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Antimicrobial Agents and Chemotherapy, September 2003, p. 2897-2902, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.2897-2902.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Aminoglycoside 6'-N-Acetyltransferase Type Ib Encoded by Tn1331 Is Evenly Distributed within the Cell's Cytoplasm

Ken J. Dery,^1, Britta Søballe,² Mavee S. L. Witherspoon,¹ Duyen Bui,^1,2 Robert Koch,¹ David J. Sherratt,² and Marcelo E. Tolmasky^1,2*

Department of Biological Science, College of Natural Science and Mathematics, California State University Fullerton, Fullerton, California 92834-6850,¹ Division of Molecular Genetics, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom²

Received 4 March 2003/ Returned for modification 9 June 2003/ Accepted 14 June 2003

The multiresistance transposon Tn1331, which mediates resistance to several aminoglycosides and ß-lactams, includes the aac(6')-Ib, aadA1, bla_OXA-9, and bla_TEM-1 genes. The nucleotide sequence of aac(6')-Ib includes a region identical to that of the bla_TEM-1 gene. This region encompasses the promoter and the initiation codon followed by 15 nucleotides. Since there were three possible translation initiation sites, the amino acid sequence at the N terminus of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] was determined and was found to be SIQHF. This result indicated that aac(6')-Ib includes a translational fusion: the first five amino acids of the leader peptide of the TEM ß-lactamase are fused to the rest of the AAC(6')-Ib protein. This gene fusion could have formed during the genesis of Tn1331 as a consequence of the generation of a 520-nucleotide duplication (M. E. Tolmasky, Plasmid 24:218-226, 1990). An identical gene isolated from a Serratia marcescens strain has been previously described (G. Tran van Nhieu and E. Collatz, J. Bacteriol. 169:5708-5714, 1987). Extraction of the periplasmic proteins of E. coli harboring aac(6')-Ib by spheroplast formation showed that most of the AAC(6')-Ib protein is present in the cytoplasm. A genetic fusion to phoA confirmed these results. AAC(6')-Ib was shown to be evenly distributed inside the cell's cytoplasm by fluorescent microscopy with an AAC(6')-Ib-cyan fluorescent protein fusion.

Present address: Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010-3011.

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