Transposon mutagenesis.

The transposon EZTn5<KAN-2>Tnp (Epicenter, Madison, WI) was used for all experiments. To create an insertional library, A. baumannii strain AB900 was grown to an optical density at 600 nm (OD₆₀₀) of 0.5 in 30 ml LB medium and pelleted by centrifugation. Cells were washed 3 times with cold 10% glycerol and resuspended at 1/100 the original volume. Cells (60 μl) were electroporated with 1 μl EZ::Tn5<KAN-2>Tnp, plated on LB agar plates with kanamycin (40 μg/ml), and incubated overnight at 37°C. The resulting individual colonies were saved in 96-well microtiter plates containing 150 μl of LB plus 20% glycerol and stored at −80°C until further use. Colonies were tested for ceftriaxone sensitivity by dropping 1 μl of each cell suspension on LB plates containing ceftriaxone at either 1 μg/ml or 2 μg/ml.