Mark Dopson

Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments.

ABSTRACT Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum YT by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate “Ferroplasma acidarmanus” Fer1T required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42°C and the pH optima were 1.0 to 1.7, and “F. acidarmanus” Fer1T was capable of growing at pH 0. The optimum yeast extract concentration for “F. acidarmanus” Fer1T was higher than that for the other three isolates. Phenotypic results suggested that isolate “F. acidarmanus” Fer1T is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and YT group as a single species. “F. acidarmanus” Fer1T groups separately, and we propose the new species “F. acidarmanus” Fer1T sp. nov.

Biodiversity, metabolism and applications of acidophilic sulfur-metabolizing microorganisms.

Extremely acidic, sulfur-rich environments can be natural, such as solfatara fields in geothermal and volcanic areas, or anthropogenic, such as acid mine drainage waters. Many species of acidophilic bacteria and archaea are known to be involved in redox transformations of sulfur, using elemental sulfur and inorganic sulfur compounds as electron donors or acceptors in reactions involving between one and eight electrons. This minireview describes the nature and origins of acidic, sulfur-rich environments, the biodiversity of sulfur-metabolizing acidophiles, and how sulfur is metabolized and assimilated by acidophiles under aerobic and anaerobic conditions. Finally, existing and developing technologies that harness the abilities of sulfur-oxidizing and sulfate-reducing acidophiles to extract and capture metals, and to remediate sulfur-polluted waste waters are outlined.

Analysis of Community Composition during Moderately Thermophilic Bioleaching of Pyrite, Arsenical Pyrite, and Chalcopyrite.

An analysis of the community composition of three previously undefined mixed cultures of moderately thermophilic bioleaching bacteria grown at 45°C on pyrite, arsenical pyrite, and chalcopyrite has been carried out. The bacterial species present were identified by comparative sequence analysis of the 16S rRNA gene isolated from the bioleaching vessels and analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The mixed cultures leached all three minerals, as shown by the increase in iron released from the mineral concentrates. The species identified from the mixed cultures during bioleaching of pyrite, arsenical pyrite, and chalcopyrite were clones closely related to Acidithiobacillus caldus C-SH12, Sulfobacillus thermosulfidooxidans AT-1, “Sulfobacillus montserratensis” L15, and an uncultured thermal soil bacterium YNP. It was also found that the same mixed culture maintained for over a year on chalcopyrite mineral selected approximately the same consortia of bacteria as the original mixed culture grown on chalcopyrite.

Sulfur metabolism in the extreme acidophile Acidithiobacillus caldus

Given the challenges to life at low pH, an analysis of inorganic sulfur compound (ISC) oxidation was initiated in the chemolithoautotrophic extremophile Acidithiobacillus caldus. A. caldus is able to metabolize elemental sulfur and a broad range of ISCs. It has been implicated in the production of environmentally damaging acidic solutions as well as participating in industrial bioleaching operations where it forms part of microbial consortia used for the recovery of metal ions. Based upon the recently published A. caldus type strain genome sequence, a bioinformatic reconstruction of elemental sulfur and ISC metabolism predicted genes included: sulfide–quinone reductase (sqr), tetrathionate hydrolase (tth), two sox gene clusters potentially involved in thiosulfate oxidation (soxABXYZ), sulfur oxygenase reductase (sor), and various electron transport components. RNA transcript profiles by semi quantitative reverse transcription PCR suggested up-regulation of sox genes in the presence of tetrathionate. Extensive gel based proteomic comparisons of total soluble and membrane enriched protein fractions during growth on elemental sulfur and tetrathionate identified differential protein levels from the two Sox clusters as well as several chaperone and stress proteins up-regulated in the presence of elemental sulfur. Proteomics results also suggested the involvement of heterodisulfide reductase (HdrABC) in A. caldus ISC metabolism. A putative new function of Hdr in acidophiles is discussed. Additional proteomic analysis evaluated protein expression differences between cells grown attached to solid, elemental sulfur versus planktonic cells. This study has provided insights into sulfur metabolism of this acidophilic chemolithotroph and gene expression during attachment to solid elemental sulfur.

Anaerobic Sulfur Metabolism Coupled to Dissimilatory Iron Reduction in the Extremophile Acidithiobacillus ferrooxidans

ABSTRACT 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 Cu2+ included in anaerobically incubated cultures was precipitated (seemingly as CuS). Since H2S 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 S0 to Fe3+ 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.

Draft Genome Sequence of the Extremely Acidophilic Bacterium Acidithiobacillus caldus ATCC 51756 Reveals Metabolic Versatility in the Genus Acidithiobacillus

Acidithiobacillus caldus is an extremely acidophilic, moderately thermophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and reduced inorganic sulfur compounds. Here we present the draft genome sequence of Acidithiobacillus caldus ATCC 51756 (the type strain of the species), which has permitted the prediction of genes for survival in extremely acidic environments, including genes for sulfur oxidation and nutrient assimilation.

Draft Genome of the Psychrotolerant Acidophile Acidithiobacillus ferrivorans SS3

Acidithiobacillus ferrivorans SS3 is a psychrotolerant acidophile capable of growth in the range of 5° to 30°C (optimum, ≈25°C). It gains energy from the oxidation of ferrous iron and inorganic sulfur compounds and obtains organic carbon from carbon dioxide. Here, we present the draft genome sequence of A. ferrivorans SS3 that will permit investigation of genes involved in growth in acidic environments at low temperatures.

Draft Genome Sequence of the Extremely Acidophilic Biomining Bacterium Acidithiobacillus thiooxidans ATCC 19377 Provides Insights into the Evolution of the Acidithiobacillus Genus

Acidithiobacillus thiooxidans is a mesophilic, extremely acidophilic, chemolithoautotrophic gammaproteobacterium that derives energy from the oxidation of sulfur and inorganic sulfur compounds. Here we present the draft genome sequence of A. thiooxidans ATCC 19377, which has allowed the identification of genes for survival and colonization of extremely acidic environments.

Regulation of a Novel Acidithiobacillus caldus Gene Cluster Involved in Metabolism of Reduced Inorganic Sulfur Compounds

ABSTRACT 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.

Biofilm development in the extremely acidophilic archaeon ‘Ferroplasma acidarmanus’ Fer1

Abstract‘Ferroplasma acidarmanus’ Fer1 is an iron-oxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA. This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies. Bioinformatic analysis of the ‘F. acidarmanus’ Fer1 genome identified genes annotated as involved in attachment and biofilm formation. No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in ‘F. acidarmanus’ Fer1 biofilm supernatant. Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth. Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations. Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth. This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.