The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis.
Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.
Molenaar
D
D
Laboratory for Microbiology, University of Groningen, Haren, The Netherlands.
Bolhuis
H
H
Abee
T
T
Poolman
B
B
Konings
W N
WN
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism*; D006358:Hot Temperature; D013294:Lactococcus lactis / Q000187:drug effects / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism; D009154:Mutation; D014638:Vanadates / Q000494:pharmacology
D005452:Fluoresceins; D014638:Vanadates; D004024:Dicyclohexylcarbodiimide; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D000255:Adenosine Triphosphate; D007785:Lactose