Abstract
An 84 bp in-frame duplication (K370_A396dup) within the rpoC subunit of RNA polymerase was found in two independent mutants selected during an adaptive laboratory evolution experiment under osmotic stress in Escherichia coli, suggesting that this mutation confers improved osmotic tolerance. To determine the role this mutation in rpoC plays in osmotic tolerance, we reconstructed the mutation in BW25113, and found it to confer improved tolerance to hyperosmotic stress. Metabolite analysis, exogenous supplementation assays, and cell membrane damage analysis suggest that the mechanism of improved osmotic tolerance by this rpoC mutation may be related to the higher production of acetic acid and amino acids such as proline, and increased membrane integrity in the presence of NaCl stress in exponential phase cells. Transcriptional analysis led to the findings that the overexpression of methionine related genes metK and mmuP improves osmotic tolerance in BW25113. Furthermore, deletion of a stress related gene bolA was found to confer enhanced osmotic tolerance in BW25113 and MG1655. These findings expand our current understanding of osmotic tolerance in E. coli, and have the potential to expand the utilization of high saline feedstocks and water sources in microbial fermentation.
Highlights
One of the challenges in the microbial production of chemicals is the low tolerance of the microbial host to inhibitors present in the feedstock
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 on osmotic stress response [15,38], we hypothesized that there are additional amino acids that play a role in osmotic tolerance in E. coli
This may be partially due to the decreased ATP requirements for amino acid biosynthesis when these amino acids are supplemented [39], which 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
Summary
One of the challenges in the microbial production of chemicals is the low tolerance of the microbial host to inhibitors present in the feedstock. Improved osmotic tolerance in microbial systems can potentially increase their productivity when using high saline feedstocks, and expand the utilization of these alternate feedstocks and water sources. We had previously identified an 84 bp in-frame duplication (K370_A396dup) in the RNA polymerization domain of RpoC of the RNA polymerase in two independently evolved osmotolerant mutants [24], which suggests that this rpoC mutation confers osmotic tolerance and warrants further study. We reconstructed this rpoC mutation in a wild-type BW25113 background and confirmed the beneficial effect of this mutation on osmotic tolerance. Subsequent metabolite analysis, membrane damage analysis and transcriptional analysis of the mutant revealed potential mechanisms of how this specific rpoC mutation confers tolerance to osmotic stress
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