Violaine Bonnefoy

Extending the models for iron and sulfur oxidation in the extreme Acidophile Acidithiobacillus ferrooxidans

BackgroundAcidithiobacillus 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.ResultsBioinformatic-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.ConclusionBioinformatic 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.

Bioenergetic challenges of microbial iron metabolisms

Before cyanobacteria invented oxygenic photosynthesis and O(2) and H(2)O began to cycle between respiration and photosynthesis, redox cycles between other elements were used to sustain microbial metabolism on a global scale. Today these cycles continue to occur in more specialized niches. In this review we focus on the bioenergetic aspects of one of these cycles - the iron cycle - because iron presents unique and fascinating challenges for cells that use it for energy. Although iron is an important nutrient for nearly all life forms, we restrict our discussion to energy-yielding pathways that use ferrous iron [Fe(II)] as an electron donor or ferric iron [Fe(III)] as an electron acceptor. We briefly review general concepts in bioenergetics, focusing on what is known about the mechanisms of electron transfer in Fe(II)-oxidizing and Fe(III)-reducing bacteria, and highlight aspects of their bioenergetic pathways that are poorly understood.

A tale of two oxidation states: bacterial colonization of arsenic-rich environments.

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth.

Nitrate reductase of Escherichia coli: Completion of the nucleotide sequence of the nar operon and reassessment of the role of the α and β subunits in iron binding and electron transfer

SummaryThe nucleotide sequence of the narGHJI operon that encodes the nitrate reductase of Escherichia coli was completed. It encodes four polypeptides NarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5 and 25.5 kDa, respectively. The analysis of deduced amino acid sequence failed to reveal any structure capable of binding iron within the NarG polypeptide. In contrast, cysteine arrangements typical of iron-sulfur centers were found in the NarH polypeptide. This suggested that the latter is an electron transfer unit of the nitrate reductase complex. Such a view is opposite to the current description of the nitrate reductase. The findings allowed us to propose a model for the electron transfer steps that occur during nitrate reduction. The NarG polypeptide was found to display a high degree of homology with numerous E. coli molybdoproteins. Moreover, the same genetic and functional organizations as well as the presence of highly conserved stretches of amino acids were noted between both NarG/NarH and DmsA/DmsB (encoding the dimethyl sulfoxide reductase) pairs.

Bacterial immobilization and oxidation of arsenic in acid mine drainage (Carnoulès creek, France).

The acid waters (pH=2.73-3.37) originating from the Carnoulès mine tailings contain high dissolved concentrations of arsenic (1-3.5 mmol l(-1)) and iron (20-40 mmol l(-1)). At the outlet, arsenite predominates. During the first 30 m of downflow, 20-60% is removed by coprecipitation with Fe(III). This process results from bacterially mediated As- and Fe-oxidation. The precipitation rates in the creek depend on the oxygen concentration in spring water and are lower during the dry summer period when the anoxic character of the spring water inhibits the activity of oxidizing bacteria. Ex situ experiments show that the presence of bacteria-rich precipitates increases the As- and Fe-removal rates. Three strains of bacteria promoting the oxidation of As have been isolated, and two of them have the characteristics of Thiomonas ynys1. The third strain, which is not identified yet, also catalyzes the oxidation of Fe.

Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon.

SummaryThe structural genes for NRZ, the second nitrate reductase of Escherichia coli, have been sequenced. They are organized in a transcription unit, narZYWV, encoding four subunits, NarZ, NarY, NarW and NarV. The transcription unit is homologous (73% identity) to the narGHJI operon which encodes the genes for NRA, the better characterized nitrate reductase of this organism. The level of homology between the corresponding polypeptides ranges from 69% for the NarW/NarJ pair to 86% for the NarV/ Narl pair. The NarZ polypeptide contains the five conserved regions present in all other known molybdoproteins of E. coli and their relative order is the same. The NarY polypeptide, which contains the same four cysteine clusters in the same order as NarH, is probably an electron transfer unit of the complex. Upstream of narZ, an open reading frame, ORFA, is present which could encode a product which has homology (73% identity) with the COON-terminal end of NarK. The ORFA-narZ intergenic region, however, is about 80 nucleotides long and does not contain the cis-acting elements, NarL and Fnr boxes, nor the terC4 terminator sequence present in the 500 nucleotide narK-narG intergenic region. This might explain why the nar-ZYWV and the narGHJI operons are regulated differently. Our results tend to support the hypothesis that a DNA fragment larger than that encompassing the narGHJI genes has been duplicated.

The high-molecular-weight cytochrome c Cyc2 of Acidithiobacillus ferrooxidans is an outer membrane protein.

A high-molecular-weight c-type cytochrome, Cyc2, and a putative 22-kDa c-type cytochrome were detected in the membrane fraction released during spheroplast formation from Acidithiobacillus ferrooxidans. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.

Genomic insights into microbial iron oxidation and iron uptake strategies in extremely acidic environments.

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.

Metabolic diversity among main microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics

By their metabolic activities, microorganisms have a crucial role in the biogeochemical cycles of elements. The complete understanding of these processes requires, however, the deciphering of both the structure and the function, including synecologic interactions, of microbial communities. Using a metagenomic approach, we demonstrated here that an acid mine drainage highly contaminated with arsenic is dominated by seven bacterial strains whose genomes were reconstructed. Five of them represent yet uncultivated bacteria and include two strains belonging to a novel bacterial phylum present in some similar ecosystems, and which was named ‘Candidatus Fodinabacter communificans.’ Metaproteomic data unravelled several microbial capabilities expressed in situ, such as iron, sulfur and arsenic oxidation that are key mechanisms in biomineralization, or organic nutrient, amino acid and vitamin metabolism involved in synthrophic associations. A statistical analysis of genomic and proteomic data and reverse transcriptase–PCR experiments allowed us to build an integrated model of the metabolic interactions that may be of prime importance in the natural attenuation of such anthropized ecosystems.

Insight into the evolution of the iron oxidation pathways

Iron is a ubiquitous element in the universe. Ferrous iron (Fe(II)) was abundant in the primordial ocean until the oxygenation of the Earths atmosphere led to its widespread oxidation and precipitation. This change of iron bioavailability likely put selective pressure on the evolution of life. This element is essential to most extant life forms and is an important cofactor in many redox-active proteins involved in a number of vital pathways. In addition, iron plays a central role in many environments as an energy source for some microorganisms. This review is focused on Fe(II) oxidation. The fact that the ability to oxidize Fe(II) is widely distributed in Bacteria and Archaea and in a number of quite different biotopes suggests that the dissimilatory Fe(II) oxidation is an ancient energy metabolism. Based on what is known today about Fe(II) oxidation pathways, we propose that they arose independently more than once in evolution and evolved convergently. The iron paleochemistry, the phylogeny, the physiology of the iron oxidizers, and the nature of the cofactors of the redox proteins involved in these pathways suggest a possible scenario for the timescale in which each type of Fe(II) oxidation pathways evolved. The nitrate dependent anoxic iron oxidizers are likely the most ancient iron oxidizers. We suggest that the phototrophic anoxic iron oxidizers arose in surface waters after the Archaea/Bacteria-split but before the Great Oxidation Event. The neutrophilic oxic iron oxidizers possibly appeared in microaerobic marine environments prior to the Great Oxidation Event while the acidophilic ones emerged likely after the advent of atmospheric O(2). This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.