Summary Integrons are genetic elements able to capture anti‐biotic resistance and other genes and to promote their transcription. Here, we have investigated integron‐dependent translation of an aminoglycoside 6′‐N‐acetyltransferase gene (aac(6′)‐Ib7) inserted at the attI1 site. N‐terminal sequencing revealed that translation of this gene was initiated at a GTG codon, which is not part of a plausible translation initiation region (TIR). A short open reading frame (called ORF‐11) overlapping the attI1 site was probed by site‐directed mutagenesis for its contribution to aac(6′)‐Ib7 translation. When ORF‐11 and its TIR were deleted en bloc, translational efficiency dropped by over 80%, as determined with an acetyltransferase– luciferase fusion product. Invalidation of the ATG start codon of ORF‐11 or its putative Shine–Dalgarno sequence resulted in a decrease of over 60%, whereas the decrease was much less pronounced when the amino acid sequence of the putative ORF‐11‐encoded peptide was altered or when the distance between ORF‐11 and aac(6′)‐Ib7 was doubled. This demonstrates that aac(6′)‐Ib7 translation is dependent upon the translation of ORF‐11, but almost certainly not upon the corresponding peptide. These results lead us to conclude that an intrinsic short ORF present in the 5′‐conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR‐deficient anti‐biotic resistance genes.

We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 ␤-lactamase-producing strain of Salmonella enterica serovar Typhimurium previously isolated in Turkey. This gene, called aac(6)-Ib 11 , was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the ␤-lactamase OXA-1 and the acetyltransferase. The gene lacked a plausible Shine-Dalgarno (SD) sequence and was located 45 nucleotides downstream from a small open reading frame, ORF-18, with a coding capacity of 18 amino acids and a properly spaced SD sequence likely to direct the initiation of aac(6)-Ib 11 translation. AAC(6)-Ib 11 had Leu118 and Ser119 as opposed to Gln and Leu or Gln and Ser, respectively, which were observed in all previously described enzymes of this type. We have evaluated the effect of Leu or Gln at position 118 by site-directed mutagenesis of aac(6)-Ib 11 and two other acetyltransferase gene variants, aac(6)-Ib 7 and -Ib 8 , which naturally encode Gln118. Our results show that the combination of Leu118 and Ser119 confers an extended-spectrum aminoglycoside resistance, with the MICs of all aminoglycosides in clinical use, including gentamicin, being two to eight times higher for strains with Leu118 and Ser119 than for those with Gln118 and Ser119.