Light modulated enzymatic activities of the LOV histidine Kinase photoreceptor in Brucella abortus (Póster)

JUAN M. PAZ; IGNACIO FERNÁNDEZ; SEBASTIÁN KLINKE; FERNANDO A. GOLDBAUM; GABRIELA SYCZ; JIMENA RINALDI

Paraná

Congreso; LIV Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2018

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

Brucella abortus virulence increases when bacteria are previously illuminated with blue light, and this effect is mediated by LOV-HK. This enzyme is the sensor element of a two component system together with its response regulator, PhyR. This work is focused on understanding how light modulates B. abortus LOV-HK enzymatic activities. Firstly, we determined in vitro autokinase activity by evaluating the phosphorylation level of LOV-HK in the presence of ATP[gamma-32-P] and Mg2+. Our results showed that the phosphorylation level is greater when the enzyme is previously illuminated than when it is kept in the dark. Furthermore, a blind mutant does not show light activation. Secondly, we evaluated in vitro phosphotransferase activity by incubating LOV-HK with ATP, Mg2+ and PhyR. Samples were run in a SDS-PAGE containing Phostag, a reagent that delays the migration of phosphorylated proteins. Gel quantification revealed a greater level of phosphotransferase activity when illuminated. Additionally, in vitro phosphatase activity was assessed with previously phosphorylated PhyR, ADP and Mg2+. We found that LOV-HK has phosphatase activity independent of light. Finally, we analyzed in vivo phosphotransferase activity. To this end, we used B. abortus wt, lovhk::km and the complemented strains with lovhk and lovhk C69S. After SDS-PAGE-Phostag and subsequent WB analysis using anti-PhyR antibodies, we observed a greater level of phosphorylation when bacteria were previously illuminated. In conclusion, we found that LOV-HK is capable of acting as a phosphatase and that both autophosphorylation and phosphotransferase activities are increased upon light exposure.