Abstract
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.
Highlights
Acidithiobacillus ferrooxidans is chemolithoautotrophic g-proteobacterium that thrives at extremely low pH
These observations taken together, suggest that, in A. ferrooxidans, CbbR can regulate the expression of RubisCO and the carboxysome genes and is likely to be involved in the regulation of carbon fixation as has been observed in other autotrophic bacteria including: Xanthobacter flavus [9], Ralstonia eutropha H16 [10], Chromatium vinosum [11], Nitrobacter vulgaris [12], Halothiobacillus neapolitanus [13], Thiobacillus denitrificans [14], Rhodobacter sphaeroides [15], Rhodobacter capsulatus [16], Rhodospirillum rubrum [17], Hydrogenovibrio marinus [18], Nitrosomonas europaea [19] and Thiomicrospira crunogena XCL-2 [20]
The genome of A. ferrooxidans American Type Culture Collection. AmpR (ATCC) 23270 encodes CbbR, a LysR-type transcription factor A gene cbbR was predicted in the genome of A. ferrooxidans ATCC 23270 that potentially encodes a protein with significant amino acid sequence similarity and domain structure to other well-documented CbbRs of the LysR family of transcription factors (Additional file 1). cbbR is divergently transcribed from cbbL1, a gene predicted to encode the large subunit of form I RubisCO
Summary
Acidithiobacillus ferrooxidans is chemolithoautotrophic g-proteobacterium that thrives at extremely low pH (pH 1-2). Microarray transcript profiling experiments have detected differential expression of several genes in A. ferrooxidans potentially involved in the CBB cycle depending on the growth substrate used [8] These observations taken together, suggest that, in A. ferrooxidans, CbbR can regulate the expression of RubisCO and the carboxysome genes and is likely to be involved in the regulation of carbon fixation as has been observed in other autotrophic bacteria including: Xanthobacter flavus [9], Ralstonia eutropha H16 [10], Chromatium vinosum [11], Nitrobacter vulgaris [12], Halothiobacillus neapolitanus [13], Thiobacillus denitrificans [14], Rhodobacter sphaeroides [15], Rhodobacter capsulatus [16], Rhodospirillum rubrum [17], Hydrogenovibrio marinus [18], Nitrosomonas europaea [19] and Thiomicrospira crunogena XCL-2 [20]. No coherent model has been developed for A. ferrooxidans to explain all the data and little experimental evidence has been provided to support several of the aforementioned observations, prompting the current investigation
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