Sensitive biological detection method for tetracyclines using a tetA-lacZ fusion system

Chopra, Ian; Hacker, K J; Misulovin, Ziva; Rothstein, David M.

doi:10.1128/aac.34.1.111

Cited by 19 publications

(19 citation statements)

References 26 publications

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“…Chelocardin, a derivative that binds poorly to the tetracycline repressor (7), was the single exception. The spectrum of tetracyclines detected is similar to that detected in previously published screenings (4,12). The tetracycline assay was also effective in detecting tetracycline from fermentation broths of tetracycline-producing bacteria (data not shown).…”

Section: Resultssupporting

confidence: 68%

Detection of tetracyclines and efflux pump inhibitors

Rothstein¹,

McGlynn²,

Bernan³

et al. 1993

Antimicrob Agents Chemother

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Screening assays for the detection of tetracyclines and inhibitors of tetracycline efflux pumps are described. The tetracycline assay is based on the observation that the tetA(B) gene encoding the efflux pump of transposon Tn10 is induced by tetracycline. The Escherichia coli strain designed to detect tetracyclines contains a single copy of a tetA(B)-lacZ transcriptional fusion integrated into the chromosome and the tetR gene encoding the tetracycline repressor on a plasmid. The assay specifically detects tetracyclines of distinct structures, but not other classes of drugs. A strain capable of detecting inhibitors of the TetA(B) efflux pump contained the tetA(B)-lacZ fusion and, in addition, a tetA(B) structural gene lacking its transcriptional regulatory signals which mediated resistance to only 5 micrograms of tetracycline per ml. This strain was more refractory to induction by tetracycline because of the action of the pump. Inhibitors were detected in two ways: (i) beta-galactosidase induction in the presence of 5 ng of tetracycline per ml, a subinducing concentration, and (ii) growth inhibition in the presence of 5 micrograms of tetracycline per ml. A strain designed to detect inhibitors of the Tet(K) efflux pump from Staphylococcus aureus was constructed by substituting the tet(K) structural gene for the tetA(B) gene. Nocardamine and other siderophores were found to interfere with the action of tetracycline efflux pumps.

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Section: Resultssupporting

confidence: 68%

Detection of tetracyclines and efflux pump inhibitors

Rothstein¹,

McGlynn²,

Bernan³

et al. 1993

Antimicrob Agents Chemother

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“…Therefore it was of interest to determine whether these analogs were able to enter the cytoplasm. Entry of tetracyclines into the cytoplasm can be assessed by examining ,-galactosidase induction in a strain carrying a tetA-lacZ fusion, e.g., pUB3610 in which expression of P-galactosidase from a tetA-lacZ translational fusion is controlled by the pSC101 tetR gene (4). Thus, induction of P-galactosidase synthesis by a tetracycline derivative reflects its entry into the cell and inactivation of the cytoplasmically located repressor encoded by tetR.…”

Section: Resultsmentioning

confidence: 99%

“…The procedures used were as described previously (4). Preparation of ribosomes and structural analysis of 16S rRNA.…”

Section: Methodsmentioning

confidence: 99%

Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome

Rasmussen¹,

Noller²,

Daubresse³

et al. 1991

Antimicrob Agents Chemother

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Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6-deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not.The tetracyclines are a group of broad-spectrum antibiotics which are generally considered to prevent bacterial growth by inhibiting protein synthesis. This results from binding of antibiotic to a single site in the 30S ribosomal subunit which prevents attachment of aminoacyl tRNA to the ribosomal acceptor site (3). In order to reach the ribosome, these antibiotics must traverse the hydrophobic lipid bilayer of the bacterial cytoplasmic membrane (3). At physiological pH tetracyclines can exist as an equilibrium mixture of two free base forms: a low-energy, lipophilic nonionized species and a high-energy, hydrophilic zwitterionic structure (7). A solvent-dependent equilibrium between the two forms has been demonstrated with oxytetracycline free base and is supported by X-ray analyses of tetracyclines crystallized from aqueous and nonaqueous solvents (7,15). Both forms are believed to be important for the antibacterial activity of tetracyclines, the low-energy, lipophilic conformational form (Fig. 1A) for uptake across the cytoplasmic membrane and the hydrophilic, zwitterionic structure (Fig. 1B) for binding to the ribosome (7).Chelocardin (14) is a naturally occurring anhydrotetracycline derivative with a modified A ring (Fig. 2). In contrast to the solvent-dependent equilibrium of the two tetracycline species mentioned above, chelocardin apparently exists in the same conformation in both polar and nonpolar solvents, as evidenced by circular dichroism measurements (6). We believe this is related to the planarity of the BCD rings in chelocardin and that a lipophilic form, perhaps related to that of tetracycline, is the preferred species. Thes...

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“…Biosensors have enabled the detection and quanti¢cation of various compounds, such as metals [1], xenobiotic compounds [2], and antibiotics [3].…”

Section: Introductionmentioning

confidence: 99%

“…Most biosensor organisms used so far carry the biosensor gene cassettes on plasmids [3,10,11]. Problems with plasmid based systems can be varying copy numbers and loss of sensitivity [12].…”

Section: Introductionmentioning

confidence: 99%

Detection and quantification of tetracyclines by whole cell biosensors

Hansen

2000

FEMS Microbiology Letters

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Three different mini-Tn5 plasmids, containing a tetracycline-inducible promoter, P tet and a regulatory gene, tetR, in operon fusions with a reporter gene system (lacZYA, luxCDABE or gfp), were constructed. These biosensor constructs responded to low levels of tetracyclines by producing L-galactosidase, light or green fluorescent protein. They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism. All three constructs were transferred successfully to different Gramnegative bacteria by conjugation. An Escherichia coli strain containing the P tet -lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors. ß

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Sensitive biological detection method for tetracyclines using a tetA-lacZ fusion system

Cited by 19 publications

References 26 publications

Detection of tetracyclines and efflux pump inhibitors

Detection of tetracyclines and efflux pump inhibitors

Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome

Detection and quantification of tetracyclines by whole cell biosensors

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