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

1 Laboratoire de Chimie Bactérienne, IBSM, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France 2 Laboratoire de Bioénergétique et Ingénierie des Protéines, IBSM, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France 3 Universidad Autonoma de Madrid, Centro de Biologia Molecular, Cantoblanco, Madrid, Spain Correspondence Violaine Bonnefoy bonnefoy{at}ibsm.cnrs-mrs.fr 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 tatC 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. Abbreviations: DOP PCR, degenerate oligonucleotide primers PCR; HiPIP, high-potential iron–sulfur protein The GenBank/EMBL/DDBJ accession numbers for the partial iro gene sequences from A. ferrooxidans strains CC1, ATCC 19859 and BRGM-1 are AJ621386–AJ621388 and the accession numbers of the iro locus from strains BRGM and ATCC 33020 are AJ621560 and AJ320262 . Details of primers, an alignment of iro gene sequences from Fe-1 and ATCC 23270 and the sequence of the iro locus of ATCC 33020 are available as supplementary material with the online version of this paper.