolerance. _MmuP_ is a transporter of S-methylmethionine, which can be used by _E. coli_ as a source of methionine when externally provided [69]. S-methylmethionine is widely produced by plants and can be used as an osmoprotectants in plants [70], but it has not been reported as an osmoprotecant in _E. coli_. As _E. coli_ is not known to produce S-methylmethionine, nor did we supplement S-methylmethionine into the media, in this case, the impact of _mmuP_ on osmotic tolerance is not obvious. Stress response genes _bolA_ and _hdeA_ are downregulated in the BW25113 _rpoC_ mutant, and the deletion of _bolA_ and _hdeA_ improved the growth of BW25113 in the presence of 0.6 M NaCl (Figure 9).