Yuichi Someya

A novel glycylcycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, is neither transported nor recognized by the transposon Tn10-encoded metal-tetracycline/H+ antiporter.

A novel tetracycline derivative, DMG-DMDOT [9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline] , is one of the glycylcyclines which have a broad antibacterial spectrum, including many tetracyclineresistant bacteria (R.T. Testa, P.J. Petersen, N.V. Jacobus, P.-E. Sum, V.J. Lee, and F.P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). The mechanism by which DMG-DMDOT overcomes efflux-based tetracycline resistance was investigated. Tetracycline-resistant Escherichia coli cells carrying an R plasmid encoding the tet(B) gene, which encodes the typical tetracycline efflux pump [TetA(B)] of gram-negative bacteria, were as susceptible to DMG-DMDOT as was the tetracycline-susceptible host. When mid-log-phase cells carrying the tet(B) gene were incubated with a subbactericidal concentration of DMG-DMDOT (0.5 micrograms/ml) for 2 h, a significant amount of the TetA(B) protein was detected in the cell membrane by Western blotting (immunoblotting) with an anti-carboxyl-terminal antibody, similar to the case in which tetracycline was used as the inducer, indicating that the tet repressor, TetR, can recognize DMG-DMDOT as an efficient inducer. Everted membrane vesicles prepared from cells producing the TetA(B) protein showed absolutely no transport activity for DMG-DMDOT. Furthermore, the presence of excess DMG-DMDOT had no effect on the tetracycline transport activity of the everted vesicles, indicating that DMG-DMDOT is not recognized as a substrate by the TetA(B) protein.

Role of the Charge Interaction between Arg70 and Asp120 in the Tn10-encoded Metal-Tetracycline/H+ Antiporter of Escherichia coli

We reported that the positive charge of Arg70 is mandatory for tetracycline transport activity of Tn10-encoded metal-tetracycline/H+ antiporter (TetA(B)) (Someya, Y., and Yamaguchi, A. (1996)Biochemistry 35, 9385–9391). Arg70 may function through a charge-pairing with a negatively charged residue in close proximity. Therefore, we mutated Asp66 and Asp120, which are only two negatively charged residues located close to Arg70 in putative secondary structure of TetA(B) and highly conserved throughout transporters of the major facilitator superfamily. Site-directed mutagenesis studies revealed that Asp66 is essential, but Asp120 is important for TetA(B) function. Surprisingly, when Asp120was replaced by a neutral residue, the R70A mutant recovered tetracycline resistance and transport activity. There was no such effect in the Asp66 mutation. The charge-exchanged mutant, R70D/D120R, also showed significant drug resistance and transport activity (about 50% of the wild type), although the R70D mutant had absolutely no activity, and the D120R mutant retained very low activity (about 10% of the wild type). Both the R70C and D120C mutants were inactivated by N-ethylmaleimide. Mercuric ion (Hg2+), which gives a positive charge to a SH group of a Cys residue through mercaptide formation, had an opposite effect on the R70C and D120C mutants. The activity of the R70C mutant was stimulated by Hg2+; however, on the contrary, the D120C mutant was partially inhibited. On the other hand, the R70C/D120C double mutant was almost completely inactivated by Hg2+, probably because the side chains at positions 70 and 120 are bridged with Hg2+. The close proximity of positions 70 and 120 were confirmed by disulfide cross-linking formation of the R70C/D120C double mutant when it was oxidized by copper-(1,10-phenanthroline). These results indicate that the positive charge of Arg70 requires the negative charge of Asp120 for neutralization, probably for properly positioning transmembrane segments in the membrane.

A novel compound, 1,1-dimethyl-5(1-hydroxypropyl)-4,6,7-trimethylindan, is an effective inhibitor of the tet(K) gene-encoded metal-tetracycline/H+ antiporter of Staphylococcus aureus.

A novel indan derivative, 1,1‐dimethyl‐5‐(1‐hydroxypropyl)‐4,6,7‐trimethylindan (Ro 07‐3149), was found to be a strong inhibitor of the tet(K) gene‐encoded tetracycline/H+ antiporter of Staphylococcus aureus. One micromole of this compound per mg membrane protein was enough for complete inhibition of the Tet(K)‐mediated tetracycline transport and tetracycline‐coupled proton transport, without the energy state of the membrane being affected. The mode of inhibition was non‐competitive. Although this compound caused membrane de‐energization at a high concentration, the IC50 value for de‐energization (7.3 μmol/mg membrane protein) was about 17 times and 33 times higher than the values for Tet(K)‐mediated proton/tetracycline antiport and [3H]tetracycline transport, respectively, indicating that the inhibitory action of Ro 07‐3149 is not due to the uncoupling effect of the inhibitor.

Second-site suppressor mutations for the Arg70 substitution mutants of the Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli

The positive charge of the Arg70 residue in the cytoplasmic loop of the Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) of Escherichia coli is essential for the tetracycline transport function (Y. Someya and A. Yamaguchi, Biochemistry 35, 9385-9391 (1996)). In this study, we found that the R70A mutation was suppressed by the second-site mutation of Thr171 to Ser. The T171S mutation suppressed any mutations at position 70 regardless of the amino acid residue introduced. The R70A and R70C mutations were also suppressed by the T171A or T171C mutations, but not by the T171Y mutation, indicating that the decrease in the side chain volume at position 171 is essential for the suppression. Tetracycline transport activity of the R70C mutant was stimulated by Hg2+ because mercaptide formed between the SH group of Cys70 and Hg2+ worked as a functional positively-charged side chain. The activity of the R70A/R71C/T171S mutant was also stimulated by Hg2+, whereas those of the R70A/R71C, R71C, and R71C/T171S mutants were not, indicating that the T171S mutation causes the switching of the functional positive charge at position 70 to 71. Since Thr171 is in the middle of the transmembrane helix VI, the switching may be based on a remote conformational effect.