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Antimicrobial Agents and Chemotherapy, May 2003, p. 1577-1583, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1577-1583.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Aminoglycoside Resistance Resulting from Tight Drug Binding to an Altered Aminoglycoside Acetyltransferase

Sophie Magnet,1 Terry-Ann Smith,2 Renjian Zheng,1 Patrice Nordmann,3 and John S. Blanchard1*

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461,1 Cornell University, Ithaca, New York 14853,2 Service de Bactériologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France3

Received 28 October 2002/ Returned for modification 17 December 2002/ Accepted 10 February 2003

The aacA29b gene, which confers an atypical aminoglycoside resistance pattern to Escherichia coli, was identified on a class 1 integron from a multidrug-resistant isolate of Pseudomonas aeruginosa. On the basis of amino acid sequence homology, it was proposed that the gene encoded a 6'-N-acetyltransferase. The resistance gene was cloned into the pET23a(+) vector, and overexpression conferred high-level resistance to the usual substrates of the aminoglycoside N-acetyltransferase AAC(6')-I, except netilmicin. The level of resistance conferred by aacA29b correlated perfectly with the level of expression of the gene. The corresponding C-terminal six-His-tagged AAC(6')-29b protein was purified and found to exist as a dimer in solution. With a spectrophotometric assay, an extremely feeble AAC activity was detected with acetyl coenzyme A (acetyl-CoA) as an acetyl donor. Fluorescence titrations of the protein with aminoglycosides demonstrated the very tight binding of tobramycin, dibekacin, kanamycin A, sisomicin (Kd, <=1 µM) and a weaker affinity for amikacin (Kd, {approx}60 µM). The binding of netilmicin and acetyl-CoA could not be detected by either fluorescence spectroscopy or isothermal titration calorimetry. The inability of AAC(6')-29b to efficiently bind acetyl-CoA is supported by an alignment analysis of its amino acid sequence compared with those of other AAC(6')-I family members. AAC(6')-29b lacks a number of residues involved in acetyl-CoA binding. These results lead to the conclusion that AAC(6')-29b is able to confer aminoglycoside resistance by sequestering the drug as a result of tight binding.

* Corresponding author. Mailing address: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., 10461 Bronx, NY. Phone: (718) 430-3096. Fax: (718) 430-8565. E-mail: blanchar{at}aecom.yu.edu.

Antimicrobial Agents and Chemotherapy, May 2003, p. 1577-1583, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1577-1583.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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