Figure 4.

S. aureus dacA suppressors with mutations in opuD and alsT have improved growth in TSB. A and B, bacterial growth curves. WT LAC*, the dacA mutant, and suppressor strains S7 (opuD), S10 (alsT), and S18 (qoxB/ΔopuD) (or short S18 (ΔopuD)) were propagated in CDM (A) or TSB (B) medium, and their growth was monitored by taking A₆₀₀ readings. The average values and standard deviations from three independent experiments were plotted. C, genetic complementation of opuD mutants. Bacterial suspensions were prepared for WT, the dacA mutant, as well as suppressor strains S7 (opuD) and S18 (qoxB/ΔopuD) (or short S18 (ΔopuD)) containing the empty piTET vector or the complementation plasmid piTET-opuD. Appropriate culture dilutions were plated on TSA plates containing 200 ng/ml Atet and CFUs per ml culture per A₆₀₀ unit determined and plotted. The average values and standard deviations from three experiments were plotted. D, opuD mutants are defective in glycine betaine uptake. WT, the dacA mutant, and the suppressor strains S7 (opuD) and S18 (qoxB/ΔopuD) (or short S18 (ΔopuD)) containing the empty piTET vector or the plasmid piTET-opuD were grown to mid-log phase in CDM supplemented with 200 ng/ml Atet. For uptake assays, radiolabeled glycine betaine was added to culture aliquots; samples were removed and filtered at the indicated time points, and the radioactivity accumulated in the cells was measured. The average values and standard deviations from four experiments were plotted. E, alanine uptake assays. WT, dacA, and S10 (alsT) strains were grown to mid-log phase in CDM containing half the l-alanine concentration as in the standard medium. Bacterial suspensions were prepared, and radiolabeled alanine was added to the cultures. Sample aliquots were removed at the indicated time points and filtered, and the radioactivity accumulated in the cells was measured. The average values and standard deviations from four experiments were plotted.