Fig 4. Complementation of an A. baumannii mutant by the four plasmids.

A-B. The coding region of the histinol dehydrogenase gene hisD was inserted into each of the four vectors and the resulting plasmids, pJL01::HisD, pJL02::HisD, pJL03::HisD and pJL04::HisD were introduced into the hisD deletion mutant of A. baumannii. Cells from overnight cultures in LB broth supplemented with appropriate antibiotics were washed with PBS and diluted 1000-fold. Aliquots of 7-μL cells were spotted onto VBS medium and images were acquired after incubation at 37°C for 16 h. C. The expression of Flag-HisD in the complementation strains. Overnight cultures diluted in LB broth were grown to OD₆₀₀ = 0.7 and arabinose and IPTG were added to the cultures at the indicated concentrations. After incubation at 37°C for 4 h, proteins solubilized by SDS sample buffer were separated by SDS-PAGE and Flag-HisD was detected by immunoblotting with the Flag-specific antibody. The metabolic enzyme isocitrate dehydrogenase (ICDH) was detected as a loading control.