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 H 2 S production came from the observation that soluble Cu 2؉ included in anaerobically incubated cultures was precipitated (seemingly as CuS). Since H 2 S 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 S 0 to Fe 3؉ 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.
Fluorescence measurements (emission scan, synchronous scan, and excitation-emission matrix [EEM] scan) were used to compare characteristics of two sources of dissolved organic carbon (DOC) from distinctly different origins: (i) a standard fulvic acid from the Suwannee River (SRF sample) and (ii) an unfractionated DOC sample from a tertiary wastewater treatment plant (MWW sample). Two methods were demonstrated that quantitatively differentiated allochthonous DOC (e.g., SRF) from autochthonous DOC (e.g., MWW). The MWW sample exhibited fluorescence peaks undetected in the SRF sample, at shorter wavelength pairs (e.g., 220 nm:300 to 350 nm) than the dominant peaks in the SRF sample (e.g., 220 nm:450 nm). These peaks may be associated with base or neutral fractions, potentially enriched in organic nitrogen. Effects of DOC concentration and solution pH were discussed. A simple procedure was recommended (pH = 3; DOC = 1 mg/L; dilution with 0.01 M KCl) that minimizes the need to correct spectra for inner-filter absorbance effects. A method, using synchronous fluorescence, to estimate the percentage of DOC from different sources when mixed together was also presented. Further work to understand the structural properties of DOC that fluoresce in wastewater samples, especially at shorter EEM wavelength pairs, will enable water managers to better understand the influence of wastewater on DOC in receiving waters (e.g., rivers, lakes).
BackgroundAcidithiobacillus ferrooxidans is chemolithoautotrophic γ-proteobacterium that thrives at extremely low pH (pH 1-2). Although a substantial amount of information is available regarding CO2 uptake and fixation in a variety of facultative autotrophs, less is known about the processes in obligate autotrophs, especially those living in extremely acidic conditions, prompting the present study.ResultsFour gene clusters (termed cbb1-4) in the A. ferrooxidans genome are predicted to encode enzymes and structural proteins involved in carbon assimilation via the Calvin-Benson-Bassham (CBB) cycle including form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO, EC 4.1.1.39) and the CO2-concentrating carboxysomes. RT-PCR experiments demonstrated that each gene cluster is a single transcriptional unit and thus is an operon. Operon cbb1 is divergently transcribed from a gene, cbbR, encoding the LysR-type transcriptional regulator CbbR that has been shown in many organisms to regulate the expression of RubisCO genes. Sigma70-like -10 and -35 promoter boxes and potential CbbR-binding sites (T-N11-A/TNA-N7TNA) were predicted in the upstream regions of the four operons. Electrophoretic mobility shift assays (EMSAs) confirmed that purified CbbR is able to bind to the upstream regions of the cbb1, cbb2 and cbb3 operons, demonstrating that the predicted CbbR-binding sites are functional in vitro. However, CbbR failed to bind the upstream region of the cbb4 operon that contains cbbP, encoding phosphoribulokinase (EC 2.7.1.19). Thus, other factors not present in the assay may be required for binding or the region lacks a functional CbbR-binding site. The cbb3 operon contains genes predicted to encode anthranilate synthase components I and II, catalyzing the formation of anthranilate and pyruvate from chorismate. This suggests a novel regulatory connection between CO2 fixation and tryptophan biosynthesis. The presence of a form II RubisCO could promote the ability of A. ferrooxidans to fix CO2 at different concentrations of CO2.ConclusionsA. ferrooxidans has features of cbb gene organization for CO2-assimilating functions that are characteristic of obligate chemolithoautotrophs and distinguish this group from facultative autotrophs. The most conspicuous difference is a separate operon for the cbbP gene. It is hypothesized that this organization may provide greater flexibility in the regulation of expression of genes involved in inorganic carbon assimilation.
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