FIG 3.

Expression of ABUW_0982 reduces chloramphenicol burden in A. baumannii. (A) Analysis of chloramphenicol uptake in E. coli MG1655 over time. Overnight cultures were diluted in fresh LB medium and grown to exponential phase. Cells were harvested by centrifugation and resuspended in PBS to an OD of 2.0. Cells were either left untreated or incubated with 20 mM glucose or 20 μM CCCP for 15 min at 37°C with shaking. After the initial incubation, 2.5 μM [¹⁴C]chloramphenicol was added to each sample. At each time point, cells were removed and collected by centrifugation, and recovered cells were added to liquid scintillation cocktail. Cell-associated chloramphenicol was measured, and results are displayed as counts per minute (cpm). Notice the increase in cell-associated CHL following CCCP treatment and the decrease in cell-associated CHL after glucose pretreatment relative to that for untreated cells. These results are representative of multiple independent experiments. (B) Analysis of chloramphenicol uptake by wild-type Ab5075. The experimental protocol was the same as that for panel A. Cells were either left untreated or incubated with 20 mM sodium succinate or 20 μM CCCP. Note that the addition of succinate does not reduce the levels of cell-associated CHL. Preincubation with CCCP increases cell-associated CHL. These results are representative of multiple independent experiments. (C) ABUW_0982 is necessary for optimal CHL efflux in A. baumannii cells. The Tn:0982 mutant expressing empty vector (pEmpty) or p0982 was evaluated for chloramphenicol efflux as described above. Over time, less cell-associated chloramphenicol or more efflux is observed for the mutant cells expressing p0982. The error bars represent the standard deviations for three independent experiments. *, P < 0.1; **, P < 0.05. Significance was determined using Student's t test.