A straightforward assay for measuring glycogen levels and RpoS

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Cellular glycogen levels are indicators of RpoS activity, a key bacterial stress-response regulator.
• RpoS influences stress-resistance genes like katE (hydroperoxidase II) and glg genes (glycogen synthesis) in Escherichia coli.
• Existing methods for measuring glycogen and RpoS activity can be complex.

Purpose of the Study:

• To develop a straightforward and cost-effective assay for measuring cellular glycogen levels.
• To establish a reliable method for assessing bacterial RpoS activity.
• To validate the assay's performance across different bacterial strains and human cells.

Main Methods:

• A simple assay using a 2% iodine solution (2% iodine/1M NaOH) was developed.
• The assay relies on the colorimetric iodine-glycogen reaction, measured spectrophotometrically.
• Calibration with known glycogen amounts demonstrated linearity (R²=0.994) over 10-300μg/assay.

Main Results:

• The assay provided reproducible measurements of glycogen levels in wild-type E. coli K-12.
• Glycogen levels were accurately assessed in RpoS- and glycogen-deficient mutants.
• The assay successfully measured glycogen in clinical bacterial isolates and human cells, reflecting RpoS activity.

Conclusions:

• A straightforward, cost-effective assay for measuring glycogen levels was successfully developed.
• This assay serves as a reliable indicator of RpoS activity in bacteria.
• The method's applicability extends to diverse bacterial samples and human cells.