The effects of copper ions on the synthesis of periplasmic and membrane proteins in Acidithiobacillus ferrooxidans as analyzed by SDS-PAGE and 2D-PAGE Ana Paula Felı ´cio a , Oswaldo Garcia Jr. a , Maria Ce ´lia Bertolini a , Laura M. Mariscal Ottoboni b , Maria Teresa Marques Novo a, * a Departamento de Bioquı ´mica e Tecnologia Quı ´mica, Instituto de Quı ´mica, Universidade Estadual Paulista, CP 355, 14801-970, Araraquara SP, Brazil b Centro de Biologia Molecular e Engenharia Gene ´tica, Universidade Estadual de Campinas, 13083-970, CP 6010, Campinas SP, Brazil Abstract The effects of 200 mM copper ions on the synthesis of membrane and periplasmic proteins were investigated in iron-grown cells of Acidithiobacillus ferrooxidans (At. ferrooxidans). Total membrane protein profiles of cells grown in the absence of copper ions (unadapted cells) and in the presence of copper ions (copper-adapted cells) were compared by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Crude preparations of outer membrane and periplasmic proteins were analyzed by SDS-PAGE. The synthesis of proteins was diminished or increased in the presence of copper ions. Low molecular weight proteins ( < 14 kDa) were significantly repressed by copper. These proteins are probably acidic proteins located in the outer membrane. An over-expression of a periplasmic protein of about 17 kDa was detected in the copper-adapted cells and was assumed to be rusticyanin, a 16.5-kDa periplasmic copper protein present in At. ferrooxidans cells and involved in the electron- transport chain of the iron oxidation pathway. To our knowledge, this is the first report of a possible involvement of the rusticyanin and outer membrane proteins in the mechanism of copper resistance in At. ferrooxidans. D 2003 Elsevier B.V. All rights reserved. Keywords: Copper resistance; Outer membrane protein; Periplasmic protein; Acidithiobacillus ferrooxidans; 2D-PAGE; Metal resistance; Rusticyanin 1. Introduction Acidithiobacillus ferrooxidans (At. ferrooxidans), formerly Thiobacillus ferrooxidans (Kelly and Wood, 2000), is a Gram-negative, acidophilic and chemoautotrophic bacterium that utilizes ferrous iron, sulfur and reduced sulfur compounds as ener- gy sources. It is commonly isolated from a wide range of sulfide-containing mineral deposits, and is industrially used in the recovery of gold, copper and uranium (McCready and Gould, 1990; Brierley and Brierley, 1999; Nyashanu et al., 1999). Copper is the most extensively recovered metal in biohydrometallurgical operations, mainly via dump or heap leaching systems. In recent years, bioreactors have been used in the recovery of copper from copper concentrates and in the pretreatment of gold ore con- centrates. In bioreactors, bioleaching processes are 0304-386X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0304-386X(03)00153-1 * Corresponding author. E-mail address: marinovo@iq.unesp.br (M.T.M. Novo). www.elsevier.com/locate/hydromet Hydrometallurgy 71 (2003) 165 – 171