Biochemical characterization of DesK, the membrane fluidity sensor of Bacillus subtilis

MARTÍN, M.; DE MENDOZA, D.

Mar del Plata- Argentina

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2007

The Des pathway of Bacillus subtillis, entirely unveiled in our laboratory, is composed of the Ä5-acyl lipid desaturase that introduces double bounds into saturated membrane phospholipids and the two component system DesK/DesR which stringently controls des expression. The induction of the pathway is brought about by the ability of the bifunctional histidine kinase/phosphatase DesK to assume different signalling states in response to changes in membrane lipid fluidity. This is accomplished in vivo by regulating the ratio of kinase to phosphatase activities of DesK over its response regulator, DesR. To gain insight into the mechanism by which the DesK sensor protein adjusts its signaling state in response to membrane fluidity changes, we wished to biochemically characterize DesK integrated into proteoliposomes. To this end, selected detergents or lipids of a comprehensive variety have been systematically evaluated with respect to their impact on the in vitro expression of DesK and their efficiency to keep synthesized DesK in solution when produced by means of a cell-free expression system. We found conditions in which DesK can be expressed in a soluble and functional form at high yields. Besides, DesK proteoliposomes were characterized in relation to its autokinase, phosphotransfer and phosphatase activities in order to identify which is the activity regulated by membrane lipids.