Panaite D M, Tolmasky M E
Department of Biological Science, School of Natural Sciences and Mathematics, California State University Fullerton, California 92834-6850, USA.
Plasmid. 1998;39(2):123-33. doi: 10.1006/plas.1997.1330.
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.
肺炎克雷伯菌质粒pJHCMW1含有转座子Tn1331的一个拷贝，Tn1331是一种多耐药转座子，包含编码6'-N-氨基糖苷乙酰转移酶的aac(6')-Ib基因。利用诱变大肠杆菌XL1-Red对该基因进行诱变。选择两个在aac(6')-Ib中发生单核苷酸突变的质粒进行进一步分析：pDP1和pDP6。质粒pDP1编码一种突变酶AAC(6')-IbDP1，其第171位苯丙氨酸被亮氨酸残基取代。在30℃进行的酶活性测定中，当以卡那霉素（Km）或其半合成衍生物阿米卡星（Ak）作为底物时，这种突变衍生物的比活性低于野生型酶。此外，在42℃孵育时，AAC(6')-IbDP1的底物特异性发生了变化。虽然其在该温度下对Km的乙酰化活性高于30℃时，但利用Ak作为乙酰化底物的能力却大大降低。因此，最低抑菌浓度测定表明，大肠杆菌（pDP1）在37℃时对Ak耐药，但在4℃时敏感。同样的测定表明，大肠杆菌（pDP1）在37℃或42℃时对Km都具有高度耐药性。在37℃或42℃时，携带野生型AAC(6')-Ib的大肠杆菌（pJHCMW1）对Ak表现出高水平耐药性。将分析扩展到其他氨基糖苷类药物表明，与Ak的情况一样，在42℃时，AAC(6')-IbDP1的酶活性以及大肠杆菌（pDP1）对奈替米星的最低抑菌浓度均下降。这些结果可能表明，在42℃时，该突变体采取了一种构象，使其无法有效地乙酰化在脱氧链霉胺部分C-1氨基上被取代的氨基糖苷分子。质粒pDP6编码突变体AAC(6')-IbDP6，其第80位酪氨酸被半胱氨酸残基取代。大肠杆菌（pDP6）对Ak、Km、妥布霉素和奈替米星的最低抑菌浓度降低。当以Ak、Km、妥布霉素或奈替米星作为底物时，对AAC(6')-IbDP6的乙酰化活性分析表明其活性仅处于边缘水平。
