Tobramycin uptake in Escherichia coli membrane vesicles

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Aminoglycoside antibiotics are crucial for treating bacterial infections.
• The precise mechanism of aminoglycoside uptake and bactericidal action remains incompletely understood.
• Previous studies suggested a role for membrane potential in aminoglycoside transport.

Purpose of the Study:

• To elucidate the role of the electrical potential (delta psi) in tobramycin uptake by Escherichia coli.
• To investigate the nature of the tobramycin transporter.
• To propose a mechanism for aminoglycoside-induced bacterial killing.

Main Methods:

• Measuring tobramycin uptake in energized and de-energized E. coli membrane vesicles.
• Manipulating membrane potential (delta psi) using ionophores (nigericin, valinomycin) and protonophores.
• Assessing the effect of N-ethylmaleimide on tobramycin uptake.
• Investigating tobramycin efflux from preloaded vesicles.

Main Results:

• Tobramycin uptake was dependent on a highly negative delta psi (-120 mV) and was not observed at lower potentials (-95 mV).
• Uptake was modulated by changes in delta psi, confirming it is gated by electrical potential, not proton motive force or delta pH.
• N-ethylmaleimide inhibited uptake, indicating the transporter is a cytoplasmic membrane protein with accessible sulfhydryl groups.
• Aminoglycosides decreased delta psi in susceptible cells, leading to channel closure and irreversible drug trapping.

Conclusions:

• Tobramycin uptake into E. coli is primarily mediated by a voltage-gated channel.
• The magnitude of the electrical potential across the cytoplasmic membrane is the critical factor governing tobramycin influx.
• Aminoglycoside-induced cell death may result from the drug's ability to decrease delta psi, leading to channel closure and lethal intracellular accumulation.