Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system

Abstract

The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153-->Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116-->Leu in AdeR at the first residue of the alpha(5) helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system.

FIG. 1.

Schematic representation of the ade gene cluster of BM4454 and recombinant plasmids. Open arrows represent coding sequences and indicate the direction of transcription. Closed arrowheads indicate the positions and orientations of the primers used to generate PCR fragments (represented by thin lines), which were used as probes: probe A (positions 3477 to 4369), probe B (positions 6197 to 7175), probe C (positions 8110 to 8737), probe R (positions 2548 to 2846), and probe S (positions 2193 to 2512) (all positions are according to accession no. AF370885 in GenBank). The inserts in recombinant plasmids are represented by horizontal thick lines, and the vectors are indicated in parentheses under the name of the plasmid.

FIG. 2.

Transcription analysis of the adeA, adeB, and adeC genes by Northern hybridization. Total RNA depleted of 16S and 23S RNA from BM4454 (lane 1), CIP 70-10 (lane 2), BM4546 (CIP 70-10 AdeS_T153M) (lane 3), and BM4547 (CIP 70-10 AdeR_P116L) (lane 4) was hybridized with the probes indicated at the top. The sizes of the transcripts relative to RNA molecular size marker I (Boehringer) are indicated.

FIG. 3.

Identification of the transcriptional start site for the adeABC operon in BM4454 by primer extension analysis. (Left panel) Lanes T, G, C, and A, results of sequencing reactions performed with primer EA-adeA; lane 1, control without RNA; lane 2, primer elongation product obtained with oligonucleotide EA-adeA and 25 μg of total RNA from BM4454 (arrowhead); (right panel) sequence from nucleotide positions 2951 to 3539 according to numbering of the sequence with GenBank accession no. AF370885. The +1 transcriptional start site for adeABC and the potential −35 and −10 promoter sequences are indicated and underlined. The ATG start codon of the adeA gene is boxed, and the ribosome-binding site is underlined.

FIG. 4.

Transcription analysis of adeR and adeS genes. (Left) Agarose gel electrophoresis of the product obtained by RT-PCR with primers RS-av and RS-am and corresponding Southern hybridizations with probes specific for adeS (center) and adeR (right). Lane 1, 100-bp DNA ladder (Gibco BRL). Incubations were carried out in the presence (lane 2) or in the absence (lane 3) of reverse transcriptase.