A straightforward assay for measuring glycogen levels and RpoS

Abstract

Cellular glycogen levels reflect the activity of RpoS, an important stress-inducible bacterial sigma factor known to regulate several stress-resistance related genes, such as katE, encoding hydroperoxidase II (HPII), and the glg genes, encoding glycogen synthesis enzymes, in Escherichia coli. In this study, a straightforward assay for measuring glycogen levels and RpoS activity was developed combining the ease and simplicity of qualitative approaches. The assay reagent was a 2% iodine solution (2% iodine/1M NaOH), and the basic principle of this assay is the iodine-glycogen reaction, which produces a reddish brown color that can be measured using a spectrophotometer. A calibration plot using a known amount of glycogen yielded the best linear fit over a range of 10-300μg/assay (R²=0.994). The applicability of the assay for measuring the glycogen level of various samples was assessed using a wild type (WT) E. coli K-12 strain, glycogen- and RpoS-deficient isogenic mutants, and clinical bacterial isolates with or without RpoS activity; the assay generated reproducible results. Additionally, the assay was successfully applied for measuring glycogen levels in human cells. In conclusion, we developed a straightforward and cost-effective assay for measuring glycogen levels, which can be applied for measuring RpoS activity.

Keywords: Escherichia coli; Glycogen assay; Quantification method; RpoS activity; Stress sensitivity.