Hydrogen can serve as an electron donor for chemolithotrophic acidophiles, especially in the deep terrestrial subsurface and geothermal ecosystems. Nevertheless, the current knowledge of hydrogen ...utilization by mesophilic acidophiles is minimal. A multi-omics analysis was applied on
growing on hydrogen, and a respiratory model was proposed. In the model, NiFe hydrogenases oxidize hydrogen to two protons and two electrons. The electrons are used to reduce membrane-soluble ubiquinone to ubiquinol. Genetically associated iron-sulfur proteins mediate electron relay from the hydrogenases to the ubiquinone pool. Under aerobic conditions, reduced ubiquinol transfers electrons to either cytochrome
oxidase via cytochrome
complex and cytochrome
or the alternate directly to cytochrome
oxidase, resulting in proton efflux and reduction of oxygen. Under anaerobic conditions, reduced ubiquinol transfers electrons to outer membrane cytochrome
(ferrireductase) via cytochrome
complex and a cascade of electron transporters (cytochrome
, cytochrome
, rusticyanin, and high potential iron-sulfur protein), resulting in proton efflux and reduction of ferric iron. The proton gradient generated by hydrogen oxidation maintains the membrane potential and allows the generation of ATP and NADH. These results further clarify the role of extremophiles in biogeochemical processes and their impact on the composition of the deep terrestrial subsurface.
A comparative analysis of the protein composition of Acidithiobacillus ferrooxidans cells grown on elemental sulfur and ferrous iron was performed. A newly developed protocol involving immobilized pH ...gradients, improved protein reduction, mass spectrometry protein identification and full genome sequence information was applied. This approach resulted in more than 1300 protein spots displayed in broad and basic pH ranges, the best A. ferrooxidans proteome resolution to date. A comparative image analysis revealed that the proteome was significantly influenced by the growth type, and allowed for the detection of many physiologically important proteins. Among them were sulfate adenylyltransferase and sulfide dehydrogenase, which are involved in sulfate assimilation and sulfide metabolism, respectively. Many other proteins were related to important processes like cell attachment and electron transport. Co‐migration of phosphate and sulfate transport proteins was also observed.
According to the literature, pyrite (FeS
) oxidation has been previously determined to involve thiosulfate as the first aqueous intermediate sulfur product, which is further oxidized to sulfate. In ...the present study, pyrite oxidation by
was studied using electrochemical and metabolic approaches in an effort to extend existing knowledge on the oxidation mechanism. Due to the small surface area, the reaction rate of a compact pyrite electrode in the form of polycrystalline pyrite aggregate in
suspension was very slow at a spontaneously formed high redox potential. The slow rate made it possible to investigate the oxidation process in detail over a term of 100 days. Using electrochemical parameters from polarization curves and levels of released iron, the number of exchanged electrons per pyrite molecule was estimated. The values close to 14 and 2 electrons were determined for the oxidation with and without bacteria, respectively. These results indicated that sulfate was the dominant first aqueous sulfur species formed in the presence of bacteria and elemental sulfur was predominantly formed without bacteria. The stoichiometric calculations are consistent with high iron-oxidizing activities of bacteria that continually keep the released iron in the ferric form, resulting in a high redox potential. The sulfur entity of pyrite was oxidized to sulfate by Fe
without intermediate thiosulfate under these conditions. Cell attachment on the corroded pyrite electrode surface was documented although pyrite surface corrosion by Fe
was evident without bacterial participation. Attached cells may be important in initiating the oxidation of the pyrite surface to release iron from the mineral. During the active phase of oxidation of a pyrite concentrate sample, the ATP levels in attached and planktonic bacteria were consistent with previously established ATP content of iron-oxidizing cells. No significant upregulation of three essential genes involved in energy metabolism of sulfur compounds was observed in the planktonic cells, which represented the dominant biomass in the pyrite culture. The study demonstrated the formation of sulfate as the first dissolved sulfur species with iron-oxidizing bacteria under high redox potential conditions. Minor aqueous sulfur intermediates may be formed but as a result of side reactions.
Elemental sulfur oxidation by ferric iron in
Acidithiobacillus ferrooxidans
was investigated. The apparent Michaelis constant for ferric iron was 18.6 mM. An absence of anaerobic ferric iron ...reduction ability was observed in bacteria maintained on elemental sulfur for an extended period of time. Upon transition from ferrous iron to elemental sulfur medium, the cells exhibited similar kinetic characteristics of ferric iron reduction under anaerobic conditions to those of cells that were originally maintained on ferrous iron. Nevertheless, a total loss of anaerobic ferric iron reduction ability after the sixth passage in elemental sulfur medium was demonstrated. The first proteomic screening of total cell lysates of anaerobically incubated bacteria resulted in the detection of 1599 protein spots in the master two-dimensional electrophoresis gel. A set of 59 more abundant and 49 less abundant protein spots that changed their protein abundances in an anaerobiosis-dependent manner was identified and compared to iron- and sulfur-grown cells, respectively. Proteomic analysis detected a significant increase in abundance under anoxic conditions of electron transporters, such as rusticyanin and cytochrome c
552
, involved in the ferrous iron oxidation pathway. Therefore we suggest the incorporation of
rus
-operon encoded proteins in the anaerobic respiration pathway. Two sulfur metabolism proteins were identified, pyridine nucleotide-disulfide oxidoreductase and sulfide-quinone reductase. The important transcription regulator, ferric uptake regulation protein, was anaerobically more abundant. The anaerobic expression of several proteins involved in cell envelope formation indicated a gradual adaptation to elemental sulfur oxidation.
Wide ranges of growth yields on sulfur (from 2.4 x 10¹⁰ to 8.1 x 10¹¹ cells g⁻¹) and maximum sulfur oxidation rates (from 0.068 to 1.30 mmol liter⁻¹ h⁻¹) of an Acidithiobacillus ferrooxidans strain ...(CCM 4253) were observed in 73 batch cultures. No significant correlation between the constants was observed. Changes of the Michaelis constant for sulfur (from 0.46 to 15.5 mM) in resting cells were also noted.
To clarify the pathway of anaerobic sulfur oxidation coupled with dissimilatory ferric iron reduction in Acidithiobacillus ferrooxidans strain CCM 4253 cells, we monitored their energy metabolism ...gene transcript profiles. Several genes encoding electron transporters involved in aerobic iron and sulfur respiration were induced during anaerobic growth of ferrous iron-grown cells. Most sulfur metabolism genes were either expressed at the basal level or their expression declined. However, transcript levels of genes assumed to be responsible for processing of elemental sulfur and other sulfur intermediates were elevated at the beginning of the growth period. In contrast, genes with predicted functions in formation of hydrogen sulfide and sulfate were significantly repressed. The main proposed mechanism involves: outer membrane protein Cyc2 (assumed to function as a terminal ferric iron reductase); periplasmic electron shuttle rusticyanin; c4-type cytochrome CycA1; the inner membrane cytochrome bc1 complex I; and the quinone pool providing connection to the sulfur metabolism machinery, consisting of heterodisulfide reductase, thiosulfate:quinone oxidoreductase and tetrathionate hydrolase. However, an alternative mechanism seems to involve a high potential iron-sulfur protein Hip, c4-type cytochrome CycA2 and inner membrane cytochrome bc1 complex II. Our results conflict with findings regarding the type strain, indicating strain- or phenotype-dependent pathway variation.
In extremely acidic environments, ferric iron can be a thermodynamically favorable electron acceptor during elemental sulfur oxidation by some Acidithiobacillus spp. under anoxic conditions. ...Quantitative 2D-PAGE proteomic analysis of a resting cell suspension of a sulfur-grown Acidithiobacillus ferrooxidans CCM 4253 subculture that had lost its iron-reducing activity revealed 147 protein spots that were downregulated relative to an iron-reducing resting cell suspension of the antecedent sulfur-oxidizing culture and 111 that were upregulated. Tandem mass spectrometric analysis of strongly downregulated spots identified several physiologically important proteins that apparently play roles in ferrous iron oxidation, including the outer membrane cytochrome Cyc2 and rusticyanin. Other strongly repressed proteins were associated with sulfur metabolism, including heterodisulfide reductase, thiosulfate:quinone oxidoreductase and sulfide:quinone reductase. Transcript-level analyses revealed additional downregulation of other respiratory genes. Components of the iron-oxidizing system thus apparently play central roles in anaerobic sulfur oxidation coupled with ferric iron reduction in the studied microbial strain.
Recombinant rusticyanin was produced in Pichia pastoris, then purified and immobilized on Sepharose CL-4B with periodate activation. Cellular lysate of acidophilic Acidithiobacillus ferrooxidans was ...applied to an affinity column with immobilized rusticyanin. Rusticyanin-binding proteins, separated using 1D PAGE and identified by mass spectrometry, included anticipated interacting partners, such as cytochromes Cyc1 and Cyc2, which are involved in the downhill electron pathway from ferrous iron to oxygen. However, the results indicate that rusticyanin’s functional protein-protein interaction (PPI) network could be more complex than expected, including various proteins involved in different cellular processes. Although affinity purification coupled to mass spectrometry should mostly detect proteins that bind stably, and thus are likely participants in functional in vivo PPIs, further verification is needed to exclude non-functional interactants. Nevertheless, our preliminary PPI data confirm some previous experimental findings and indicate potentially fruitful directions for probing additional roles of rusticyanin in sulfur metabolism, copper resistance, anaerobic iron reduction, iron transport, and oxidative stress in extreme acidophiles.
In contrast to iron-oxidizing
Acidithiobacillus ferrooxidans,
A. ferrooxidans
from a stationary phase elemental sulfur-oxidizing culture exhibited a lag phase in pyrite oxidation, which is similar to ...its behaviour during ferrous iron oxidation. The ability of elemental sulfur-oxidizing
A. ferrooxidans
to immediately oxidize ferrous iron or pyrite without a lag phase was only observed in bacteria obtained from growing cultures with elemental sulfur. However, these cultures that shifted to ferrous iron oxidation showed a low rate of ferrous iron oxidation while no growth was observed. Two-dimensional gel electrophoresis was used for a quantitative proteomic analysis of the adaptation process when bacteria were switched from elemental sulfur to ferrous iron. A comparison of total cell lysates revealed 39 proteins whose increase or decrease in abundance was related to this phenotypic switching. However, only a few proteins were closely related to iron and sulfur metabolism. Reverse-transcription quantitative PCR was used to further characterize the bacterial adaptation process. The expression profiles of selected genes primarily involved in the ferrous iron oxidation indicated that phenotypic switching is a complex process that includes the activation of genes encoding a membrane protein, maturation proteins, electron transport proteins and their regulators.
PDF
