Figure 7. RpoS negatively regulates glycogen synthesis in V. cholerae.

(A) Glycogen accumulation in WT and an rpoS mutant was quantified by enzymatically hydrolyzing glycogen stores into glucose monomers using amyloglucosidase during growth on low nitrogen M9 agar. The median and interquartile range of two independent experiments performed in quadruplicate are shown. An rpoS mutant was found to store significantly more glycogen than WT using a two-tailed student’s t-test (p=0.0014). (B) Quantitative RT-PCR (qRT-PCR) analysis of glgC1 and glgC2 expression during growth of WT and an rpoS mutant on M9-glucose under nitrogen limitation. Samples were collected during late- logarithmic growth (OD₆₀₀ = 0.8). All samples were prepared in triplicate and expression levels were normalized to rpoB expression in each sample and expressed relative to glgC1 or glgC2 expression in WT. The mean and standard deviation of a representative experiment is shown. (C) M9 minimal media with no carbon source or pond water were inoculated with a 1:1 ratio of rpoS:wt that were grown under nitrogen limiting conditions. At the indicated time-points, bacteria were removed and differentiated by blue:white screening. Each data point is the geometric mean of the CI obtained from samples tested in quadruplicate. A representative experiment is shown.