Purification and Characterization of an ATPase GsiA from Salmonella enterica.

ABSTRACT

The coding sequence of Salmonella enterica gsiA was cloned and expressed in E. coli. The protein was purified and ATPase activity was characterized by NADH oxidation method. GsiA exhibited optimum activity at 30°C and at pH 8 in Tris/HCl buffer. GsiA protein was stable at 20°C. 66% and 44% activity remained after incubation at 30°C and 40°C for 30 min. pH 7 and pH 9 incubation would obviously reduce the ATPase activity. In vivo functionality of gsiA was determined by constructing gene deletion strains. gsiA was shown to be essential for GSI mediated glutathione uptake and gsiA deletion could decrease the virulence of Salmonella enterica. Interactions of glutathione import proteins GsiA, GsiB, GsiC, and GsiD were investigated by using bacterial two-hybrid system. GsiA could interact with itself and inner membrane proteins GsiC and GsiD. This report provides the first description of gsiA functions in Salmonella enterica. The results could help elucidating the glutathione uptake mechanism and glutathione functions in bacteria.