Abstract
The presence of intracellular glycogen can be detected by the following iodine staining technique. Cells with glycogen stain dark brown, whereas in its absence they remain with a pale yellowish color. It is hypothesized that iodine atoms fit into helical coils formed by the α-polyglucan to form a coloured glycogen-iodine complex. Here, we have studied the expression of Streptococcus mutans (S. mutans) genes that control the biosynthesis of this polysaccharide (Asencion Diez et al., 2013). Thus, we expressed glgC and glgD genes coding for both ADP-Glc pyrophosphorylase subunits in Escherichia coli (E. coli) AC70R1-504 cells to complement the deficient accumulation of glycogen by this strain (Iglesias et al., 1993). In control cells or in those where an inactive protein was expressed, the synthesis of the polysaccharide was undetectable by this iodine staining technique.
Keywords: Reserve polysaccharide, Bacterial metabolism, Qualitative analysis, ADP-glucose, Glycogenesis
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Iodine staining of cells accumulating different amounts of glycogen. Iodine staining of cell pellets from E. coli AC70RI-504 (from left to right): Untransformed cells (lacking ADP-Glc PPase, negative control), transformed to express GlgC (catalytic subunit of ADP-Glc PPase forming homotetrameric enzyme, low positive sample), transformed to express GlgD (non-catalytic, inactive, subunit of ADP-Glc PPase, negative sample), or to express GlgC/GlgD (heterotetrameric ADP-Glc PPase, high positive sample). The staining is mainly qualitative and as much it could be established a semi-quantitative scale based on the visualization by eye (or scanner).
Notes
Acknowledgments
This work was supported by grants to AAI from CONICET [PIP 2519 and CONICET-NSF 19537/28/06/12], UNL [CAI+D Orientado and Redes], and ANPCyT [PICT’08 1754]; and to MAB from the NSF [MCB 1024945]. MDAD is fellow from CONICET; SAG and AAI are investigators from the same institution.
References
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