The Klebsiella pneumoniae plasmid pJHCMW1 harbors a copy of Tn1331, a multiresistance transposon that includes the aac(6')-Ib gene which encodes a 6'-N-aminoglycoside acetyltransferase. This gene was mutagenized using the mutator Escherichia coli XL1-Red. Two plasmids with a single nucleotide mutation in aac(6')-Ib were selected for further analysis: pDP1 and pDP6. Plasmid pDP1 codes for a mutant enzyme, AAC(6')-IbDP1, that has the Phe171 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 degrees C. Furthermore, AAC(6')-IbDP1 showed a change of specificity of substrate when incubated at 42 degrees C. While its acetylating activity for Km was higher at this temperature than at 30 degrees C, it had its ability to utilize Ak as substrate for acetylation considerably reduced. Accordingly, minimal inhibitory concentration assays showed that E. coli(pDP1) was resistant to Ak at 37 degrees C but susceptible at 42 degrees C. The same assays showed that E. coli(pDP1) was highly resistant to Km at either 37 degrees C or 42 degrees C. A high level of resistance to Ak was observed for E. coli(pJHCMW1) which harbors the wild-type AAC(6')-Ib at either 37 or 42 degrees C. Extension of the analyses to other aminoglycosides showed that the enzymatic activity of AAC(6')-IbDP1 as well as the E. coli(pDP1) MICs for netilmicin dropped at 42 degrees C as was the case for Ak. These results could indicate that at 42 degrees C the mutant adopts a conformation that makes it unable to efficiently acetylate aminoglycoside molecules substituted in the C-1amino group of the deoxystreptamine moiety. Plasmid pDP6 encodes the mutant AAC(6')-IbDP6 which has the Tyr80 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')-IbDP6 showed only marginal levels of activity when either Ak, Km, tobramycin, or netilmicin were used as substrates.