Differential Effects of Sucrose or NaCl Osmotic Shock on β-galactosidase Activity in Escherichia coli BW25993 is Dependent on Normalization Method by Total Viable Count or Total Protein Content

Abstract

The purpose of this study was to determine if the osmoregulatory response in Escherichia coli BW25993 varied depending on the solute used to cause osmotic stress, as measured by β-galactosidase enzyme activity. β-galactosidase enzyme activity was measured in E. coli BW25993 after osmotic shock with NaCl or sucrose for 2 hours. β-galactosidase activity was normalized against number of colony forming units or to total protein content, to demonstrate comparable, standardized activities. The normalization of the results eliminated the issue of variable culture concentration and viability. By offering several normalization methods, this study has provided a more comprehensive assessment of the complexity of osmoregulation. With either normalization method, β-galactosidase activity showed no significant difference compared to the Luria-Bertani Lennox medium control after a 0.3 M NaCl stress condition, but β-galactosidase activity increased at 0.6 M NaCl. When normalized against total protein, β-galactosidase activity decreased with increasing sucrose concentration (0, 0.6 and 1.2 M stress conditions). In contrast, β-galactosidase activity normalized against colony forming units decreased at 0.6 M sucrose, but increased at 1.2 M sucrose, relative to the control. To investigate the cause of differing results between normalizations, a sucrose dose-response curve was performed and it was determined that both cell viability and culture turbidity decreased with increasing sucrose concentrations. The discrepancy between the two different β-galactosidase activity normalization methods at 1.2 M sucrose is therefore likely a mathematical result of low cell viability, but somewhat higher protein content, due to the presence of dead cells. In the sucrose dose-response, the turbidity decreased faster than the viable cell count, which may be explained by higher bacterial aggregations at higher sucrose concentrations. Therefore, the β-galactosidase activity normalized against total protein or viable cell counts differs when E. coli BW25993 is osmotically shocked with NaCl or sucrose. This suggests that osmoregulation is dependent on the source of the osmotic stress, and is not restricted to a single physiological response.