Eugenia Jedlicki

Extending the models for iron and sulfur oxidation in the extreme Acidophile Acidithiobacillus ferrooxidans

BackgroundAcidithiobacillus 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.ResultsBioinformatic-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.ConclusionBioinformatic 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.

Identification of a gene cluster for the formation of extracellular polysaccharide precursors in the chemolithoautotroph Acidithiobacillus ferrooxidans.

ABSTRACT 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.

Bioinformatic prediction and experimental verification of Fur-regulated genes in the extreme acidophile Acidithiobacillus ferrooxidans

The γ-proteobacterium Acidithiobacillus ferrooxidans lives in extremely acidic conditions (pH 2) and, unlike most organisms, is confronted with an abundant supply of soluble iron. It is also unusual in that it oxidizes iron as an energy source. Consequently, it faces the challenging dual problems of (i) maintaining intracellular iron homeostasis when confronted with extremely high environmental loads of iron and (ii) of regulating the use of iron both as an energy source and as a metabolic micronutrient. A combined bioinformatic and experimental approach was undertaken to identify Fur regulatory sites in the genome of A. ferrooxidans and to gain insight into the constitution of its Fur regulon. Fur regulatory targets associated with a variety of cellular functions including metal trafficking (e.g. feoPABC, tdr, tonBexbBD, copB, cdf), utilization (e.g. fdx, nif), transcriptional regulation (e.g. phoB, irr, iscR) and redox balance (grx, trx, gst) were identified. Selected predicted Fur regulatory sites were confirmed by FURTA, EMSA and in vitro transcription analyses. This study provides the first model for a Fur-binding site consensus sequence in an acidophilic iron-oxidizing microorganism and lays the foundation for future studies aimed at deepening our understanding of the regulatory networks that control iron uptake, homeostasis and oxidation in extreme acidophiles.

Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis

BackgroundAcidithiobacillus 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.ResultsTwo 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.ConclusionsA 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.

Second Acyl Homoserine Lactone Production System in the Extreme Acidophile Acidithiobacillus ferrooxidans

ABSTRACT 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 biogeochemical 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.

Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: Case study with the extremophile Acidithiobacillus ferrooxidans

BackgroundNormalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data.ResultsHere, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes map, rpoC, alaS and era results in improved interpretation of gene expression profiles in A. ferrooxidans.ConclusionThis investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems.

A Lux-like Quorum Sensing System in the Extreme Acidophile Acidithiobacillus ferrooxidans

The genome of the acidophilic, proteobacterium Acidithiobacillusferrooxidans, contains linked but divergently oriented genes, termed afel and afeR, whose predicted protein products are significantly similar to the LuxI and LuxR families of proteins. A possible promoter and Lux box are predicted upstream of afel. A cloned copy of afel, expressed in E. coli, encodes an enzyme that catalyzes the production of a diffusible compound identified by gas chromatography and mass spectrometry as an unsubstituted N-acyl homoserine lactone (AHL) of chain length C14. This AHL can be detected by a reporter strain of Sinorhizobium meliloti Rm41 suggesting that it is biologically active. The reporter strain also responds to extracts of the supernatant of A. ferrooxidans grown to early stationary phase in sulfur medium indicating that a diffusible AHL is produced by this microorganism. Semi-quantitative RT-PCR experiments indicate that afeI and afeR are expressed maximally in early stationary phase and are more expressed when A. ferrooxidans is grown in sulfur--rather than iron-containing medium. Given the predicted amino acid sequence and functional properties of AfeI and AfeR it is proposed that A. ferrooxidans has a quorum sensing system similar to the LuxI-LuxR paradigm.

Aspects of the predicted physiology of Acidithiobacillus ferrooxidans deduced from an analysis of its partial genome sequence

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.

SEQUENCE AND EXPRESSION OF THE RUSTICYANIN STRUCTURAL GENE FROM THIOBACILLUS FERROOXIDANS ATCC33020 STRAIN

The periplasmic blue copper protein rusticyanin is thought to play an important role in iron oxidation by Thiobacillus ferrooxidans. We present the sequence of the gene, rus, encoding rusticyanin together with about 1.4 kb of upstream and 0.3 kb of downstream DNA. The rus gene is unique to T. ferrooxidans. Evidence is presented that it is the last gene of an operon and that it can be transcribed from its own promoter. In ATCC33020 strain, rusticyanin is synthesized in ferrous iron but also in sulfur growth conditions suggesting that it could play a role in both energetic metabolisms. The rus gene transcribed from a vector promoter in Escherichia coli leads to the production of a processed aporusticyanin in the periplasmic space, indicating that its signal sequence is correctly recognized by the secretion machinery and the signal peptidase of E. coli.

Stereospecificity of isopentenylpyrophosphate isomerase and prenyl transferase from Pinus and Citrus

Abstract Isopentenylpyrophosphate isomerases in cell-free extracts from Pinus radiata and from Citrus sinensis eliminate the C-4 pro-S proton from mevalonic acid. Prenyl transferase from Pinus forms geranyl pyrophosphate, neryl pyrophosphate, 2,6 trans, trans farnesyl pyrophosphate and 2 cis, 6 trans farnesyl pyrophosphate from mevalonic acid. The 4-pro-S proton of mevalonic acid is eliminated in the formation of both trans and of cis double-bonds. The pattern is similar for the Citrus enzyme. Isopentenyl pyrophosphate isomerase from Pinus is completely inhibited by 15 m m iodoacetamide, which does not affect prenyl transferase. The pattern is the same for the Citrus isomerase, but less clear for the transferase. Previous work has ruled out direct trans-cis isomerization of C10 or C15 prenylpyrophosphates. Alternative mechanisms are proposed for the stereospecific elimination of the 4-pro-S proton of mevalonic acid in the biosynthesis of both cis and trans prenyl pyrophosphates.