Figure 1. Kinetics of intracellular cAMP levels and expression of ACs in Mtb during in vitro growth.

A) Estimation of intracellular cAMP levels in Mtb. Wild-type Mtb CDC1551 was cultured in Middlebrook 7H9 broth supplemented with OADC, glycerol, and tween-80 at 37°C with shaking and growth was monitored at regular intervals by measuring optical density of the cultures at 600 nm (OD₆₀₀). An aliquot of culture suspension was used at each time point up to 8 days for cAMP measurement, as described in materials and methods, and final cAMP concentration was calculated as nmol/gm wet weight. Data are the averages of two independent experiments and the mean values ± standard deviations are shown. B) Analysis of the expression of AC-encoding genes at different time points during in vitro growth of Mtb. Total RNA was isolated from Mtb CDC1551 cultures at regular intervals as shown in the graph. Quantitative RT-PCR (qRT-PCR) was performed, and the relative expression of each gene was calculated using values of sigA transcript for normalizing the RNA amounts and day-1 expression value as a control. The graph represents an average of two independent experiments and the mean values ± standard deviations are shown.