The Aminoglycoside 6′- N -Acetyltransferase Type Ib Encoded by Tn 1331 Is Evenly Distributed within the Cell's Cytoplasm

Abstract
The multiresistance transposon Tn1331, which mediates resistance to several aminoglycosides and β-lactams, includes the aac(6′)-Ib, aadA1, blaOXA-9, and blaTEM-1 genes. The nucleotide sequence of aac(6′)-Ib includes a region identical to that of the blaTEM-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 β-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.