The sod gene from Rhodobacter capsulatus. Negative modulatio by RegA

ANA BORTOLOTTI; LEANDRO C. TABARES; MATÍAS TEMPERINI; NESTOR CORTEZ

Rosario

Congreso; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Rhodobacter capsulatus is a phototrophic -proteobacteria capable of heterotrophic (aerobic) or photosynthetic (autotrophic) growth. A single sod gene encodes a cambialistic superoxide dismutase (RcSOD) that exchanges its cofactor, Fe or Mn, depending on oxygen tension. A sod insertional mutant was isolated on minimal medium plates under light in anaerobiosis. The Rhodobacter SOD- strain displayed a 2-times slower duplication time than the wild-type and undergoes intracellular superoxide increase, as deduced by lucigenin promoted luminescence. Lowering of aconitase activity reveals disintegration of 4Fe-4S clusters while DNA oxidative damage was visualized as a 4-fold increase of spontaneous mutation frequency in the sod mutant. The conserved RegA/RegB regulatory system provides an overlying layer of redox control of metabolism in bacteria. Three putative RegA binding sites (-10, -53 and -129) upstream the gene were identified by means of reported consensus. The expression of gene was analyzed by western blot, in-situ SOD-activity PAGE and galactosidase activity measuring from a ::lacZ translational fusions. Although the amount of SOD increases with the oxygen tension, the A mutant displayed always higher levels of dismutase than wild-type, suggesting a repressor role for RegA in the expression of sod in Rhodobacter capsulatus.