Search

The type strain of the mineral-oxidizing acidophilic bacterium Acidithiobacillus ferridurans was grown in liquid medium containing elevated concentrations of sodium chloride with hydrogen as electron donor. While it became more tolerant to chloride, after about 1 year, the salt-stressed acidophile was found to have lost its ability to oxidize iron, though not sulfur or hydrogen. Detailed molecular examination revealed that this was due to an insertion sequence, IS Afd1 , which belongs to the IS Pepr1 subgroup of the IS 4 family, having been inserted downstream of the two promoters PI and PII of the rus operon (which codes for the iron oxidation pathway in this acidophile), thereby preventing its transcription. The ability to oxidize iron was regained on protracted incubation of the culture inoculated onto salt-free solid medium containing ferrous iron and incubated under hydrogen. Two revertant strains were obtained. In one, the insertion sequence IS Afd1 had been excised, leaving an 11-bp signature, while in the other an ∼2,500-bp insertion sequence (belonging to the IS 66 family) was detected in the downstream inverted repeat of IS Afd1 The transcriptional start site of the rus operon in the second revertant strain was downstream of the two ISs, due to the creation of a new "hybrid" promoter. The loss and subsequent regaining of the ability of A. ferridurans T to reduce ferric iron were concurrent with those observed for ferrous iron oxidation, suggesting that these two traits are closely linked in this acidophile. IMPORTANCE Iron-oxidizing acidophilic bacteria have primary roles in the oxidative dissolution of sulfide minerals, a process that underpins commercial mineral-processing biotechnologies ("biomining"). Most of these prokaryotes have relatively low tolerance to chloride, which limits their activities when only saline or brackish waters are available. The study showed that it was possible to adapt a typical iron-oxidizing acidophile to grow in the presence of salt concentrations similar to those in seawater, but in so doing they lost their ability to oxidize iron, though not sulfur or hydrogen. The bacterium regained its capacity for oxidizing iron when the salt stress was removed but simultaneously reverted to tolerating lower concentrations of salt. These results suggest that the bacteria that have the main roles in biomining operations could survive but become ineffective in cases where saline or brackish waters are used for irrigation., (Copyright © 2018 American Society for Microbiology.)

Acidithiobacillus ferrooxidans is a strict acidophilic chemolithoautotrophic bacterium that obtains its energy from reduced inorganic sulfur species or ferrous iron oxidation under aerobic conditions. Carbon felt electrodes were pre-colonized by A. ferrooxidansATCC 23270 T using ferrous iron or sulfur as electron donors, via the addition (or not) of a mixture of C14 acyl-homoserine lactones (C14-AHLs). Electrode coverage during pre-colonization was sparse regardless of the electron donor source, whereas activation of quorum sensing significantly enhanced it. Microbial fuel cells (MFCs) inoculated with pre-colonized electrodes (which behaved as biocathodes) were more efficient in terms of current production when iron was used as an electron donor. Biocathode coverage and current output were remarkably increased to -0.56 A m -2 by concomitantly using iron-based metabolism and C14-AHLs. Cyclic voltammetry displayed different electrochemical reactions in relation to the nature of the electron donor, underlying the implication of different electron transfer mechanisms., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Considering the importance of tRNAs in the translation machinery, scant attention has been paid to tRNA array units defined as genomic regions containing at least 20 tRNA genes with a minimal tRNA gene density of two tRNA genes per kilobase. Our analysis of Acidithiobacillus ferrivorans CF27 and Acidithiobacillus ferrooxidans ATCC 23270(T) genomes showed that both display a tRNA array unit with syntenic conservation which mainly contributed to the tRNA gene redundancy in these two organisms. Our investigations into the occurrence and distribution of tRNA array units revealed that 1) this tRNA organization is limited to few phyla and mainly found in Gram-positive bacteria; and 2) the presence of tRNA arrays favors the redundancy of tRNA genes, in particular those encoding the core tRNA isoacceptors. Finally, comparative array organization revealed that tRNA arrays were acquired through horizontal gene transfer (from Firmicutes or unknown donor), before being subjected to tRNA rearrangements, deletions, and duplications. In Bacilli, the most parsimonious evolutionary history involved two common ancestors and the acquisition of their arrays arose late in evolution, in the genera branches. Functional roles of the array units in organism lifestyle, selective genetic advantage and translation efficiency, as well as the evolutionary advantages of organisms harboring them were proposed. Our study offers new insight into the structural organization and evolution of tRNA arrays in prokaryotic organisms., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The iron-oxidizing acidithiobacilli cluster into at least four groups, three of which (Acidithiobacillus ferrooxidans, Acidithiobacillus ferridurans and Acidithiobacillus ferrivorans) have been designated as separate species. While these have many physiological traits in common, they differ in some phenotypic characteristics including motility, and pH and temperature minima. In contrast to At. ferrooxidans and At. ferridurans, all At. ferrivorans strains analysed to date possess the iro gene (encoding an iron oxidase) and, with the exception of strain CF27, the rusB gene encoding an iso-rusticyanin whose exact function is uncertain. Strain CF27 differs from other acidithiobacilli by its marked propensity to form macroscopic biofilms in liquid media. To identify the genetic determinants responsible for the oxidation of ferrous iron and sulfur and for the formation of extracellular polymeric substances, the genome of At. ferrivorans CF27 strain was sequenced and comparative genomic studies carried out with other Acidithiobacillus spp.. Genetic disparities were detected that indicate possible differences in ferrous iron and reduced inorganic sulfur compounds oxidation pathways among iron-oxidizing acidithiobacilli. In addition, strain CF27 is the only sequenced Acidithiobacillus spp. to possess genes involved in the biosynthesis of fucose, a sugar known to confer high thickening and flocculating properties to extracellular polymeric substances., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Gene transcription (microarrays) and protein levels (proteomics) were compared in cultures of the acidophilic chemolithotroph Acidithiobacillus ferrooxidans grown on elemental sulfur as the electron donor under aerobic and anaerobic conditions, using either molecular oxygen or ferric iron as the electron acceptor, respectively. No evidence supporting the role of either tetrathionate hydrolase or arsenic reductase in mediating the transfer of electrons to ferric iron (as suggested by previous studies) was obtained. In addition, no novel ferric iron reductase was identified. However, data suggested that sulfur was disproportionated under anaerobic conditions, forming hydrogen sulfide via sulfur reductase and sulfate via heterodisulfide reductase and ATP sulfurylase. Supporting physiological evidence for H2S production came from the observation that soluble Cu(2+) included in anaerobically incubated cultures was precipitated (seemingly as CuS). Since H(2)S reduces ferric iron to ferrous in acidic medium, its production under anaerobic conditions indicates that anaerobic iron reduction is mediated, at least in part, by an indirect mechanism. Evidence was obtained for an alternative model implicating the transfer of electrons from S(0) to Fe(3+) via a respiratory chain that includes a bc(1) complex and a cytochrome c. Central carbon pathways were upregulated under aerobic conditions, correlating with higher growth rates, while many Calvin-Benson-Bassham cycle components were upregulated during anaerobic growth, probably as a result of more limited access to carbon dioxide. These results are important for understanding the role of A. ferrooxidans in environmental biogeochemical metal cycling and in industrial bioleaching operations.

This minireview presents recent advances in our understanding of iron oxidation and homeostasis in acidophilic Bacteria and Archaea. These processes influence the flux of metals and nutrients in pristine and man-made acidic environments such as acid mine drainage and industrial bioleaching operations. Acidophiles are also being studied to understand life in extreme conditions and their role in the generation of biomarkers used in the search for evidence of existing or past extra-terrestrial life. Iron oxidation in acidophiles is best understood in the model organism Acidithiobacillus ferrooxidans. However, recent functional genomic analysis of acidophiles is leading to a deeper appreciation of the diversity of acidophilic iron-oxidizing pathways. Although it is too early to paint a detailed picture of the role played by lateral gene transfer in the evolution of iron oxidation, emerging evidence tends to support the view that iron oxidation arose independently more than once in evolution. Acidic environments are generally rich in soluble iron and extreme acidophiles (e.g. the Leptospirillum genus) have considerably fewer iron uptake systems compared with neutrophiles. However, some acidophiles have been shown to grow as high as pH 6 and, in the case of the Acidithiobacillus genus, to have multiple iron uptake systems. This could be an adaption allowing them to respond to different iron concentrations via the use of a multiplicity of different siderophores. Both Leptospirillum spp. and Acidithiobacillus spp. are predicted to synthesize the acid stable citrate siderophore for Fe(III) uptake. In addition, both groups have predicted receptors for siderophores produced by other microorganisms, suggesting that competition for iron occurs influencing the ecophysiology of acidic environments. Little is known about the genetic regulation of iron oxidation and iron uptake in acidophiles, especially how the use of iron as an energy source is balanced with its need to take up iron for metabolism. It is anticipated that integrated and complex regulatory networks sensing different environmental signals, such as the energy source and/or the redox state of the cell as well as the oxygen availability, are involved., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Autotrophic acidophilic iron- and sulfur-oxidizing bacteria of the genus Acidithiobacillus constitute a heterogeneous taxon encompassing a high degree of diversity at the phylogenetic and genetic levels, though currently only two species are recognized (Acidithiobacillus ferrooxidans and Acidithiobacillus ferrivorans). One of the major functional disparities concerns the biochemical mechanisms of iron and sulfur oxidation, with discrepancies reported in the literature concerning the genes and proteins involved in these processes. These include two types of high-potential iron-sulfur proteins (HiPIPs): (i) Iro, which has been described as the iron oxidase; and (ii) Hip, which has been proposed to be involved in the electron transfer between sulfur compounds and oxygen. In addition, two rusticyanins have been described: (i) rusticyanin A, encoded by the rusA gene and belonging to the well-characterized rus operon, which plays a central role in the iron respiratory chain; and (ii) rusticyanin B, a protein to which no function has yet been ascribed. Data from a multilocus sequence analysis of 21 strains of Fe(II)-oxidizing acidithiobacilli obtained from public and private collections using five phylogenetic markers showed that these strains could be divided into four monophyletic groups. These divisions correlated not only with levels of genomic DNA hybridization and phenotypic differences among the strains, but also with the types of rusticyanin and HiPIPs that they harbour. Taken together, the data indicate that Fe(II)-oxidizing acidithiobacilli comprise at least four distinct taxa, all of which are able to oxidize both ferrous iron and sulfur, and suggest that different iron oxidation pathways have evolved in these closely related bacteria.

Background: Acidithiobacillus ferrooxidans gains energy from the oxidation of ferrous iron and various reduced inorganic sulfur compounds at very acidic pH. Although an initial model for the electron pathways involved in iron oxidation has been developed, much less is known about the sulfur oxidation in this microorganism. In addition, what has been reported for both iron and sulfur oxidation has been derived from different A. ferrooxidans strains, some of which have not been phylogenetically characterized and some have been shown to be mixed cultures. It is necessary to provide models of iron and sulfur oxidation pathways within one strain of A. ferrooxidans in order to comprehend the full metabolic potential of the pangenome of the genus., Results: Bioinformatic-based metabolic reconstruction supported by microarray transcript profiling and quantitative RT-PCR analysis predicts the involvement of a number of novel genes involved in iron and sulfur oxidation in A. ferrooxidans ATCC23270. These include for iron oxidation: cup (copper oxidase-like), ctaABT (heme biogenesis and insertion), nuoI and nuoK (NADH complex subunits), sdrA1 (a NADH complex accessory protein) and atpB and atpE (ATP synthetase F0 subunits). The following new genes are predicted to be involved in reduced inorganic sulfur compounds oxidation: a gene cluster (rhd, tusA, dsrE, hdrC, hdrB, hdrA, orf2, hdrC, hdrB) encoding three sulfurtransferases and a heterodisulfide reductase complex, sat potentially encoding an ATP sulfurylase and sdrA2 (an accessory NADH complex subunit). Two different regulatory components are predicted to be involved in the regulation of alternate electron transfer pathways: 1) a gene cluster (ctaRUS) that contains a predicted iron responsive regulator of the Rrf2 family that is hypothesized to regulate cytochrome aa3 oxidase biogenesis and 2) a two component sensor-regulator of the RegB-RegA family that may respond to the redox state of the quinone pool., Conclusion: Bioinformatic analysis coupled with gene transcript profiling extends our understanding of the iron and reduced inorganic sulfur compounds oxidation pathways in A. ferrooxidans and suggests mechanisms for their regulation. The models provide unified and coherent descriptions of these processes within the type strain, eliminating previous ambiguity caused by models built from analyses of multiple and divergent strains of this microorganism.

Acidithiobacillus caldus has been proposed to play a role in the oxidation of reduced inorganic sulfur compounds (RISCs) produced in industrial biomining of sulfidic minerals. Here, we describe the regulation of a new cluster containing the gene encoding tetrathionate hydrolase (tetH), a key enzyme in the RISC metabolism of this bacterium. The cluster contains five cotranscribed genes, ISac1, rsrR, rsrS, tetH, and doxD, coding for a transposase, a two-component response regulator (RsrR and RsrS), tetrathionate hydrolase, and DoxD, respectively. As shown by quantitative PCR, rsrR, tetH, and doxD are upregulated to different degrees in the presence of tetrathionate. Western blot analysis also indicates upregulation of TetH in the presence of tetrathionate, thiosulfate, and pyrite. The tetH cluster is predicted to have two promoters, both of which are functional in Escherichia coli and one of which was mapped by primer extension. A pyrrolo-quinoline quinone binding domain in TetH was predicted by bioinformatic analysis, and the presence of an o-quinone moiety was experimentally verified, suggesting a mechanism for tetrathionate oxidation.

Three strains of the strict acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans, including the type strain ATCC 23270, contain a petIIABC gene cluster that encodes the three proteins, cytochrome c1, cytochrome b and a Rieske protein, that constitute a bc1 electron-transfer complex. RT-PCR and Northern blotting show that the petIIABC cluster is co-transcribed with cycA, encoding a cytochrome c belonging to the c4 family, sdrA, encoding a putative short-chain dehydrogenase, and hip, encoding a high potential iron-sulfur protein, suggesting that the six genes constitute an operon, termed the petII operon. Previous results indicated that A. ferrooxidans contains a second pet operon, termed the petI operon, which contains a gene cluster that is similarly organized except that it lacks hip. Real-time PCR and Northern blot experiments demonstrate that petI is transcribed mainly in cells grown in medium containing iron, whereas petII is transcribed in cells grown in media containing sulfur or iron. Primer extension experiments revealed possible transcription initiation sites for the petI and petII operons. A model is presented in which petI is proposed to encode the bc1 complex, functioning in the uphill flow of electrons from iron to NAD(P), whereas petII is suggested to be involved in electron transfer from sulfur (or formate) to oxygen (or ferric iron). A. ferrooxidans is the only organism, to date, to exhibit two functional bc1 complexes.

Hip is a high-potential iron-sulfur protein (HiPIP) isolated from the acidophilic bacterium, Acidithiobacillus ferrooxidans. In the present work, a structural model of Hip suggests that the role of proline residues is essential to stabilize the protein folding at very low pH. The presence of an unusual disulfide bridge in Hip is demonstrated using mass spectrometry and nuclear magnetic resonance. This disulfide bridge is necessary to anchor the N-terminal extremity of the protein, but is not involved in the acid stability of Hip. The structural parameters correlated with the pH dependence of Hip redox potential are also analysed on the basis of this model. Given that the same structural features can enhance acidic stability and lead to elevated redox potentials, modulation of the redox potentials of electron carriers may be necessary to achieve electron transfer at very low pH.

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 DeltatatC 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 c1 from the second A. ferrooxidans cytochrome bc1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

The regulation of the expression of the rus operon, proposed to encode an electron transfer chain from the outer to the inner membrane in the obligate acidophilic chemolithoautroph Acidithiobacillus ferrooxidans, has been studied at the RNA and protein levels. As observed by Northern hybridization, real-time PCR and reverse transcription analyses, this operon was more highly expressed in ferrous iron- than in sulfur-grown cells. Furthermore, it was shown by immunodetection that components of this respiratory chain are synthesized in ferrous iron- rather than in sulfur-growth conditions. Nonetheless, weak transcription and translation products of the rus operon were detected in sulfur-grown cells at the early exponential phase. The results strongly support the notion that rus-operon expression is induced by ferrous iron, in agreement with the involvement of the rus-operon-encoded products in the oxidation of ferrous iron, and that ferrous iron is used in preference to sulfur.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe(2+), or reducing sulfur compounds that provide more energy for growth than Fe(2+). We have previously shown that the uphill electron transfer pathway between Fe(2+) and NAD(+) involved a bc(1) complex that functions only in the reverse direction [J. Bacteriol. 182, (2000) 3602]. In the present work, we demonstrate both the existence of a bc(1) complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba(3) type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa(3) that was found to be expressed only in iron-grown cells. Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc(1)-->ba(3) pathway. We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo(3) type oxidase. A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O(2). Possible reasons for these apparently redundant pathways are discussed.

Weathering of the As-rich pyrite-rich tailings of the abandoned mining site of Carnoulès (southeastern France) results in the formation of acid waters heavily loaded with arsenic. Dissolved arsenic present in the seepage waters precipitates within a few meters from the bottom of the tailing dam in the presence of microorganisms. An Acidithiobacillus ferrooxidans strain, referred to as CC1, was isolated from the effluents. This strain was able to remove arsenic from a defined synthetic medium only when grown on ferrous iron. This A. ferrooxidans strain did not oxidize arsenite to arsenate directly or indirectly. Strain CC1 precipitated arsenic unexpectedly as arsenite but not arsenate, with ferric iron produced by its energy metabolism. Furthermore, arsenite was almost not found adsorbed on jarosite but associated with a poorly ordered schwertmannite. Arsenate is known to efficiently precipitate with ferric iron and sulfate in the form of more or less ordered schwertmannite, depending on the sulfur-to-arsenic ratio. Our data demonstrate that the coprecipitation of arsenite with schwertmannite also appears as a potential mechanism of arsenite removal in heavily contaminated acid waters. The removal of arsenite by coprecipitation with ferric iron appears to be a common property of the A. ferrooxidans species, as such a feature was observed with one private and three collection strains, one of which was the type strain.