Advanced Materials Research Vols. 71-73

Abstract: Advances in DNA sequencing technologies have promoted the use of genome information as a key component in most of biological studies. In the case of biomining microorganisms, partial and complete genome information has provided critical clues for unraveling their physiology. This information has also provided genetic material for the generation of functional and biodiversity directed markers. In this work, we present a compilation of the most relevant findings based on genomic analysis of the model organism Acidithiobacillus ferrooxidans ATCC23270 that were extended and compared to the recently sequenced genomes of Acithiobacillus thiooxidans and Acidithiobacillus caldus. The phylogenetic relatedness of these three microorganisms has permitted the identification of a shared genomic core that encodes the common metabolic and regulatory functions critical for survival and proliferation in extremely acidic environments. We also identified microorganism-specific genomic components that are predicted to be responsible for the metabolic speciation of these microorganisms. Finally, we evaluated the impact of lateral gene transfer on these genomes in order to determine the functional contribution of this phenomenon to the fitness of these microbial representatives. The information gathered by genomic analyses in the Acidithiobacillus genus will be presented in conjunction with other biomining genomic and metagenomic information in order to generate a more comprehensive picture of the biodiversity, metabolism and ecophysiology of the bioleaching niche.

Abstract: Previous physiological studies of the genus Ferroplasma have indicated that these microorganisms are capable of fixing CO2 in the presence of ferrous iron and low concentrations of yeast extract. Analysis of the gene complement of Ferroplasma acidarmanus fer1 and two partial genomes of Ferroplasma type I and II derived from the Iron Mountain acid mine drainage metagenome revealed the absence of several functional marker genes encoding key enzymes of three know alternative CO2 fixation routes present in archaea, i.e. the 3-hydroxypropionate cycle, the Ljungdahl–Wood pathway and the reverse TCA cycle. It is thus intriguing how these chemoautotrophic archaeal species deal with their requirements for carbon and suggests that they might have a distinct CO2 fixation route, as yet unreported. Using comparative genomics and metabolic reconstruction strategies, a putative pathway was detected for C1 fixation consisting of four main steps: 1) conversion of carbon monoxide to carbon dioxide with gain of energy and/or 2) reduction of carbon dioxide to formate, 3) incorporation of formate to tetrahydrofolate and 4) donation of the carbon moiety of tetrahydrofolate to glycine to produce serine. Steps 1 to 3 involve enzymes that correspond to some of the Ljungdahl–Wood pathway proteins, whereas step 4 resembles the well known “serine cycle”, utilized by methylotrophic microorganisms for formaldehyde fixation. Thus, this chimaeric pathway might represent the missing carbon fixation route in Ferroplasmatales. Herein, we discuss the implications of these findings in the context of central carbon metabolism requirements for biomass production in acidic environments.

Abstract: Acidithiobacillus ferrooxidans, A. thiooxidans, and A. caldus are acidophilic Gram-negative -proteobacteria involved in the bioleaching of metal sulfides. Bacterial attachment to mineral surface and biofilm development play a pivotal role in this process. Therefore, the understanding of biofilm formation has relevance to the design of biological strategies to improve the efficiency of bioleaching processes. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in biomining bacteria. In many bacteria, the intracellular level of c-di-GMP molecules regulates the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development. Thus, we recently started the study of c-di-GMP pathway in biomining bacteria. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). So far, two kinds of effectors have been identified, including three protein families (pilZ, PleD and FleQ) and a conserved RNA domain (GEMM) which acts as a riboswitch. We previously reported the existence of different molecular players involved in c-di-GMP pathway in A. ferrooxidans ATCC 23270. Here, we expanded our work to other Acidithioibacillus species: A. thiooxidans ATCC 19377 and A. caldus ATCC 51756. In both, we identified several putative-ORFs encoding DGC, PDE and effector proteins. By using total RNA extracted from A. ferrooxidans and A. caldus cells in RT-PCR and qPCR experiments, we demonstrated that these genes are expressed. In addition, we characterized the presence of c-di-GMP in A. ferrooxidans ATCC 23270 and A. caldus ATCC 51756 cell extracts. Taken together, these results strongly suggest that A. ferrooxidans, A. caldus and A. thiooxidans possess functional c-di-GMP pathways. As it occurs in other Gram-negative bacteria, this pathway should be involved in the regulation of the planktonic/biofilm switch. In the future, we have to integrate this new biological dimension to improve the biological understanding of bioleaching.

Abstract: Heap bioleaching is presently the most successful technology for copper extraction from low-grade sulfide ores. Metabolic behaviors and adaptation mechanisms of microbial communities to this system remain unknown. A combination of molecular approaches has been used to determine the key microorganisms in the Escondida Sulphide Heap. This work reports the global gene expression in industrial bioleaching samples by using a specific DNA microarray of Leptospirillum ferrooxidans to better understand the metabolic functions displayed by Leptospirillum spp. iron oxidizers as mineral leaching proceeds. Quantitative real-time PCR analysis showed low cell numbers of Leptospirillum species during the first part of the irrigation period, however, it became the most abundant and active in the second part of the cycle. Transcriptome analysis by DNA microarray could provide specifics transcription patterns in the different stages of the industrial process. We studied the gene expression from bioleaching heap samples with variable Leptospirillum cell numbers, operation times and with different chemical and physical conditions. The results showed induction of genes involved in energetic metabolism, electron transport, ribosomal proteins, DNA repair and response to heavy metals when Leptospirillum species were most abundant and active. We suggest that up- and down-regulation of specifics genes could be used as indicators of physiological responses to operational conditions in the bioleaching systems.

Abstract: A strategy for the monitoring and control of genetic expression in an industrial bioleaching process of copper sulphide minerals is developed in order to understand more fully this process and investigate optimization possibilities. The aim of this research is to find a group of housekeeping genes to normalize the genetic expression data associated to the metabolic functions from industrial bioleaching samples obtained through specific real-time PCR. The data includes the quantification of the previously identified genes in the industrial process in samples from different strips (heap sections) and times. Two studies were carried out, one with the gene expression data associated to each species and the other with the expression data from all the genes detected in the industrial process, during a period of 18 months, using the VBA applet geNorm [1]. The first analysis showed that for the archaea Ferroplasma acidiphilum, the gene with the most stable expression codifies to the ribosomal protein S4 (rpS4) and secondly the mercury reductase gene (merA). In the case of Acidithiobacillus ferrooxidans DM and D2 strains, genes merA and pyruvate dehydrogenase (pdhA) presented similar factors of stability. In the community analysis it was concluded that the gene with the most stable expression was pdhA of A.ferrooxidans. A new analysis was done including other genes using geNorm to define the most stable genes for the whole community. In the community analysis it was concluded that the gene with the most stable expression was pdhA of A.ferrooxidans and merA of F.acidiphilum.

Abstract: The composition and distribution of elements within cells of two species of Fe and S oxidising microbes utilising S, Fe2+ or chalcopyrite (CuFeS2) as an energy source were compared to determine possible sites of oxidation and function of intracellular granules. The bacterium Acidithiobacillus ferrooxidans and the archaeon Metallosphaera hakonensis were examined using energy filtered transmission electron microscopy (EFTEM), TEM energy dispersive X-ray spectroscopy (EDS), Scanning TEM (STEM) and electron energy-loss spectroscopy (EELS). Both species have intracellular granules and we show that these store Fe, S and P. The microbes slowly lost Fe from granules when switched to an Fe-free medium. EELS showed that the Fe in the granules of both species was consistent with Fe3+. Both microbes sometimes contain Cu and Si on their walls and intracellularly. Si concentrations have been shown to affect bioleach performance, so element deposition on the microbial catalyst may be a reason for this. Bands of Fe and S were present close to, or in, the cell membrane of M. hakonensis, as might be expected for the site of oxidation, and S also occurred throughout the cytoplasm. These are the first element maps of M. hakonensis, and these early results demonstrate that advanced characterisation and microanalysis techniques can provide insights into microbial processes involved in bioleaching.

Abstract: The elemental sulfur oxidising enzyme Sulfur Oxygenase Reductase (SOR) is very well investigated in acidothermophilic archaea, such as Acidianus brierleyi and Sulfolobus metallicus. In contrast, not much is known about the biochemistry of elemental sulfur oxidation in acidophilic bacteria. Recently, however, the SOR-encoding gene has been found also in a bacterial strain closely related to the moderate thermophile Acidithiobacillus caldus. Confusingly, for the latter species, also the involvement of the SOX system as well as thiosulfate:quinone oxidoreductase (TQO) and tetrathionate hydrolase (TTH) in sulfur compound oxidation has been proposed based on genome analysis. In this study, we have detected the sor-gene in other Acidithiobacillus caldus-like strains, isolated from various bioleaching habitats, indicating that SOR plays an important role in sulfur oxidation in this species. Based on sequence comparison, the new bacterial sor-genes are closely related and distant from the known archaeal sequences as well as from the SOR found in the neutrophilic bacterium Aquifex aeolicus. In addition, SOR activity has been detected in crude cell extracts from all Acidithiobacillus caldus-like strains tested. The enzyme is truly thermophilic as highest activities were achieved at 65 °C, which is far beyond the growth optimum of Acidithiobacillus caldus. This finding may give rise to the question whether the presence of SOR in Acidithiobacillus caldus is only relevant while growing at elevated temperatures. Currently, experiments are performed for testing this hypothesis (comparing growth and enzyme activities at 30 vs. 45 °C).

Abstract: It is mysterious that, when A. ferrooxidans ATCC 23270 cells grow on elemental sulfur, they have high iron oxidase activity comparable to that of iron-grown cells as well as high activities of sulfide:ferric ion oxidoreductase (SFORase) and tetrathionate hydrolase. To clarify this interesting phenomenon, cytochrome c and tetrathionate hydrolase were purified from sulfur-grown A. ferrooxidans cells using ammonium sulfate precipitation, Phenyl column chromatography, and SuperdexTM 75 and Sephadex G-100 size exclusion column chromatographies. The purified cytochrome c was reduced by tetrathionate in the presence of purified tetrathionate hydrolase, but not in the absence of the enzyme. When the partially purified cytochrome c fraction containing aa3-type cytochrome oxidase was used, both cytochrome c and aa3-type cytochrome oxidase were reduced by tetrathionate in the presence of purified tetrathionate hydrolase. These results indicate that tetrathionate in the presence of tetrathionate hydrolase can reduce iron oxidase enzyme system containing cytochrome c and aa3-type cytochrome oxidase as tetrathionate hydrolase decomposes tetrathionate to produce thiosulfate, elemental sulfur, and sulfate; and the formed thiosulfate can chemically reduce cytochrome c and Fe3+.