Expression of the Resistance-Nodulation-Cell Division Pump AdeIJK in Acinetobacter baumannii Is Regulated by AdeN, a TetR-Type Regulator

Resistance-nodulation-division efflux system AdeIJK contributes to intrinsic resistance to various drug classes in Acinetobacter baumannii. By whole-genome sequencing, we have identified in clinical isolate BM4587 the adeN gene, located 813 kbp upstream from adeIJK, which encodes a TetR transcriptional regulator. In one-step mutant BM4666 overexpressing adeIJK, the deletion of cytosine 582 (C-582) in the 3' portion of this gene was responsible for a frameshift mutation resulting in the deletion of the seven C-terminal residues. trans-Complementation of this BM4587 derivative with a plasmid expressing adeN restored antibiotic susceptibility to the host associated with the loss of adeJ overexpression. The inactivation of adeN in BM4587 led to a diminished susceptibility to antibiotics that are substrates for AdeIJK and to a 5-fold increase in adeJ expression. Taken together, these results indicate that AdeN represses AdeIJK expression. Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that AdeN is constitutively expressed in BM4587 and does not regulate its own expression. Deletion of cytosine 582 and a 394-bp deletion of the 3' part of adeN were found in independent one-step adeIJK-overexpressing mutants selected from clinical isolates BM4667 and BM4651, respectively. The corresponding alterations were located in the alpha 9 helix, which is known to be involved in dimerization, a process essential for the activity of TetR regulators. The adeN gene was detected in all of the 30 A. baumannii strains tested and in Acinetobacter calcoaceticus, Acinetobacter nosocomialis, and Acinetobacter pittii.