Acidithiobacillus ferrooxidans oxidizes ferrous iron before sulfur likely through transcriptional regulation by the global redox responding RegBA signal transducing system ☆ José Sandoval Ponce a , Danielle Moinier a , Deborah Byrne b , Agnès Amouric a , Violaine Bonnefoy a, ⁎ a Aix-Marseille Université and C.N.R.S., Institut de Microbiologie de la Méditerranée, Laboratoire de Chimie Bactérienne UMR-CNRS 7283, 31 chemin Joseph Aiguier, Cedex 20, 13402, Marseille, France b Aix-Marseille Université and C.N.R.S., Institut de Microbiologie de la Méditerranée,Protein Expression Facility, 31 chemin Joseph Aiguier, Cedex 20, 13402, Marseille, France abstract article info Available online 4 August 2012 Keywords: Acidithiobacillus ferrooxidans Iron oxidation Reduced inorganic sulfur compound oxidation Redox state RegBA two-component signal transducing system The mechanisms of ferrous iron (Fe(II)) and reduced inorganic sulfur compounds (RISC) oxidation under oxic conditions of the bioleaching bacterium Acidithiobacillus ferrooxidans have been deciphered. The genes involved in these pathways are more transcribed in the presence than in the absence of the corresponding electron donor, i.e. the genes involved in Fe(II) oxidation are expressed preferentially in the presence of Fe(II) than sulfur (S 0 ) and those involved in RISC oxidation are more transcribed in S 0 - than in Fe(II)-grown cells. In this study, oxida- tion of Fe(II) and S 0 and expression of the main genes involved in these pathways have been followed when both electron donors are present in the medium. The results obtained clearly indicate that (i) S 0 oxidation takes place only when iron is totally oxidized and (ii) the genes involved in Fe(II) are expressed before those involved in RISC oxidation, even in the presence of S 0 . Therefore, a transcriptional regulator is likely inducing the genes involved in Fe(II) oxidation and repressing those involved in RISC oxidation when Fe(II) is present. Data presented in this paper suggest that the redox sensing sensor/regulator two-component signal transducing system RegBA could be responsible for this regulation since (i) the redox potential increases during Fe(II) oxidation but remains stable during S 0 oxidation and (ii) the regulator RegA is able to bind to the regulatory region of a number of genes/operons of which the expression is regulated by Fe(II). The understanding of how RegBA controls the en- ergetic pathways depending on the overall redox state of the cell is of primordial importance since it could help in controlling the dynamics of Fe(II) and RISC oxidation during bioleaching processes. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Bioleaching heap and acid mine drainage water environments are characterized by a fluctuating redox potential. Acidithiobacillus (At.) ferrooxidans is a well-known mesophilic Gram negative proteobacterium frequently detected in these environments where it thrives and plays a key role in controlling the geochemistry of these extreme habitats. This acidophilic microorganism uses exclusively inorganic substrates as carbon and energy sources, i.e. it is a chemolithoautotroph. Indeed, this bacterium is able to fix carbon dioxide using the oxidation of ferrous iron (Fe(II)) and reduced inorganic sulfur compounds (RISC) present in sulfide ores as electron donors. The products of these oxidations, namely ferric iron (Fe(III)) and sulfuric acid respectively, chemically attack the mineral thus releasing the metal (for example, copper in the case of chalcopyrite), Fe(II) and RISC. Therefore, the role of this prokaryote in the “biomining” process is to recycle these chemical agents. Most progress to understand the molecular mechanisms underlying the Fe(II) and RISC oxidation pathways in the type strain (ATCC 23270) of At. ferrooxidans has been made using biochemistry, molecular ge- netics and functional genomic tools (see reviews and references there- in (Bonnefoy, 2010; Bonnefoy and Holmes, 2012; Holmes and Bonnefoy, 2007; Quatrini et al., 2009)). The At. ferrooxidans respiratory system is flexible allowing it to adapt efficiently to environmental changes by modulating gene expression according to the prevailing growth conditions (Quatrini et al., 2009). Indeed, in the At. ferrooxidans type strain, genes involved in the Fe(II) oxidation pathways are induced in the presence of Fe(II) and those involved in the RISC oxidation are more expressed in sulfur (S 0 )- than in Fe(II)-grown cells (Amouric et al., 2009; Quatrini et al., 2006, 2009), indicating that the expression of the genes involved in Fe(II) and RISC oxidation is controlled depending on the electron donor available in the environment. Thus far, nothing is known about the mechanism underlying this regulation. Nevertheless, three models could be proposed (Fig. 1). In the first one (Fig. 1a), Fe(II) and RISC oxidation occur simultaneously. The genes involved in the oxidation of an electron donor are induced in the presence of Hydrometallurgy 127–128 (2012) 187–194 ☆ This paper was originally presented at the International Biohydrometallurgy Sym- posium (IBS), Changsha, China, 18-22 September 2011. ⁎ Corresponding author. Tel.: +33 491 164 146; fax: +33 491 718 914. E-mail addresses: sandovalponcejose@gmail.com (J.S. Ponce), moinier@imm.cnrs.fr (D. Moinier), byrne@imm.cnrs.fr (D. Byrne), agnesamouric@yahoo.fr (A. Amouric), bonnefoy@imm.cnrs.fr (V. Bonnefoy). 0304-386X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hydromet.2012.07.016 Contents lists available at SciVerse ScienceDirect Hydrometallurgy journal homepage: www.elsevier.com/locate/hydromet