FIGURE 2.

c-di-AMP depletion negatively affects growth of S. aureus. A, growth of S. aureus strains LAC* and LAC*idacA. Growth curves of LAC* were performed as described in Fig. 1A. An overnight culture of LAC*idacA grown in the presence of 50 ng/ml Atet was washed, diluted to an A₆₀₀ of 0.01, and grown to an A₆₀₀ of 0.5 in the presence or absence Atet. Cultures were again diluted to an A₆₀₀ of 0.01 (time = 0 h) and grown in the presence and absence of 50 ng/ml Atet for 8 h. Growth curves were performed three times, and average A₆₀₀ readings and standard deviations are plotted. B, intracellular c-di-AMP levels. The intracellular concentration of c-di-AMP in strain LAC*idacA grown in the presence or absence of Atet was determined at the 2- and 4-h time points by LC-MS/MS. As LAC*idacA cultures grown in the absence of Atet contain a large number of nonviable cells, c-di-AMP concentrations are presented as nanograms of c-di-AMP/mg of dry weight. CFU counts were obtained for LAC*idacA grown in the presence of Atet, and hence the micromolar concentration of c-di-AMP is also indicated. Average values and standard deviation of three experiments are plotted. C, TEM images of wild type LAC* and LAC*idacA grown in the absence of Atet. Samples were taken at the 4-h time point during bacterial growth and prepared for TEM as described under “Experimental Procedures.” Representative images and scale bars are shown.