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Autotrophic fixation of carbon dioxide into cellular carbon occurs via several pathways but quantitatively, the Calvin-Benson-Bassham cycle is the most important. CbbR regulates the expression of the cbb genes involved in CO2 fixation via the Calvin-Benson-Bassham cycle in a number of autotrophic bacteria. A gene potentially encoding CbbR (cbbRAF) has been predicted in the genome of the chemolithoautotrophic, extreme acidophile Acidithiobacillus ferrooxidans. However, this microorganism is recalcitrant to genetic manipulation impeding the experimental validation of bioinformatic predictions. Two novel functional assays were devised to advance our understanding of cbbRAF function using the mutated facultative autotroph Ralstonia eutropha H14 ΔcbbR as a surrogate host to test gene function: (i) cbbRAF was expressed in R. eutropha and was able to complement ΔcbbR; and (ii) CbbRAF was able to regulate the in vivo activity of four A. ferrooxidans cbb operon promoters in R. eutropha. These results open up the use of R. eutropha as a surrogate host to explore cbbRAF activity. [ABSTRACT FROM AUTHOR]

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species. [ABSTRACT FROM AUTHOR]

A cluster of five genes, proposed to be involved in the formation of extracellular polysaccharide (EPS) precursors via the Leloir pathway, have been identified in the acidophilic autotroph Acidithiobacillus ferrooxidans. The order of the genes is luxA-galE-galK-pgm-galM, encoding a LuxA-like protein, UDP-glucose 4-epimerase, galactokinase, phosphoglucomutase, and galactose mutarotase, respectively. The gal cluster forms a single transcriptional unit and is therefore an operon. Two other putative genes of the Leloir pathway, galU, potentially encoding UDP-glucose pyrophosphorylase, and a gene designated galT-like, which may encode a galactose-1-phosphate uridylyltransferase-like activity, were found unlinked in the genome. Using semiquantitative reverse transcription-PCR, the genes of the gal operon were shown to be expressed more during growth in iron medium than in growth in sulfur medium. The functions of galE, pgm, galU, and the galT-like gene were validated by complementation of Escherichia coli mutants and by in vitro enzyme assays. The data suggest that A. ferrooxidans is capable of synthesizing the EPS precursors UDP-glucose and UDP-galactose. In addition, genes rfbA, -B, -C, and -D were identified in the genome of A. ferrooxidans, suggesting that it can also synthesize the EPS precursor dTDP-rhamnose. Since EPSs constitute the major bulk of biofilms, this study may provide an initial model for the metabolic pathways involved in biofilm formation in A. ferrooxidans and aid in understanding the role of biofilms in mineral leaching and the formation of acid mine drainage. [ABSTRACT FROM AUTHOR]

Abstract: Reporter gene assays are important tools for evaluating gene expression. A frequently used assay measures the activity of β-galactosidase (β-gal) expressed from lacZ in plasmid or genomic constructions. Such constructions are often used to interrogate the ability of DNA (query DNA), potentially encoding a transcription factor, to regulate in trans the expression of a promoter fused to the reporter lacZ. Query DNA is frequently inserted into a second plasmid within the α-subunit of β-gal, interrupting its function. However, this plasmid can induce up-expression of β-gal even when void of query DNA, leading to confusion between artifact and authentic regulation. [Copyright &y& Elsevier]

The acidophilic proteobacterium Acidithiobacillus ferrooxidans is involved in the industrial biorecovery of copper. It is found in acidic environments in biofilms and is important in the biogeochemicai cycling of metals and nutrients. Its genome contains a cluster of four genes, glyQ, glysS, gph, and act, that are predicted to encode the α and β subunits of glycine tRNA synthetase, a phosphatase, and an acyltransferase, respectively (GenBank accession no. DQ149607). act, cloned and expressed in Escherichia coli, produces acyl homoserine lactones (AHLs) principally of chain length C14 according to gas chromatography and mass spectrometry measurements. The AHLs have biological activity as shown by in vivo studies using the reporter strain Sinorhizobium meliloti Rm41 SinI-. Reverse transcription-PCR (RT-PCR) experiments indicate that the four genes are expressed as a single transcript, demonstrating that they constitute an operon. According to semiquantitative RT-PCR results, act is expressed more highly when A. ferrooxidans is grown in medium containing iron than when it is grown in medium containing sulfur. Since AHLs are important intercellular signaling molecules used by many bacteria to monitor their population density in quorum-sensing control of gene expression, this result suggests that A. ferrooxidans has two quorum-sensing systems, one based on Act, as described herein, and the other based on a Lux-like quorum-sensing system, reported previously. The latter system was shown to be upregulated in A. ferrooxidans grown in sulfur medium, suggesting that the two quorum-sensing systems respond to different environmental signals that may be related to their abilities to colonize and use different solid sulfur- and iron-containing minerals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

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 FROM AUTHOR]

Abstract: An understanding of the physiology and metabolic complexity of microbial consortia involved in metal solubilization is a prerequisite for the rational improvement of bioleaching technologies. Among the most challenging aspects that remain to be addressed is how aerobic acidophiles, especially Fe(II)-oxidizers, contend with the paradoxical hazards of iron overload and iron deficiency, each with deleterious consequences for growth. Homeostatic mechanisms regulating the acquisition, utilization/oxidation, storage and intracellular mobilization of cellular iron are deemed to be critical for fitness and survival of bioleaching microbes. In an attempt to contribute to the comprehensive understanding of the biology and ecology of the microbial communities in bioleaching econiches, we have used comparative genomics and other bioinformatic tools to reconstruct the iron management strategies in newly sequenced Acidithiobacilli and other biomining genomes available in public databases. Species specific genes have been identified with distinctive functional roles in iron management as well as genes shared by several species in biomining consortia. Their analysis contributes to our understanding of the general survival strategies in acidic and iron loaded environments and suggests functions for genes with currently unknown roles that might reveal novel aspects of iron response in acidophiles. Comprehensive examination of the occurrence and conservation of regulatory functions and regulatory sites also allowed the prediction of the metal regulatory networks for these biomining microbes. [Copyright &y& Elsevier]

The article presents a study about the differential expression of cytochrome bc1 complex in the strict acidophilic chemolithoautotrophic bacterium acidithiobacillus ferroxidans. Acidithiobacillus ferroxidans is found to contain pet operon that comprises with gene cluster. The study revealed the possible transcription initiation sites for the petl and petll operons that encodes complexes for the uphill flow direction of the electrons from iron to quinone oxidoreductase.

Abstract: Acidithiobacillus ferrooxidans is a well known acidophilic, chemolithoautotrophic, Gram negative, bacterium involved in bioleaching and acid mine drainage. In aerobic conditions, it gains energy mainly from the oxidation of ferrous iron and/or reduced sulfur compounds present in ores. After initial oxidation of the substrate, electrons from ferrous iron or sulfur enter respiratory chains and are transported through several redox proteins to oxygen. However, the oxidation of ferrous iron and reduced sulfur compounds has also to provide electrons for the reduction of NAD(P) that is subsequently required for many metabolic processes including CO2 fixation. To help unravel the enzymatic pathways and the electron transfer chains involved in these processes, a genome-wide microarray transcript profiling analysis was carried out. Oligonucleotides corresponding to approximately 3000 genes of the A. ferrooxidans type strain ATCC23270 were spotted onto glass-slides and hybridized with cDNA retrotranscribed from RNA extracted from ferrous iron and sulfur grown cells. The genes which are preferentially transcribed in ferrous iron conditions and those preferentially transcribed in sulfur conditions were analyzed. The expression of a substantial number of these genes has been validated by real-time PCR, Northern blot hybridization and/or immunodetection analysis. Our results support and extend certain models of iron and sulfur oxidation and highlight previous observations regarding the possible presence of alternate electron pathways. Our findings also suggest ways in which iron and sulfur oxidation may be co-ordinately regulated. An accompanying paper (Appia-Ayme et al.) describes results pertaining to other metabolic functions. [Copyright &y& Elsevier]

Abstract: Acidithiobacillus ferrooxidans is a chemolithoautotrophic bacterium that uses iron or sulfur as an energy and electron source. Bioinformatic analysis of the A. ferrooxidans draft genome sequence was used to identify putative genes and potential metabolic pathways involved in CO2 fixation, 2P-glycolate detoxification, carboxysome formation and glycogen utilization. Microarray transcript profiling was carried out to compare the relative expression of the predicted genes of these pathways when the microorganism was grown in the presence of iron versus sulfur. Several gene expression patterns were confirmed by real-time PCR. Genes for each of the above-predicted pathways were found to be organized into discrete clusters. Clusters exhibited differential gene expression depending on the presence of iron or sulfur in the medium. Concordance of gene expression within each cluster suggested that they are operons. Most notably, clusters of genes predicted to be involved in CO2 fixation, carboxysome formation, 2P-glycolate detoxification and glycogen biosynthesis were upregulated in sulfur medium, whereas genes involved in glycogen utilization were preferentially expressed in iron medium. These results can be explained in terms of models of gene regulation that suggest how A. ferrooxidans can adjust its central carbon management to respond to changes in its environment. [Copyright &y& Elsevier]

Candidate genes have been identified in the almost complete genome sequence of Acidithiobacillus ferrooxidans that are potentially involved in several physiological processes of relevance to the application of this microorganism to biohydrometallurgical processes. Candidates for six of the 10 missing genes in the previously described amino acid biosynthesis pathways have been located. Genes potentially involved in hydrogen utilization, conjugation, type IV pilus formation, heavy metal resistance and cation fluxes are described. [Copyright &y& Elsevier]

Examines the characterization of the petI and res operons of Acidithiobacillus ferrooxidans. Formation of a bc1 complex; Maturation of a cytochrome c-type proteins; Use of DNA sequence analysis and bioinformatic interpretations.

Studies ISAfe1, an ISL3 family insertion sequence (IS) from Acidithiobacillus ferooxidans ATCC 19859. Characterization of the putative transposase enzyme; Characterization of the DNA sequences present in ISAfe1; Distribution of copies of ISAfe1 in A. Ferooxidans and other organisms; Transposition of ISAfe1 in A. ferooxidans and Escherichia coli.