title: High-level tetracycline resistance mediated by efflux pumps Tet(A) and Tet(A)-1 with two start codons.
abstract: 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, GTG 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 µg 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 µg ml(-1) following GTG mutation, and to ≤4 µg ml(-1) following mutation of both GTG and ATG. It was presumed that class A tet determinants had two start codons, which are the primary start codon GTG 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.
authors.affiliation: Institute of Biomedical Science, Fudan University, Shanghai, PR China.|Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China.|Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China.
authors.forename: Weixia
authors.initials: W
authors.lastname: Wang
authors.affiliation: Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China.|Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China.
authors.forename: Qinglan
authors.initials: Q
authors.lastname: Guo
authors.affiliation: Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China.|Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Xu
authors.affiliation: Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China.|Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China.
authors.forename: Zi-Ke
authors.initials: ZK
authors.lastname: Sheng
authors.affiliation: Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China.|Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Ye
authors.affiliation: Institute of Biomedical Science, Fudan University, Shanghai, PR China.|Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, PR China.|Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China.
authors.forename: Minggui
authors.initials: M
authors.lastname: Wang
mesh_terms: D017920:Antiporters / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018387:Codon, Initiator; D003227:Conjugation, Genetic / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D013752:Tetracycline / Q000494:pharmacology*; D013753:Tetracycline Resistance / Q000235:genetics*
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D018387:Codon, Initiator; C079051:tetA protein, Bacteria; D013752:Tetracycline