Cysteine‐scanning mutagenesis around transmembrane segment VI of Tn10‐encoded metal‐tetracycline/H<sup>+</sup> antiporter

Konishi, Satoko; Iwaki, Shinobu; Kimura‐Someya, Tomomi; Yamaguchi, Akihito

doi:10.1016/s0014-5793(99)01490-8

Cited by 20 publications

(19 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We also intentionally chose the Thr191Cys mutant for its strategical position between Asp190 and Glu192 as another 'control' (Tamura et al, 2001). The single cysteine mutations on a low-copynumber plasmid were provided by A. Yamaguchi and their tetracycline MICs were consistent with those reported previously (Kimura-Someya et al, 2000;Konishi et al, 1999;Tamura et al, 2001). For each single cysteine mutant, the MICs and the ratio of the MIC for the two analogues to the MIC for tetracycline showed a proportional decrease in resistance to both tetracycline and to the analogues in all cases (see Table 2).…”

Section: Resultssupporting

confidence: 85%

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Sapunaric

Levy

2005

Microbiology

View full text Add to dashboard Cite

Cysteine replacement of Asp190, Glu192 and Ser201 residues in the cytoplasmic interdomain loop of the TetA(B) tetracycline efflux antiporter from Tn10 reduces tetracycline resistance [Tamura, N., Konishi, S., Iwaki, S., Kimura-Someya, T., Nada, S. & Yamaguchi, A. (2001). J Biol Chem 276, 20330–20339]. It was found that these Cys substitutions altered the substrate specificity of TetA(B), increasing the relative resistance to doxycycline and minocycline over that to tetracycline by three- to sixfold. Substitutions of Asp190 and Glu192 by Ala, Asn and Gln also impaired the ability of TetA(B) to mediate tetracycline resistance while Ser201Ala and Ser201Thr substitutions did not. A Leu9Phe substitution in the first transmembrane helix of TetA(B) suppressed the Ser201Cys mutation, undoing the alterations in resistance and specificity. That the interdomain loop might contact substrate during transport, as is suggested from its role in substrate specificity, is unexpected considering that the primary sequence in the loop is not conserved among a group of otherwise homologous TetA proteins. However, in the interdomain loop of 11 of 14 homologous TetA efflux proteins, computational analysis revealed a short α-helix, which includes some residues affecting activity and substrate specificity. Perhaps this conserved secondary structure accounts for the role of the non-conserved interdomain loop in TetA function.

show abstract

Section: Resultssupporting

confidence: 85%

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Sapunaric

Levy

2005

Microbiology

View full text Add to dashboard Cite

show abstract

“…Therefore, within the efflux proteins from gramnegative bacteria, these loops may interact functionally as the proton pump (175). The boundaries of membrane-embedded domains have been defined (126,134), and the proposed topology of the proteins developed by earlier modeling methods has been confirmed experimentally (127). Furthermore, charge interactions between key residues such as arginine-70 and aspartate-120 in the Tet(B) protein have been identified as a requirement for correct positioning of transmembrane segments in the cytoplasmic membrane (279).…”

Section: Genetic and Biochemical Mechanisms Of Tetracycline Resistancementioning

confidence: 99%

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

Chopra

Roberts

2001

Microbiol Mol Biol Rev

3,630

2,643

View full text Add to dashboard Cite

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century

show abstract

“…Resistance-We have hitherto constructed 260 cysteine-scanning mutants as to TM1 to TM6, TM9 and TM11, and the connecting loop regions of TetA(B) and reported their reactivity with sulfhydryl reagents (6,7,8,20,21,22). In this study, we constructed 140 cysteine-scanning mutants as to TM7, TM8, TM10, and TM12 and their connecting loop regions.…”

Section: Construction Of Cysteine-scanning Mutants and Their Drugmentioning

confidence: 99%

Complete Cysteine-scanning Mutagenesis and Site-directed Chemical Modification of the Tn10-encoded Metal-Tetracycline/H+ Antiporter

Tamura

Konishi

Iwaki

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Bacterial Tn10-encoded metal-tetracycline/H؉ antiporter was the first found drug exporter and has been studied as a paradigm of antiporter-type major facilitator superfamily transporters. Here the 400 amino acid residues of this protein were individually replaced by cysteine except for the initial methionine. As a result, we could obtain a complete map of the functionally or structurally important residues. In addition, we could determine the precise boundaries of all the transmembrane segments on the basis of the reactivity with Nethylmaleimide (NEM). The NEM binding results indicated the presence of a transmembrane water-filled channel in the transporter. The twelve transmembrane segments can be divided into three groups; four are totally embedded in the hydrophobic interior, four face a putative water-filled channel along their full length, and the remaining four face the channel for half their length, the other halves being embedded in the hydrophobic interior. These three types of transmembrane segments are mutually arranged with a 4-fold symmetry. The competitive binding of membrane-permeable and -impermeable SH reagents in intact cells indicates that the transmembrane water-filled channel has a thin barrier against hydrophilic molecules in the middle of the transmembrane region. Inhibition and stimulation of NEM binding in the presence of tetracycline reflects the substrate-induced protection or conformational change of the Tn10-encoded metal-tetracycline/H ؉ antiporter. The mutations protected from NEM binding by tetracycline were mainly located around the permeability barrier in the N-terminal half, suggesting the location of the substrate binding site.

show abstract

Cysteine‐scanning mutagenesis around transmembrane segment VI of Tn10‐encoded metal‐tetracycline/H⁺ antiporter

Cited by 20 publications

References 20 publications

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

Complete Cysteine-scanning Mutagenesis and Site-directed Chemical Modification of the Tn10-encoded Metal-Tetracycline/H+ Antiporter

Contact Info

Product

Resources

About

Cysteine‐scanning mutagenesis around transmembrane segment VI of Tn10‐encoded metal‐tetracycline/H+ antiporter

Cited by 20 publications

References 20 publications

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Substitutions in the interdomain loop of the Tn10 TetA efflux transporter alter tetracycline resistance and substrate specificity

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

Complete Cysteine-scanning Mutagenesis and Site-directed Chemical Modification of the Tn10-encoded Metal-Tetracycline/H+ Antiporter

Contact Info

Product

Resources

About

Cysteine‐scanning mutagenesis around transmembrane segment VI of Tn10‐encoded metal‐tetracycline/H⁺ antiporter