The recent revision of the Acidithiobacillia class using genomic taxonomy methods has shown that, in addition to the existence of previously unrecognized genera and species, some species of the class harbor levels of divergence that are congruent with ongoing differentiation processes. In this study, we have performed a subspecies-level analysis of sequenced strains of Acidithiobacillus ferrooxidans to prove the existence of distinct sublineages and identify the discriminant genomic/genetic characteristics linked to these sublineages, and to shed light on the processes driving such differentiation. Differences in the genomic relatedness metrics, levels of synteny, gene content, and both integrated and episomal mobile genetic elements (MGE) repertoires support the existence of two subspecies-level taxa within A. ferrooxidans. While sublineage 2A harbors a small plasmid related to pTF5, this episomal MGE is absent in sublineage 2B strains. Likewise, clear differences in the occurrence, coverage and conservation of integrated MGEs are apparent between sublineages. Differential MGE-associated gene cargo pertained to the functional categories of energy metabolism, ion transport, cell surface modification, and defense mechanisms. Inferred functional differences have the potential to impact long-term adaptive processes and may underpin the basis of the subspecies-level differentiation uncovered within A. ferrooxidans. Genome resequencing of iron- and sulfur-adapted cultures of a selected 2A sublineage strain (CCM 4253) showed that both episomal and large integrated MGEs are conserved over twenty generations in either growth condition. In turn, active insertion sequences profoundly impact short-term adaptive processes. The ISAfe1 element was found to be highly active in sublineage 2A strain CCM 4253. Phenotypic mutations caused by the transposition of ISAfe1 into the pstC2 encoding phosphate-transport system permease protein were detected in sulfur-adapted cultures and shown to impair growth on ferrous iron upon the switch of electron donor. The phenotypic manifestation of the △pstC2 mutation, such as a loss of the ability to oxidize ferrous iron, is likely related to the inability of the mutant to secure the phosphorous availability for electron transport-linked phosphorylation coupled to iron oxidation. Depletion of the transpositional △pstC2 mutation occurred concomitantly with a shortening of the iron-oxidation lag phase at later transfers on a ferrous iron-containing medium. Therefore, the pstII operon appears to play an essential role in A. ferrooxidans when cells oxidize ferrous iron. Results highlight the influence of insertion sequences and both integrated and episomal mobile genetic elements in the short- and long-term adaptive processes of A. ferrooxidans strains under changing growth conditions.
- Klíčová slova
- sirné bakterie, acidofilní bakterie, anaerobní prostředí, pyrit,
- MeSH
- Acidithiobacillus * chemie metabolismus MeSH
- adsorpce MeSH
- Bacteria chemie metabolismus MeSH
- biochemické jevy * MeSH
- biotechnologie MeSH
- kationty chemie MeSH
- oxidace-redukce MeSH
- proteom MeSH
- síra chemie MeSH
- těžební a zpracovatelský průmysl metody MeSH
- železo chemie MeSH
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.
- MeSH
- 2D gelová elektroforéza MeSH
- Acidithiobacillus chemie genetika metabolismus MeSH
- anaerobióza MeSH
- bakteriální proteiny analýza MeSH
- mutace MeSH
- oxidace-redukce MeSH
- proteom analýza MeSH
- síra metabolismus MeSH
- stanovení celkové genové exprese MeSH
- tandemová hmotnostní spektrometrie MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
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 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.
- MeSH
- 2D gelová elektroforéza MeSH
- Acidithiobacillus růst a vývoj metabolismus fyziologie MeSH
- bakteriální proteiny biosyntéza MeSH
- fyziologická adaptace MeSH
- genetická transkripce MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- metabolické sítě a dráhy genetika MeSH
- oxidace-redukce MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteom analýza MeSH
- regulace genové exprese * MeSH
- síra metabolismus MeSH
- stanovení celkové genové exprese MeSH
- sulfidy metabolismus MeSH
- železnaté sloučeniny metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The conventional stoichiometry of the oxidation of elemental sulfur by ferric iron in Acidithiobacillus ferrooxidans was not in agreement with our experimental data in terms of ferrous iron and proton formation. Reaction modelling under the actual conditions of bacterial activity resulted in a different stoichiometry, where additional iron species participate in the process to affect the number of released protons. The suggested reaction equation may more accurately predict the intensity of environmental acidification during the anaerobic bioprocess.
- MeSH
- Acidithiobacillus metabolismus MeSH
- anaerobióza MeSH
- energetický metabolismus MeSH
- oxidace-redukce MeSH
- síra metabolismus MeSH
- železité sloučeniny metabolismus MeSH
- železnaté sloučeniny metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- 2D gelová elektroforéza MeSH
- Acidithiobacillus metabolismus MeSH
- anaerobióza MeSH
- bakteriální proteiny analýza MeSH
- fyziologická adaptace MeSH
- kinetika MeSH
- oxidace-redukce MeSH
- proteomika MeSH
- síra metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- železité sloučeniny metabolismus MeSH
- železnaté sloučeniny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Thiosulfate dehydrogenase was purified from Acidithiobacillus ferrooxidans using three purification steps. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography, and gel permeation chromatography. Specific activity of the purified enzyme (after IEC) was 3.26 nkat/mg, and yield of the enzyme was 78%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is a tetramer composed of four probably identical subunits of relative molecular weight 45,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 3.0. The isoelectric point of the enzyme was 8.3. Enzyme activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate. Reagents selective for arginine, cysteine, and tryptophane had no effect on enzyme activity.
- MeSH
- Acidithiobacillus enzymologie MeSH
- aktivace enzymů účinky léků MeSH
- časové faktory MeSH
- chromatografie iontoměničová MeSH
- diethylpyrokarbonát farmakologie MeSH
- dodecylsíran sodný chemie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- financování organizované MeSH
- gelová chromatografie MeSH
- histidin farmakologie MeSH
- izoelektrický bod MeSH
- koncentrace vodíkových iontů MeSH
- molekulová hmotnost MeSH
- oxidoreduktasy chemie izolace a purifikace metabolismus MeSH
- síran amonný chemie MeSH
- substrátová specifita MeSH
