Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis.

Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, of Streptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.

Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.

We have used capacitance measurements with a 1-microsecond voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s-1 at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-s-1, pump transition (the E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.

Identification of the overtone of the Fe-CO stretching mode in heme proteins: a probe of the heme active site.

Two CO-isotope sensitive lines have been detected in the overtone region of the resonance Raman spectra of CO-bound hemeproteins. One line is assigned as the overtone of the Fe-CO stretching mode and is located in the 1000- to 1070-cm-1 region. The other line is found in the 1180- to 1210-cm-1 region and is assigned as a combination between a porphyrin mode, nu 7, and the Fe-CO stretching mode. The high intensities of these lines, which in the terminal oxidase class of proteins are of the same order as those of the fundamental stretching mode, indicate that the mechanism of enhancement for modes involving the Fe-CO moiety is different from that for the modes of the porphyrin macrocycle and call for reexamination of Raman theory of porphyrins as applied to axial ligands. The anharmonicity of the electronic potential function was evaluated, revealing that in the terminal oxidases the anharmonicity is greater than in the other heme proteins that were examined, suggesting a distinctive interaction of the bound CO with its distal environment in this family. Furthermore, the anharmonicity correlates with the frequency of the C-O stretching mode, demonstrating that both of these parameters are sensitive to the Fe-CO bond energy. The overtone and combination lines involving the bound CO promise to be additional probes of heme protein structural properties.