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Abstract Background Acidithiobacillus 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. Results Four 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. Conclusions A. 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.

Abstract Background Acidithiobacillus ferrooxidans gains energy from the oxidation of ferrous iron and various reduced inorganic sulfur compounds at very acidic pH. Although an initial model for the electron pathways involved in iron oxidation has been developed, much less is known about the sulfur oxidation in this microorganism. In addition, what has been reported for both iron and sulfur oxidation has been derived from different A. ferrooxidans strains, some of which have not been phylogenetically characterized and some have been shown to be mixed cultures. It is necessary to provide models of iron and sulfur oxidation pathways within one strain of A. ferrooxidans in order to comprehend the full metabolic potential of the pangenome of the genus. Results Bioinformatic-based metabolic reconstruction supported by microarray transcript profiling and quantitative RT-PCR analysis predicts the involvement of a number of novel genes involved in iron and sulfur oxidation in A. ferrooxidans ATCC23270. These include for iron oxidation: cup (copper oxidase-like), ctaABT (heme biogenesis and insertion), nuoI and nuoK (NADH complex subunits), sdrA1 (a NADH complex accessory protein) and atpB and atpE (ATP synthetase F0 subunits). The following new genes are predicted to be involved in reduced inorganic sulfur compounds oxidation: a gene cluster (rhd, tusA, dsrE, hdrC, hdrB, hdrA, orf2, hdrC, hdrB) encoding three sulfurtransferases and a heterodisulfide reductase complex, sat potentially encoding an ATP sulfurylase and sdrA2 (an accessory NADH complex subunit). Two different regulatory components are predicted to be involved in the regulation of alternate electron transfer pathways: 1) a gene cluster (ctaRUS) that contains a predicted iron responsive regulator of the Rrf2 family that is hypothesized to regulate cytochrome aa3 oxidase biogenesis and 2) a two component sensor-regulator of the RegB-RegA family that may respond to the redox state of the quinone pool. Conclusion Bioinformatic analysis coupled with gene transcript profiling extends our understanding of the iron and reduced inorganic sulfur compounds oxidation pathways in A. ferrooxidans and suggests mechanisms for their regulation. The models provide unified and coherent descriptions of these processes within the type strain, eliminating previous ambiguity caused by models built from analyses of multiple and divergent strains of this microorganism.

Abstract Background Acidithiobacillus ferrooxidans is a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences of A. ferrooxidans are available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism. Results Two candidate mechanisms for sulfate uptake from the environment were detected but both belong to large paralogous families of membrane transporters and their identification remains tentative. Prospective genes, pathways and regulatory mechanisms were identified that are likely to be involved in the assimilation of sulfate into cysteine and in the formation of Fe-S centers. Genes and regulatory networks were also uncovered that may link sulfur assimilation with nitrogen fixation, hydrogen utilization and sulfur reduction. Potential pathways were identified for sulfation of extracellular metabolites that may possibly be involved in cellular attachment to pyrite, sulfur and other solid substrates. Conclusions A bioinformatic analysis of the genome sequence of A. ferrooxidans has revealed candidate genes, metabolic process and control mechanisms potentially involved in aspects of sulfur metabolism. Metabolic modeling provides an important preliminary step in understanding the unusual physiology of this extremophile especially given the severe difficulties involved in its genetic manipulation and biochemical analysis.

Three strains of the strict acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans, including the type strain ATCC 23270, contain a petllABC gene cluster that encodes the three proteins, cytochrome [c.sub.1], cytochrome b and a Rieske protein, that constitute a [bc.sub.1] electron-transfer complex. RT-PCR and Northern blotting show that the petllABC cluster is co-transcribed with cycA, encoding a cytochrome c belonging to the [c.sub.4] family, sdrA, encoding a putative short-chain dehydrogenase, and hip, encoding a high potential iron-sulfur protein, suggesting that the six genes constitute an operon, termed the petll operon. Previous results indicated that A. ferrooxidans contains a second pet operon, termed the petl operon, which contains a gene cluster that is similarly organized except that it lacks hip. Real-time PCR and Northern blot experiments demonstrate that petl is transcribed mainly in cells grown in medium containing iron, whereas petll is transcribed in cells grown in media containing sulfur or iron. Primer extension experiments revealed possible transcription initiation sites for the petl and petll operons. A model is presented in which petl is proposed to encode the [bc.sub.1] complex, functioning in the uphill flow of electrons from iron to NAD(P), whereas petll is suggested to be involved in electron transfer from sulfur (or formate) to oxygen (or ferric iron). A. ferrooxidans is the only organism, to date, to exhibit two functional [bc.sub.1] complexes.

A cluster of five genes, proposed to be involved in the formation of extracellular polysaccharide (EPS) precursors were identified in the acidophilic autotroph Acidithiobacillus ferrooxidans. This study might provide an initial model for the metabolic pathways involved in biofilm formation in Acidithiobacillus ferrooxidans and help in understanding the role of biofilms in mineral leaching and the formation of acid mine drainage.

Research has been conducted on the gene clusters involved in the bc1 complex formation and cytochrome c-type protein maturation in the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans. The use of the DNA sequence analysis and bioinformatic interruptions in identifying these clusters is described.

An analysis of the bacterial populations in bioleaching systems by analysis of DNA from ores, leaching systems, or laboratory cultures indicates the predominance of different bacteria depending on the growth conditions. PCR amplification and characterization of the spacer regions between the 16 and 23S genes in the rRNA genetic loci indicates that Thiobacillus ferrooxidans predominates at high ferrous iron concentrations. T. thiooxidans and Leptospirillum ferrooxidans predominate at low ferrous iron levels.

Characterization of isolated tyrosyl-tRNA synthetase gene (tyrZ) from Thiobacillus ferroxidans to determine the amino acid sequence involves the bioleaching of minerals. The sequence is 44% similar to the product from tyrZ gene of Bacillus subtilis. Southern blot analysis reveals tyrZ gene as a single copy gene located upstream from rrnT2 ribosomal RNA operon. A 24% limited similarity is observed between tyrZ and Escherichia coli TyrRS.

An effort is made to understand quorum sensing in Acidithiobacillus ferrooxidants and evidence for the role of Act in the tranformation of acyl homoserine lactones in Acidithiobacillus ferrooxidants is provided.

This study was motivated by surprising gaps in the current knowledge of microbial inorganic carbon (Ci) uptake and assimilation at acidic pH values (pH < 3). Particularly striking is the limited understanding of the differences between Ci uptake mechanisms in acidic versus circumneutral environments where the Ci predominantly occurs either as a dissolved gas (CO2) or as bicarbonate (HCO3-), respectively. In order to gain initial traction on the problem, the relative abundance of transcripts encoding proteins involved in Ci uptake and assimilation was studied in the autotrophic, polyextreme acidophile Acidithiobacillus ferrooxidans whose optimum pH for growth is 2.5 using ferrous iron as an energy source, although they are able to grow at pH 5 when using sulfur as an energy source. The relative abundance of transcripts of five operons (cbb1-5) and one gene cluster (can - sulP) was monitored by RT-qPCR and, in selected cases, at the protein level by Western blotting, when cells were grown under different regimens of CO2 concentration in elemental sulfur. Of particular note was the absence of a classical bicarbonate uptake system in A. ferrooxidans. However, bioinformatic approaches predict that sulP , previously annotated as a sulfate transporter, is a novel type of bicarbonate transporter. A conceptual model of CO2 fixation was constructed from combined bioinformatic and experimental approaches that suggests strategies for providing ecological flexibility under changing concentrations of CO2 and provides a portal to elucidating Ci uptake and regulation in acidic conditions. The results could advance the understanding of industrial bioleaching processes to recover metals such as copper at acidic pH. In addition, they may also shed light on how chemolithoautotrophic acidophiles influence the nutrient and energy balance in naturally occurring low pH environments. [ABSTRACT FROM AUTHOR]

The genome of Thiobacillus ferrooxidans contains at least two different repetitive DNA elements. One of these elements, termed IST2 has been sequenced and shown to exhibit the characteristics of a typical prokaryotic insertion sequence. Furthermore, preiliminary evidence has implicated IST2 in genomic rearrangements, although the mechanism of rearrangement, whether by transposition or recombination, has not been established. In this report we provide evidence from detailed restriction enzyme analyses and DNA sequencing data that support a model of transposition, consistent with the notion that IST2 is a mobile insertion sequence. [ABSTRACT FROM AUTHOR]