Twelve-Transmembrane-Segment (TMS) Version (ΔTMS VII-VIII) of the 14-TMS Tet(L) Antibiotic Resistance Protein Retains Monovalent Cation Transport Modes but Lacks Tetracycline Efflux Capacity

Abstract

A “Tet(L)-12” version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na+/H+antiport and antiport with K+ as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me2+/H+ antiport inEscherichia coli vesicles.

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

“…The P156A and C mutants may retain some electrogenic monovalent cation/proton antiport activity although they were negative for Tc-Co 2+ efflux. Several earlier mutants led to the hypothesis that although the efflux substrates share a binding site and translocation pathway, the most complicated substrate, the Tc-Co 2+ complex, required more elements to position it properly than the simpler monovalent cation efflux substrates (40,41,49). Together, the current observations suggest that Pro 156 is not essential for antiport but is very important in tight packing and "leak-proofing" in this critical region in both Tet(L) and Tet(K).…”

“…This suggested that these mutant Tet(L) proteins promote leakage of K + into the vesicles in response to the generation of the transmembrane potential, ΔΨ, while such a leak is not observed in wild type Tet(L) vesicles or with other Tet(L) mutants (e.g. see Figure 3) (40,41,49). The activity in the complete assay, with both electron donor and intravesicular K + , was lower than wild type in each Pro 156 mutant ( Figure 4).…”

“…The A 600 after 15 hours was plotted for determination of MIC (49). All assays were carried out in duplicate in at least two independent experiments.…”

“…After everted membrane samples (50 μg protein) were fractionated on sodium dodecyl sulfatepolyacrylamide gels and transferred to nitrocellulose membranes, Western analyses were carried out using antibodies raised to synthetic peptides corresponding to the N-terminus of Tet(L) or Tet(K), as described earlier (49). The signals were quantified by using ImageQuant software (Molecular Dynamics).…”

Tet(L) and Tet(K) Tetracycline-Divalent Metal/H + Antiporters: Characterization of Multiple Catalytic Modes and a Mutagenesis Approach to Differences in Their Efflux Substrate and Coupling Ion Preferences

“…For assays of Tc r , 10 l of stationary-phase cultures of the E. coli DH5␣ transformants with various tet genes expressed from pBK15 was inoculated into 2 ml of LBK medium containing a TC concentration of 0, 2, 4, 8, 10, 12, 16, or 32 g/ml. The data for growth (A 600 ) at 15 h were plotted for determinations of the MIC (26). For assays of complementation of the K ϩ uptake defect of E. coli TK2420, 10 l of a stationary-phase culture was inoculated into defined medium (9) containing KCl concentrations from 5 to 25 mM.…”