The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon Metallosphaera sedula Provides Insights into Bioleaching-Associated Metabolism

ABSTRACT Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ∼2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.

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Respiratory gene clusters of Metallosphaera sedula – differential expression and transcriptional organization

Microbiology ◽

10.1099/mic.0.27515-0 ◽

2005 ◽

Vol 151 (1) ◽

pp. 35-43 ◽

Cited By ~ 42

Author(s):

Ulrike Kappler ◽

Lindsay I. Sly ◽

Alastair G. McEwan

Keyword(s):

Differential Expression ◽

Gene Cluster ◽

Iron Oxidation ◽

Gene Clusters ◽

Terminal Oxidase ◽

Difference Spectra ◽

Oxidase Gene ◽

Expression Levels ◽

Metallosphaera Sedula ◽

Complex Analogue

Metallosphaera sedula is a thermoacidophilic Crenarchaeon which is capable of leaching metals from sulfidic ores. The authors have investigated the presence and expression of genes encoding respiratory complexes in this organism when grown heterotrophically or chemolithotrophically on either sulfur or pyrite. The presence of three gene clusters, encoding two terminal oxidase complexes, the quinol oxidase SoxABCD and the SoxM oxidase supercomplex, and a gene cluster encoding a high-potential cytochrome b and components of a bc 1 complex analogue (cbsBA–soxL2N gene cluster) was established. Expression studies showed that the soxM gene was expressed to high levels during heterotrophic growth of M. sedula on yeast extract, while the soxABCD mRNA was most abundant in cells grown on sulfur. Reduced-minus-oxidized difference spectra of cell membranes showed cytochrome-related peaks that correspond to published spectra of Sulfolobus-type terminal oxidase complexes. In pyrite-grown cells, expression levels of the two monitored oxidase gene clusters were reduced by a factor of 10–12 relative to maximal expression levels, although spectra of membranes clearly contained oxidase-associated haems, suggesting the presence of additional gene clusters encoding terminal oxidases in M. sedula. Pyrite- and sulfur-grown cells contained high levels of the cbsA transcript, which encodes a membrane-bound cytochrome b with a possible role in iron oxidation or chemolithotrophy. The cbsA gene is not co-transcribed with the soxL2N genes, and therefore does not appear to be an integral part of this bc 1 complex analogue. The data show for the first time the differential expression of the Sulfolobus-type terminal oxidase gene clusters in a Crenarchaeon in response to changing growth modes.

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Comparative Genome Analysis of the Genus Thiothrix Involving Three Novel Species, Thiothrix subterranea sp. nov. Ku-5, Thiothrix litoralis sp. nov. AS and “Candidatus Thiothrix anitrata” sp. nov. A52, Revealed the Conservation of the Pathways of Dissimilatory Sulfur Metabolism and Variations in the Genetic Inventory for Nitrogen Metabolism and Autotrophic Carbon Fixation

Frontiers in Microbiology ◽

10.3389/fmicb.2021.760289 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nikolai V. Ravin ◽

Tatyana S. Rudenko ◽

Dmitry D. Smolyakov ◽

Alexey V. Beletsky ◽

Andrey L. Rakitin ◽

...

Keyword(s):

Core Genome ◽

Carbon Fixation ◽

Tricarboxylic Acid Cycle ◽

Sulfur Metabolism ◽

Sulfide Oxidation ◽

Calvin Cycle ◽

Sulfur Oxidation ◽

Rrna Gene ◽

Reduced Sulfur Compounds ◽

Hybridization Data

Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (Thiothrix sp. Ku-5) and on the seashore of the White Sea (Thiothrix sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phylogenetic analysis based on the 16S rRNA gene sequences showed that all genomes represented the genus Thiothrix. Based on their average nucleotide identity and digital DNA-DNA hybridization data these new isolates and the MAG represent three species within the genus Thiothrix with the proposed names Thiothrix subterranea sp. nov. Ku-5T, Thiothrix litoralis sp. nov. AST, and “Candidatus Thiothrix anitrata” sp. nov. A52. The complete genome sequences of Thiothrix fructosivorans QT and Thiothrix unzii A1T were determined. Complete genomes of seven Thiothrix isolates, as well as two MAGs, were used for pangenome analysis. The Thiothrix core genome consisted of 1,355 genes, including ones for the glycolysis, the tricarboxylic acid cycle, the aerobic respiratory chain, and the Calvin cycle of carbon fixation. Genes for dissimilatory oxidation of reduced sulfur compounds, namely the branched SOX system (SoxAXBYZ), direct (soeABC) and indirect (aprAB, sat) pathways of sulfite oxidation, sulfur oxidation complex Dsr (dsrABEFHCEMKLJONR), sulfide oxidation systems SQR (sqrA, sqrF), and FCSD (fccAB) were found in the core genome. Genomes differ in the set of genes for dissimilatory reduction of nitrogen compounds, nitrogen fixation, and the presence of various types of RuBisCO.

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Oxidation of Copper Sulfides in Aqueous Ammonia. III. Kinetic Characteristics

Australian Journal of Chemistry ◽

10.1071/ch9792597 ◽

1979 ◽

Vol 32 (12) ◽

pp. 2597 ◽

Cited By ~ 5

Author(s):

AO Filmer ◽

AJ Parker ◽

BW Clare ◽

LGB Wadley

Keyword(s):

Aqueous Ammonia ◽

Sulfide Oxidation ◽

Metal Sulfide ◽

Diffusion Control ◽

Sulfate Ions ◽

Kinetics Of Oxidation ◽

Copper Sulfides ◽

Shrinking Core ◽

Kinetics Of ◽

And Diffusion

The kinetics of oxidation with oxygen of chalcocite, Cu2S, to CuS in buffered aqueous ammonia at pH 10.5 at 30� can be modeled approximately by a shrinking core of Cu2S within a thickening shell of CuxS (x ≥ 1). The Cu2S core offers partial cathodic protection to the CuxS and diffusion of Cu+ through CuxS controls the rate of reaction. The kinetics of oxidation of covellite, CuS, to Cu2+, sulfur and sulfate ions in the same solvent can be modeled by a shrinking core of CuS surrounded by a shrinking sphere of CuyS (y < 1) which is much less effectively protected cathodically by the CuS core. Oxidation of CuS is subject to mixed chemical and diffusion control. Rates of oxidation of NiS and of CuS, in the presence and absence of tetrachloroethene and ammonium sulfate, show that, whether sulfur is a major oxidation product or not, the presence of sulfur has very little, if any, influence on the rate or mechanism of oxidation. This is contrary to current ideas on metal sulfide oxidation.

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Copper Recovery by Bioleaching of Chalcopyrite: A Microcalorimetric Approach for the Fast Determination of Bioleaching Activity

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.825.322 ◽

2013 ◽

Vol 825 ◽

pp. 322-325

Author(s):

Beate Krok ◽

Axel Schippers ◽

Wolfgang Sand

Keyword(s):

Enrichment Culture ◽

Sulfide Oxidation ◽

Exothermic Reaction ◽

Metal Sulfide ◽

Copper Recovery ◽

Heat Output ◽

Low Grade ◽

Non Invasive ◽

Pure Cultures ◽

Invasive Method

Low grade copper ores containing chalcopyrite are increasingly used for copper recovery via biomining. Since metal sulfide oxidation is an exothememic process, bioleaching activity can be measured due to the heat output by microcalorimetry, which is a non-destructive and non-invasive method. The bioleaching activity of pure cultures ofSulfolobus metallicus,Metallosphaera hakonensisand a moderate thermophilic enrichment culture on high grade chalcopyrite was evaluated. Chalcopyrite leaching by microorganisms showed a higher copper recovery than sterile controls. Chemical chalcopyrite leaching by acid produced heat due to the exothermic reaction, the heat output was increased while metal sulfide oxidation by microorganisms.

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Transcriptomes of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula Exposed to Metal “Shock” Reveal Generic and Specific Metal Responses

Applied and Environmental Microbiology ◽

10.1128/aem.01176-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4613-4627 ◽

Cited By ~ 19

Author(s):

Garrett H. Wheaton ◽

Arpan Mukherjee ◽

Robert M. Kelly

Keyword(s):

Metal Toxicity ◽

Cellular Response ◽

Sulfur Metabolism ◽

Metal Resistance ◽

Open Reading Frames ◽

Copper Resistance ◽

Natural Environments ◽

Cluster Assembly ◽

Content Type ◽

Metallosphaera Sedula

ABSTRACTThe extremely thermoacidophilic archaeonMetallosphaera sedulamobilizes metals by novel membrane-associated oxidase clusters and, consequently, requires metal resistance strategies. This issue was examined by “shocking”M. sedulawith representative metals (Co2+, Cu2+, Ni2+, UO22+, Zn2+) at inhibitory and subinhibitory levels. Collectively, one-quarter of the genome (554 open reading frames [ORFs]) responded to inhibitory levels, and two-thirds (354) of the ORFs were responsive to a single metal. Cu2+(259 ORFs, 106 Cu2+-specific ORFs) and Zn2+(262 ORFs, 131 Zn2+-specific ORFs) triggered the largest responses, followed by UO22+(187 ORFs, 91 UO22+-specific ORFs), Ni2+(93 ORFs, 25 Ni2+-specific ORFs), and Co2+(61 ORFs, 1 Co2+-specific ORF). While one-third of the metal-responsive ORFs are annotated as encoding hypothetical proteins, metal challenge also impacted ORFs responsible for identifiable processes related to the cell cycle, DNA repair, and oxidative stress. Surprisingly, there were only 30 ORFs that responded to at least four metals, and 10 of these responded to all five metals. This core transcriptome indicated induction of Fe-S cluster assembly (Msed_1656-Msed_1657), tungsten/molybdenum transport (Msed_1780-Msed_1781), and decreased central metabolism. Not surprisingly, a metal-translocating P-type ATPase (Msed_0490) associated with a copper resistance system (Cop) was upregulated in response to Cu2+(6-fold) but also in response to UO22+(4-fold) and Zn2+(9-fold). Cu2+challenge uniquely induced assimilatory sulfur metabolism for cysteine biosynthesis, suggesting a role for this amino acid in Cu2+resistance or issues in sulfur metabolism. The results indicate thatM. sedulaemploys a range of physiological and biochemical responses to metal challenge, many of which are specific to a single metal and involve proteins with yet unassigned or definitive functions.IMPORTANCEThe mechanisms by which extremely thermoacidophilic archaea resist and are negatively impacted by metals encountered in their natural environments are important to understand so that technologies such as bioleaching, which leverage microbially based conversion of insoluble metal sulfides to soluble species, can be improved. Transcriptomic analysis of the cellular response to metal challenge provided both global and specific insights into how these novel microorganisms negotiate metal toxicity in natural and technological settings. As genetics tools are further developed and implemented for extreme thermoacidophiles, information about metal toxicity and resistance can be leveraged to create metabolically engineered strains with improved bioleaching characteristics.

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Arcobacter peruensis sp. nov., a Chemolithoheterotroph Isolated from Sulfide- and Organic-Rich Coastal Waters off Peru

Applied and Environmental Microbiology ◽

10.1128/aem.01344-19 ◽

2019 ◽

Vol 85 (24) ◽

Cited By ~ 8

Author(s):

Cameron M. Callbeck ◽

Chris Pelzer ◽

Gaute Lavik ◽

Timothy G. Ferdelman ◽

Jon S. Graf ◽

...

Keyword(s):

Coastal Waters ◽

Carbon Fixation ◽

Isotope Labeling ◽

Nitrogen Loss ◽

Sulfide Oxidation ◽

Mass Spectrometry Analysis ◽

Anthropogenic Pressures ◽

Content Type ◽

Genes Encoding ◽

Isotope Labeling Experiments

ABSTRACT Members of the epsilonproteobacterial genus Arcobacter have been identified to be potentially important sulfide oxidizers in marine coastal, seep, and stratified basin environments. In the highly productive upwelling waters off the coast of Peru, Arcobacter cells comprised 3 to 25% of the total microbial community at a near-shore station where sulfide concentrations exceeded 20 μM in bottom waters. From the chemocline where the Arcobacter population exceeded 106 cells ml−1 and where high rates of denitrification (up to 6.5 ± 0.4 μM N day−1) and dark carbon fixation (2.8 ± 0.2 μM C day−1) were measured, we isolated a previously uncultivated Arcobacter species, Arcobacter peruensis sp. nov. (BCCM LMG-31510). Genomic analysis showed that A. peruensis possesses genes encoding sulfide oxidation and denitrification pathways but lacks the ability to fix CO2 via autotrophic carbon fixation pathways. Genes encoding transporters for organic carbon compounds, however, were present in the A. peruensis genome. Physiological experiments demonstrated that A. peruensis grew best on a mix of sulfide, nitrate, and acetate. Isotope labeling experiments further verified that A. peruensis completely reduced nitrate to N2 and assimilated acetate but did not fix CO2, thus coupling heterotrophic growth to sulfide oxidation and denitrification. Single-cell nanoscale secondary ion mass spectrometry analysis of samples taken from shipboard isotope labeling experiments also confirmed that the Arcobacter population in situ did not substantially fix CO2. The efficient growth yield associated with the chemolithoheterotrophic metabolism of A. peruensis may allow this Arcobacter species to rapidly bloom in eutrophic and sulfide-rich waters off the coast of Peru. IMPORTANCE Our multidisciplinary approach provides new insights into the ecophysiology of a newly isolated environmental Arcobacter species, as well as the physiological flexibility within the Arcobacter genus and sulfide-oxidizing, denitrifying microbial communities within oceanic oxygen minimum zones (OMZs). The chemolithoheterotrophic species Arcobacter peruensis may play a substantial role in the diverse consortium of bacteria that is capable of coupling denitrification and fixed nitrogen loss to sulfide oxidation in eutrophic, sulfidic coastal waters. With increasing anthropogenic pressures on coastal regions, e.g., eutrophication and deoxygenation (D. Breitburg, L. A. Levin, A. Oschlies, M. Grégoire, et al., Science 359:eaam7240, 2018, https://doi.org/10.1126/science.aam7240), niches where sulfide-oxidizing, denitrifying heterotrophs such as A. peruensis thrive are likely to expand.

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Conversion of 4-Hydroxybutyrate to Acetyl Coenzyme A and Its Anapleurosis in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Carbon Fixation Pathway

Applied and Environmental Microbiology ◽

10.1128/aem.04146-13 ◽

2014 ◽

Vol 80 (8) ◽

pp. 2536-2545 ◽

Cited By ~ 17

Author(s):

Aaron B. Hawkins ◽

Michael W. W. Adams ◽

Robert M. Kelly

Keyword(s):

Carbon Fixation ◽

Coenzyme A ◽

Flux Analysis ◽

Central Metabolism ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Content Type ◽

Metallosphaera Sedula ◽

Acetyl Coenzyme ◽

Coa Ligase

ABSTRACTThe extremely thermoacidophilic archaeonMetallosphaera sedula(optimum growth temperature, 73°C, pH 2.0) grows chemolithoautotrophically on metal sulfides or molecular hydrogen by employing the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) carbon fixation cycle. This cycle adds two CO2molecules to acetyl coenzyme A (acetyl-CoA) to generate 4HB, which is then rearranged and cleaved to form two acetyl-CoA molecules. Previous metabolic flux analysis showed that two-thirds of central carbon precursor molecules are derived from succinyl-CoA, which is oxidized to malate and oxaloacetate. The remaining one-third is apparently derived from acetyl-CoA. As such, the steps beyond succinyl-CoA are essential for completing the carbon fixation cycle and for anapleurosis of acetyl-CoA. Here, the final four enzymes of the 3HP/4HB cycle, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_0406), 4-hydroxybutyryl-CoA dehydratase (Msed_1321), crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (Msed_0399), and acetoacetyl-CoA β-ketothiolase (Msed_0656), were produced recombinantly inEscherichia coli, combinedin vitro, and shown to convert 4HB to acetyl-CoA. Metabolic pathways connecting CO2fixation and central metabolism were examined using a gas-intensive bioreactor system in whichM. sedulawas grown under autotrophic (CO2-limited) and heterotrophic conditions. Transcriptomic analysis revealed the importance of the 3HP/4HB pathway in supplying acetyl-CoA to anabolic pathways generating intermediates inM. sedulametabolism. The results indicated that flux between the succinate and acetyl-CoA branches in the 3HP/4HB pathway is governed by 4-hydroxybutyrate-CoA ligase, possibly regulated posttranslationally by the protein acetyltransferase (Pat)/Sir2-dependent system. Taken together, this work confirms the final four steps of the 3HP/4HB pathway, thereby providing the framework for examining connections between CO2fixation and central metabolism inM. sedula.

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Draft Genome Sequence of the Phosphate-Solubilizing Rhizobacterium Burkholderia pseudomultivorans Strain MPSB1, Isolated from a Copper Mined-Out Site

Microbiology Resource Announcements ◽

10.1128/mra.01304-20 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Berna Lou L. Aba-Regis ◽

Kristel Mae P. Oliveros ◽

Cherry Ibarra-Romero ◽

Asuncion K. Raymundo ◽

Nelly S. Aggangan ◽

...

Keyword(s):

Heavy Metal ◽

Soil Sample ◽

Genome Sequence ◽

Phosphate Solubilization ◽

Metal Tolerance ◽

Draft Genome ◽

Heavy Metal Tolerance ◽

Draft Genome Sequence ◽

Content Type ◽

Phosphate Solubilizing

ABSTRACT Burkholderia pseudomultivorans MPSB1 was isolated from a copper mined-out soil sample collected from Mogpog, Marinduque, Philippines. Here, we report the draft genome sequence with predicted gene inventories supporting rhizosphere bioremediation, such as heavy metal tolerance, phosphate solubilization, and siderophore production.

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3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase, Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales

Journal of Bacteriology ◽

10.1128/jb.00068-09 ◽

2009 ◽

Vol 191 (14) ◽

pp. 4572-4581 ◽

Cited By ~ 51

Author(s):

Robin Teufel ◽

Johannes W. Kung ◽

Daniel Kockelkorn ◽

Birgit E. Alber ◽

Georg Fuchs

Keyword(s):

Alcohol Dehydrogenase ◽

Carbon Fixation ◽

Coenzyme A ◽

Chloroflexus Aurantiacus ◽

Reaction Sequence ◽

Acetyl Coenzyme A ◽

Sulfolobus Tokodaii ◽

Metallosphaera Sedula ◽

Acetyl Coenzyme ◽

Coa Reductase

ABSTRACT A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO2 fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.

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ROLE OF THIOBACILLUS FERROOXIDANS IN THE OXIDATION OF SULFIDE MINERALS

Canadian Journal of Microbiology ◽

10.1139/m67-052 ◽

1967 ◽

Vol 13 (4) ◽

pp. 397-403 ◽

Cited By ~ 39

Author(s):

D. W. Duncan ◽

J. Landesman ◽

C. C. Walden

Keyword(s):

Oxygen Uptake ◽

Sodium Azide ◽

Ferrous Iron ◽

Thiobacillus Ferrooxidans ◽

Sulfide Oxidation ◽

Iron Oxidation ◽

Rapid Rate ◽

Total Oxygen ◽

Washed Cell

Selective inhibitors of iron and sulfide oxidation, sodium azide and N-ethylmaleimide respectively, were used to demonstrate that washed cell suspensions of Thiobacillus ferrooxidans attacked both insoluble ferrous iron and sulfide during the oxidation of chalcopyrite (CuFeS2) and pyrite (FeS2). The oxidation of the two substrates occurred simultaneously and independently but the relative rates depended on how the cells were grown. When chalcopyrite-grown cells were used to oxidize chalcopyrite, 68–74% of the oxygen uptake was the result of sulfide oxidation and 25–30% the result of iron oxidation. With pyrite, all the oxygen uptake was due to sulfide oxidation. When iron-grown cells were used to oxidize chalcopyrite, two rates resulted. During the initial rapid rate, 80–90% of the oxygen uptake was due to iron oxidation, but, during the second slower rate, the result duplicated those found with chalcopyrite-grown cells. Iron-grown cells oxidized pyrite at a constant and more rapid rate than chalcopyrite-grown cells. The faster rate was due to iron oxidation; since only 20–30% of the total oxygen uptake was due to sulfide oxidation.

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