Osmotic and chill activation of glycine betaine porter II in Listeria monocytogenes membrane vesicles

Listeria monocytogenes is a foodborne pathogen known for its tolerance to c onditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organism's tolerance to both of these str esses. A procedure was developed for the purification of membranes from L, monocytogenes cells in which the putative ATP driven glycine betaine permea se gla cine betaine porter II (Gbu) is functional. As is the case for the L , monocytogenes sodium-driven glycine betaine uptake system (glycine betain e porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the gbu gene product ha d no uptake activity. Transport pv this porter did not require sodium ion a nd could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically act ivated and cryoactivated. Under conditions of osmotic activation, the syste m exhibited Arrhenius-type behavior although the uptake rates were profound ly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18 degrees C, In the absence of osmotic act ivation, the permease could be activated by decreasing temperature within t he range of 15 to 4 degrees C. Kinetic analyses of the permease at 30 degre es C revealed K-m values for glycine betaine of 1.2 and 2.9 mu M with V-max values of 2,200 and 3,700 pmol/min . mg of protein under conditions of opt imal osmotic activation as mediated by KCl and sucrose, respectively.