Advanced Materials Research Vols. 71-73

Abstract: It has long been recognized that isolates of iron- and sulfur-oxidizing acidophiles referred to as “Acidithiobacillus ferrooxidans” probably include more than one species, on the basis of differences in chromosomal GC contents and 16S rRNA gene sequences. Phylogenetic heterogeneity among these isolates was confirmed by phylogenetic analysis using the sequences of the 16S-23S intergenic spacers (ITS). Two main groups have been identified: the first includes the type strain of Acidithiobacillus ferrooxidans and a second comprises a cluster of newly isolated strains that have 98.5% 16S rRNA gene sequence identity with the type strain. Given that the new group of isolates have GC contents of 56 mol% as opposed to 58.8 mol% for At. ferrooxidansT, and that they share only 37% homologous DNA, these were given the new species name Acidithiobacillus ferrivorans. Further studies showed that, while strains of At. ferrivorans have many physiological traits in common with At. ferrooxidans, they also differ in some key characteristics. These include the ability to grow at temperatures as low as 4°C (as opposed to the lower limit of between 10 and 12°C for At. ferrooxidans) and the greater sensitivity of At. ferrivorans to low pH (minimum of 1.9 for growth as opposed to 1.3 for At. ferrooxidansT). Important genotypic differences include the fact that all strains of At. ferrivorans do not contain the archetypal rusticyanin gene (rusA), rather most contain a rusA homologue (rusB). Furthermore, the high potential iron-sulfur protein-encoding gene of all At. ferrivorans strains analyzed is more similar to the iro than to the hip gene characterized in At. ferrooxidansT. These results suggest that the iron oxidation pathways are different in At. ferrivorans and At. ferrooxidans.

Abstract: Arsenic resistance genes were isolated from the moderately thermophilic, Gram-positive iron and sulfur-oxidizing bacterium, Sulfobacillus thermosulfidooxidans. Only arsR and arsB genes were present and attempts to identify an arsC using degenerate PCR primers or dependent arsC genes as probes in Southern hybridization experiments were unsuccessful. Although enhanced resistance to arsenite was not detected when the ars genes were cloned in Escherichia coli, the kumamolisin-As and arsRB genes were induced by arsenite. RT-PCR experiments suggested that transcription of the cloned kumamolisin-As-like and arsRB genes is linked in Escherichia coli, but not in Sb. thermosulfidooxidans. The gene order kumamolisin-As precursor, arsR and arsB was maintained among three strains of Sb. thermosulfidooxidans isolated from three continents. Southern hybridization using a Sb. thermosulfidooxidans arsB gene fragment as a probe gave a positive hybridization signal using S. acidophilus but not with S. thermotolerans genomic DNA. Comparison of partial sequence data of the arsB and 16S rRNA genes suggested that the two types of genes have undergone a similar evolutionary history and therefore that the arsB genes were present in the ancestral Sulfobacillus before its divergence into species.

Abstract: Bioleaching is the extraction of metals, such as copper or gold, from ore by microorganisms. Bacterial attachment increases leaching activities due to the formation of a "reaction space" between the metal sulfide surface and the cell. This process depends on abiotic characteristics such as purity and degree of crystallization of the metal sulfide, as well as biotic ones such as the capacity of the bacteria for detecting favourable attachment sites and synthesizing a suitable cell envelope (EPS), for adhesion. Planktonic and sessile cells should differ significantly in their metabolic activities and therefore in their gene expression patterns. To help to understand At. ferrooxidans biofilm formation, microarray transcript profiling was carried out to compare planktonic and sessile cells. The high contents of EPS and ferric iron of the biofilms are interfering with RNA extraction, causing inhibition of DNAse, reverse transcriptase and/or polymerase activities required to get labelled target cDNA. In order to have sufficient high quality RNA suitable for transcriptomic analysis, we have optimized the biofilm formation of At. ferrooxidans on pyrite (FeS2) and the RNA extraction from the sessile cell population. DNA microarrays have been hybridized with labelled cDNAs from sessile and planktonic cells and preliminary data suggest that some genes are differently expressed between these two subpopulations. The understanding of these differences may help us to shift populations of leaching bacteria from the planktonic state towards the sessile state in order to influence bioleaching.

Abstract: The Acidithiobacillus ferrooxidans periplasmic space is known to have proteins involved in the respiratory chains. There are no reports about the expression of the periplasmic proteins in A. ferrooxidans cells attached to chalcopyrite. In this preliminary work, it was compared the periplasmic protein profiles of A. ferrooxidans planktonic and attached cells after exposure to chalcopyrite for 2 hours. The bacterial response to chalcopyrite was investigated by a proteomic approach (two- dimensional gel electrophoresis and mass spectrometry). Four proteins differentially expressed between planktonic and attached cells after exposure to chalcopyrite were identified. Two of these proteins, repressed in chalcopyrite- attached cells, were both identified as superoxide dismutase, whereas the single strand binding protein (SSB) and the PspA/IM30 protein were induced. These results showed that A. ferrooxidans chalcopyrite- attached and planktonic cells show differential expression of the periplasmic proteins and that a proteomic approach can provide a valuable tool to detect proteins related to the A. ferrooxidans response to attachment to the mineral substrates.

Abstract: Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile capable of catalyzing the oxidation of ferrous iron and sulfur reducing compounds. During the dissolution of ores A. ferrooxidans adheres to the solid substrate by hydrophobic and hydrophilic interactions and also extracellular polymeric substances may be involved. Nevertheless, many of these agents that participate in the process are unknown. Therefore, the identification and characterization of the extracellular proteome of A. ferrooxidans was addressed during this work. First, by searching the genome of A. ferrooxidans ATCC 23270 the putative genes coding for proteins present in most of the different secretion systems (type I, II, IV, V systems and the Usher pathway) were found. Second, when the secretion signal prediction server SubCel 1.0 was used, 26.7 % of the total open reading frames showed possible signals that would allow these proteins to leave the cytoplasm towards the internal and outer membranes, the periplasm, or the extracellular milieu. By using 2D-NEPHGE and MS sequencing we identified the eleven most abundant proteins present in the extracellular fraction. Proteins related to the transport and binding of solutes and the folding of proteins, among others were identified. Along with the non-classical pathway, the protein secretion systems identified could constitute the secretion machinery for the proteins found in the extracellular sub-proteome of A. ferrooxidans that as a whole constitute its secretome. Further studies of the extracellular proteins from this biomining microorganism will be important to find out their possible role if any, during bacteria-mineral interactions.

Abstract: The genome sequences from two strains of the acidophilic, autotrophic, chemolithotrophic proteobacterium A. ferrooxidans are available from genome databases. Bioinformatic sequence comparison revealed the existence in one strain of a putative integrative conjugative element (ICE), containing an entire set of clustered tRNA genes. ICE is missing in the other strain, suggesting that this element as well as the tRNA genes cluster is dispensable for the bacterium. Bioinformatic predictions suggest that the tRNA genes cluster might mainly contribute to the translation of ICE encoded genes.

Abstract: Bioinformatic approaches are described for the discovery of small regulatory RNAs (srRNAs) in the biomining microorganism Acidithiobacillus ferrooxidans. Intergenic regions of the annotated genome were extracted and computationally searched for srRNAs. Candidate srRNAs that were associated with predicted sigma 70 promoters and/or rho-independent terminators were chosen for further study. Experimental validation is presented for 6S srRNA and frr. srRNAs are known to control gene expression in a wide variety of microorganisms, usually at the post-transcriptional level, by acting as antisense RNAs that bind targeted mRNAs or by interacting with regulatory proteins. srRNAs are involved in the regulation of a large variety of processes. Frr is an RNA antisense to fur; the latter encodes a global regulator involved the control of a large number of genes involved in iron uptake and homeostasis. Because of the widespread occurrence and extensive repertoire of regulatory functions afforded by srRNAs, it is expected that their discovery functional analysis in biomining microorganisms will contribute to improving our understanding of the microbiology of bioleaching processes.

Abstract: Efficient bioleaching requires adequate access to oxygen to drive the biochemical reactions that underpin iron and sulfur oxidation and ultimately copper solubilization. However, microaerophilic or anaerobic conditions may occur in certain parts of the heap, especially in areas of intense microbial activity or in biofilms where oxygen gradients occur. Microaerophilic conditions have also been detected in pristine acidic environments and in abandoned bioleaching operations. An important microorganism in bioleaching at ambient temperatures is the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans. In addition to its well established role in oxidative processes, it has been shown to be capable of reducing metals such as iron and sulfur, and the products of these reactions may promote passivation of mineral surfaces and impede efficient solubilization of copper. In an effort to advance our understanding of the genetic and physiological basis of anaerobic metabolism, gene clusters controlled by the master anaerobic transcriptional regulator FNR were predicted in the genome of A. ferrooxidans using bioinformatics techniques. These clusters were found to be associated with energy metabolism, nitrogen fixation and carbohydrate metabolism. The results not only support previous evidence for proposed anaerobic metabolic pathways but also identify new genetic components and pathways that may be important for anaerobic or microaerophilic growth of this microorganism.

Abstract: Acidithiobacillus ferrooxidans is an acidophilic, chemolithotrophic bacterium that can gain energy and electrons by the oxidation of iron. PetI is an operon that encodes electron transport proteins involved in the reverse flow of electrons from iron to the NAD complex during iron oxidation. Although a substantial body of evidence describes the components and pathways for iron oxidation, little is known about their regulation. It is proposed that environmental iron concentrations and oxygen levels could influence the ability of A. ferrooxidans to oxidize iron as an energy source. We report initial investigators into the possibility that the expression of the PetI operon is regulated, inter alia, by the master transcription factor controlling iron uptake and homeostasis (Fur) and anaerobic metabolism (FNR). Potential DNA binding sites (boxes) for both regulators were predicted in the region of the PetI operon promoter and the binding of Fur to its cognate box was demonstrated experimentally. This represents the first attempt to unravel the mechanisms involved in the regulation of iron oxidation.

Abstract: A particularly challenging problem in genome annotation is to attribute function to genes annotated as “hypothetical, no known function”. These typically account for about 40% of all genes regardless of the genome. Some of these are “orphan” genes and are not found in any other genome. Some of these could encode species specific proteins and so are particularly interesting for evaluating novel metabolic potential and for understanding the evolution of genes and genomes. Several similarity and non-similarity bioinformatics tools exist that help predict function of hypotheticals, but none are able to suggest function for more than a few percent and the annotation of the others remains a formidable task. We have developed a bioinformatics tool called AlterORF (www.AlterORF.cl) that is able to identify alternate open reading frames (ORFs) embedded within annotated genes. Analysis of over 2 million genes in over 700 completely sequenced genomes reveals that alternate ORFs of substantial length (potentially encoding 70 amino acids or more) are surprisingly common, especially in G+C rich genomes. During our examination of these alternate ORFs, we uncovered hundreds of examples where the alternate ORF has a significant hit with databases of motifs and domains (e.g. CDD, Pfam) and where the actual annotated gene is described as hypothetical and has no database match. This strongly suggests that the annotated gene has been incorrectly identified and that the alternate ORF is the real gene. We describe the evaluation of the following genomes of bioleaching microorganisms and others that reside in similar ecological niches using AlterORF: Acidithiobacillus ferrooxidans (2 strains), Leptospirillum type II, Methylacidiphilum infernorum, Picrophilus torridus, Sulfolobus acidocaldarius, S. solfataricus, S. tokodaii, Thermodesulfovibrio yellowstonii, Thermoplasma acidophilum and T. volcanium. Examples of novel genes from these microorganisms and their suggested roles in metabolism will be described.