Tobramycin uptake in Escherichia coli is driven by either electrical potential or ATP

Abstract

Aminoglycoside antibiotics such as streptomycin and tobramycin must traverse the bacterial cytoplasmic membrane prior to initiating lethal effects. Previous data on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis have demonstrated that transport of aminoglycosides is regulated by delta psi, the electrical component of the proton motive force. However, several laboratories have observed that growth of bacterial cells can occur in the apparent absence of delta psi, and we wished to confirm these studies with E. coli and further investigate whether transport of aminoglycosides could occur in the absence of a membrane potential. Treatment of acrA strain CL2 with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) dissipated delta psi, decreased intracellular ATP levels, and resulted in cessation of growth; after a variable period of time (3 to 7 h), growth resumed, ultimately achieving growth rates comparable to those of untreated cells. Absence of delta psi in these cells was confirmed by absence of [3H]tetraphenyl phosphonium+ uptake as measured by membrane filtration, lack of flagellar motion, and inability of these cells to transport proline (but not methionine). Regrowth was associated with restoration of normal intracellular ATP as measured by luciferin-luciferase bioluminescence assay. Unlike unacclimatized CL2 cells treated with CCCP, these cells transported [3H]tobramycin similarly to untreated cells; aminoglycoside-induced killing was seen in association with transport. These studies suggest that under certain circumstances aminoglycoside transport can be driven by ATP (or other high-energy activated phosphate donors) alone, in the absence of a measurable delta psi. delta uncBC mutants of CL2 incapable of interconverting delta psi and ATP were treated with CCCP, resulting in dissipation of delta psi but no alteration in ATP content. Despite maintenance of normal ATP, there was no transport of [3H] bramycin, confirming that under normal growth conditions ATP has no role in the transport of aminoglycosides.

Escherichia coli is able to grow with negligible sodium ion extrusion activity at alkaline pH

“…Heefner and Harold (13) reported the passive movement of sodium ions driven by the membrane potential in streptococci and they called this CCCP may be restricted to the particular media movement "energized leak." Thus, it is possible to assume ise previous studies (9,16). that E. coli generating the membrane potential can accumut activities of proline and serine in RS1.…”

“…In previous studies (9,16), growth stopped for a time after the addition of CCCP and then resumed. However, no cessation of growth was observed in the present study (Fig.…”

“…However, it was shown that E. coli could grow with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) at alkaline pH (22) as well as at neutral pH (9,16).…”

The spatial profiles and metabolic capabilities of microbial populations impact the growth of antibiotic-resistant mutants

“…At least two possible mechanisms have been suggested (i) enhanced active uptake of aminoglycosides [36][37][38][39]; (ii) more aminoglycosides in their non-ionized, more-bactericidal form [36]. Thus, we infer that the IF inhibits mutants by increasing pH.…”

Osmotic adaptation of Escherichia coli with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone

“…Culture medium (1.5 ml) was overlaid on the oil mixture of 0. 10,000 x g for 10 min at room temperature. Since the contents of amino acids and trehalose in the supernatant were negligible, washing of cells was omitted.…”