The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Abstract

The gene encoding a putative high-potential iron-sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron-sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a DtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria. INTRODUCTIONOne of the most studied bacteria that thrives in acidic mine drainage is Acidithiobacillus ferrooxidans, formerly Thiobacillus ferrooxidans (Kelly & Wood, 2000;Leduc & Ferroni, 1994). It is characterized as a Gram-negative, acidophilic chemolithoautotroph that obtains energy for its growth mainly from the oxidation of ferrous iron (Fe 2+ ) or reduced sulfur compounds (Ingledew, 1982). Attempts to understand such an energetic metabolism, and especially the oxidation of Fe 2+ , have been initiated, leading to the proposition of several respiratory chains involving various redox proteins that have been identified and characterized in A. ferrooxidans (Fukumori et al., 1988;Blake & Shute, 1994;Yamanaka & Fukumori, 1995;Giudici-Orticoni et al., 1997Appia-Ayme, 1998;Appia-Ayme et al., 1999). Among them, a high-potential iron-sulfur protein (HiPIP) designated Iro, for iron-oxidizing enzyme, was characterized in A. ferrooxidans strains Fe-1 (Fukumori et al., 1988;Kusano et al., 1992) and BRGM (Cavazza et al., 1995) and the corresponding iro gene was also cloned and studied from strain Fe-1 (Kusano et al., 1992). The Iro protein was proposed to be the first electron acceptor in several alternative models of electron transfer chain between Fe 2+ and oxygen (Fukumori et al., 1988;Yamanaka et al., 1995). Based on genetic and subcellular localization studies with strain ATCC 33020, we proposed a model, not involving Iro, in which the electrons are passed from Fe 2+ to oxygen through a set of redox proteins expressed from an operon (Appia-Ayme, 1998;Appia-Ayme et al., 1998, 1999 Bengrine et al., 1998; Yarzábal et al., 2002) that is induced in Fe 2+ -grown cells (Yarzábal et al., 2004). To investigate the possible role of Iro, we probed for the presence of the iro Abbr...

Nonredundant Roles for Cytochrome c 2 and Two High-Potential Iron-Sulfur Proteins in the Photoferrotroph Rhodopseudomonas palustris TIE-1

“…So, what role might Rpal_4085 play? There are examples of HiPIPs in nonphototrophic organisms that interact with the cytochrome bc 1 complex during respiration, such as those in Rhodothermus marinus (35,36) and Acidithiobacillus ferrooxidans (37,38). It is possible that Rpal_4085 serves a similar function in R. palustris respiration, although the ⌬Rpal_4085 mutant had no defect when growing aerobically on acetate and succinate under standard growth conditions.…”

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system

“…3C). Substrate-dependent TatA-YFP association was also seen when an alternative Tat substrate, the Acidithiobacillus ferrooxidans high-potential iron-sulfur protein (HiPIP) (35), was overproduced and TatA association was again dependent on a functional signal peptide (Fig. 3 A-C).…”

Purification and Characterization of Sulfide:Quinone Oxidoreductase from an Acidophilic Iron-Oxidizing Bacterium,Acidithiobacillus ferrooxidans

“…It has been found that the production of periplasmic redox holoproteins from the acidophilic A. ferrooxidans in neutrophilic E. coli has presented experimental problems. 35) Rusticyanin, a blue-copper protein, has been produced in the periplasm, but the copper was not incorporated and reconstitution in vitro was required. 36) No SQR activity was detected when the recombinant protein was reconstituted in the presence of FAD.…”