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.
AllmeierH., CresnarB., GreckM., SchmittR.1992; Complete nucleotide sequence of Tn1721: gene organization and a novel gene product with features of a chemotaxis protein. Gene 111:11–20 [View Article][PubMed]
BerensC., HillenW.2003; Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. Eur J Biochem 270:3109–3121 [View Article][PubMed]
ColemanD. C., FosterT. J.1981; Analysis of the reduction in expression of tetracycline resistance determined by transposon Tn10 in the multicopy state. Mol Gen Genet 182:171–177 [View Article][PubMed]
HartmanA. B., EssietI. I., IsenbargerD. W., LindlerL. E.2003; Epidemiology of tetracycline resistance determinants in Shigella spp. and enteroinvasive Escherichia coli: characterization and dissemination of tet(A)-1.. J Clin Microbiol 41:1023–1032 [View Article][PubMed]
McMurryL., LevyS. B.1978; Two transport systems for tetracycline in sensitive Escherichia coli: critical role for an initial rapid uptake system insensitive to energy inhibitors. Antimicrob Agents Chemother 14:201–209 [View Article][PubMed]
McMurryL., PetrucciR. E.Jr, LevyS. B.1980; Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli.. Proc Natl Acad Sci U S A 77:3974–3977 [View Article][PubMed]
NguyenT. T., PostleK., BertrandK. P.1983; Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322. Gene 25:83–92 [View Article][PubMed]
SaengerW., OrthP., KiskerC., HillenW., HinrichsW.2000; The tetracycline repressor-a paradigm for a biological switch. Angew Chem Int Ed Engl 39:2042–2052 [View Article][PubMed]
SapunaricF. M., LevyS. B.2005; Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity. Microbiology 151:2315–2322 [View Article][PubMed]
SoaresG. M., FigueiredoL. C., FaveriM., CortelliS. C., DuarteP. M., FeresM.2012; Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs. J Appl Oral Sci 20:295–309 [View Article][PubMed]
TuckmanM., PetersenP. J., ProjanS. J.2000; Mutations in the interdomain loop region of the tetA(A) tetracycline resistance gene increase efflux of minocycline and glycylcyclines. Microb Drug Resist 6:277–282 [View Article][PubMed]