Efflux is the most common mechanism of tetracycline resistance. Class A tetracycline efflux pumps, which often have high prevalence in Enterobacteriaceae, are encoded by tet(A) and tet(A)-1 genes. These genes have two potential start codons, GIG and ATG, located upstream of the genes. The purpose of this study was to determine the start codon(s) of the class A tetracycline resistance (tet) determinants tet(A) and tet(A)-1, and the tetracycline resistance level they mediated. Conjugation, transformation and cloning experiments were performed and the genetic environment of tet(A)-1 was analysed. The start codons in class A tet determinants were investigated by site-directed mutagenesis of ATG and GTG, the putative translation initiation codons. High-level tetracycline resistance was transferred from the clinical strain of Klebsiella pneumoniae 10-148 containing tet(A)-1 plasmid pHS27 to Escherichia coli J53 by conjugation. The transformants harbouring recombinant plasmids that carried tet(A) or tet(A)-1 exhibited tetracycline MICs of 256-512 mu g ml(-1), with or without tetR(A). Once the ATG was mutated to a non-start codon, the tetracycline MICs were not changed, while the tetracycline MICs decreased from 512 to 64 mu g ml(-1) following GTG mutation, and to 4 mu g ml(-1) following mutation of both GIG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GIG and secondary start codon ATG. Accordingly, two putative promoters were predicted. In conclusion, class A tet determinants can confer high-level tetracycline resistance and have two start codons.
[1]Fudan Univ, Huashan Hosp, Inst Antibiot, Shanghai 200433, Peoples R China.
[2]Minist Hlth, Key Lab Clin Pharmacol Antibiot, Beijing, Peoples R China.
[3]Fudan Univ, Inst Biomed Sci, Shanghai 200433, Peoples R China.
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