TheKlebsiella pneumoniaeplasmid pJHCMW1 harbors a copy of Tn1331,a multiresistance transposon that includes theaac(6′)-Ibgene which encodes a 6′-N-aminoglycoside acetyltransferase. This gene was mutagenized using the mutatorEscherichia coliXL1-Red. Two plasmids with a single nucleotide mutation inaac(6′)-Ibwere selected for further analysis: pDP1 and pDP6. Plasmid pDP1 codes for a mutant enzyme, AAC(6′)-Ib DP1 , that has the Phe 171 replaced by a Leu residue. This mutant derivative showed a lower specific activity than the wild-type enzyme when either kanamycin (Km) or its semisynthetic derivative amikacin (Ak) were used as substrates in enzymatic assays performed at 30°C. Furthermore, AAC(6′)-Ib DP1 showed a change of specificity of substrate when incubated at 42°C. While its acetylating activity for Km was higher at this temperature than at 30°C, it had its ability to utilize Ak as substrate for acetylation considerably reduced. Accordingly, minimal inhibitory concentration assays showed thatE. coli(pDP1) was resistant to Ak at 37°C but susceptible at 42°C. The same assays showed thatE. coli(pDP1) was highly resistant to Km at either 37°C or 42°C. A high level of resistance to Ak was observed forE. coli(pJHCMW1) which harbors the wild-type AAC(6′)-Ib at either 37 or 42°C. Extension of the analyses to other aminoglycosides showed that the enzymatic activity of AAC(6′)-Ib DP1 as well as theE. coli(pDP1) MICs for netilmicin dropped at 42°C as was the case for Ak. These results could indicate that at 42°C the mutant adopts a conformation that makes it unable to efficiently acetylate aminoglycoside molecules substituted in the C-1 amino group of the deoxystreptamine moiety. Plasmid pDP6 encodes the mutant AAC(6′)-Ib DP6 which has the Tyr 80 substituted by a Cys residue.E. coli(pDP6) exhibited reduced MICs for Ak, Km, tobramycin, and netilmicin. Analysis of the acetylating activity of AAC(6′)-Ib DP6 showed only marginal levels of activity when either Ak, Km, tobramycin, or netilmicin were used as substrates.