Membrane lateral phase separations and chlortetracycline transport by Bacillus megaterium

Abstract

Chlortetracycline, a fluorescent robe of its own active transport, has been used to study lateral phase sep- Tetracyclines are broad-spectrum antibiotics that bind to the bacterial ribosome, where they specifically inhibit protein biosynthesis (1, 2). The antibiotic is most likely accumulated within bacteria by an active transport system, which may account for the specificity of the drug (3). In several recent papers, we have shown that tetracyclines can be used as fluorescent probes of their own transport through bacterial membranes (4-7). Qualities that make them ideal for transport studies are the specificity of binding of the probe to the transport site in the bacterial membrane and the large increase in the quantum yield of fluorescence when tetracyclines bind to these apolar regions of the membrane through a divalent cation complex. The latter property results in great sensitivity for detection of the probe without perturbation of membrane integrity. Monitoring the tetracycline fluorescence allows one to follow antibiotic accumulation continuously. We have previously used the fluorescence to demonstrate saturation of the tetracycline transport system in Staphylococcus aureus and to examine the role of the membrane lipid in this transport system (4, 5). The temperature dependence of the transport rate, as measured by fluorescence enhancement, and tetracycline mobility changes, as measured by fluorescence polarization, were used in these studies (5). (14). Transesterification of the fatty acids with boron trifluoride/methanol followed by gas chromatography on a diethylene glycol succinate column at 1700 showed that the major fatty acids of these cells were the branched-chain fatty acids, anteiso and iso-C13

Membrane-Active Macromolecules Resensitize NDM-1 Gram-Negative Clinical Isolates to Tetracycline Antibiotics

“…coli (ATCC 25922) to understand the mechanism of re-sensitization of tetracycline antibiotics. Tetracycline is fluorescent in aqueous solutions and it is reported that there is an enhancement in its fluorescence as it traverses the bacterial cell membranes [ 29 ]. The uptake of tetracycline (100 μg mL -1 ) alone was less and slow in bla NDM-1 E .…”

Florfenicol As a Modulator Enhancing Antimicrobial Activity: Example Using Combination with Thiamphenicol against Pasteurella multocida

“…The OTC uptake was evaluated via monitoring the fluorescence enhancement of oxytetracycline according to previous reports (Dockter et al, 1978 ; Ejim et al, 2011 ). P. aeruginosa (103-3430) grown in 10 mL TSB to OD 600 = 0.8 were harvested at 2000 g for 10 min and then resuspended in 2.5 mL of 10 mM HEPES pH7.2.…”

Bacterial resistance to the tetracyclines.

“…Although Franklin and Higginson (63) were unable to demonstrate saturation kinetics for the initial rate of uptake by whole cells, Franklin (59) showed that uptake by membrane vesicles was saturable. Data obtained more recently (45,47,158) provide convincing evidence that uptake of tetracycline and chlortetracycline by several bacteria, including E. coli, S. aureus, and B. megaterium, demonstrates saturation kinetics. Furthermore, in B. megaterium there is evidence from biophysical studies that the membrane carrier for tetracycline is a protein (47).…”

“…Data obtained more recently (45,47,158) provide convincing evidence that uptake of tetracycline and chlortetracycline by several bacteria, including E. coli, S. aureus, and B. megaterium, demonstrates saturation kinetics. Furthermore, in B. megaterium there is evidence from biophysical studies that the membrane carrier for tetracycline is a protein (47). Data of another type also support the notion of a protein carrier for chlortetracycline transport.…”