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Arsenopyrite is the most abundant arsenic-bearing sulfide mineral in the lithosphere, usually associated with sulfide gold ores. The recovery of this highly valuable metal is associated with the release of large quantities of soluble arsenic. One way to mitigate the effects of high concentrations of arsenic in solution is to immobilize it as scorodite precipitate, a more stable form. Hence, we addressed the scorodite formation capacity (under mesophilic conditions) of psychrotolerant Acidithiobacillus ferrivorans ACH isolated from the Chilean Altiplano. Bio-oxidation assays were performed with 1% arsenopyrite concentrate as unique energy source and produced solids were evaluated by X-ray diffraction (XRD) and QEMSCAN analysis. Interestingly, the results evidenced scorodite generation as the main sub-product after incubation for 15 days, due to the presence of the microorganism. Moreover, the QEMSCAN analysis support the XRD, detecting a 3.5% increase in scorodite generation by ACH strain and a 18.7% decrease in arsenopyrite matrix, implying an active oxidation. Finally, we presented the first record of arsenopyrite oxidation capacity and the stable scorodite production ability by a member of A. ferrivorans species under mesophilic conditions.

The use of microorganisms in mining processes is a technology widely employed around the world. Leaching bacteria are characterized by having resistance mechanisms for several metals found in their acidic environments, some of which have been partially described in the Acidithiobacillus genus (mainly on ferrooxidans species). However, the response to copper has not been studied in the psychrotolerant Acidithiobacillus ferrivorans strains. Therefore, we propose to elucidate the response mechanisms of A. ferrivorans ACH to high copper concentrations (0&ndash, 800 mM), describing its genetic repertoire and transcriptional regulation. Our results show that A. ferrivorans ACH can grow in up to 400 mM of copper. Moreover, we found the presence of several copper-related makers, belonging to cop and cus systems, as well as rusticyanins and periplasmatic acop protein in the genome. Interestingly, the ACH strain is the only one in which we find three copies of copB and copZ genes. Moreover, transcriptional expression showed an up-regulation response (acop, copZ, cusA, rusA, and rusB) to high copper concentrations. Finally, our results support the important role of these genes in A. ferrivorans copper stress resistance, promoting the use of the ACH strain in industrial leaching under low temperatures, which could decrease the activation times of oxidation processes and the energy costs.

Salmonella comprises over 2500 serotypes and foodborne contamination associated with this pathogen remains an important health concern worldwide. During the last decade, a shift in serotype prevalence has occurred as traditionally less prevalent serotypes are increasing in frequency of infections, especially those related to poultry meat contamination. S. Infantis is one of the major emerging serotypes, and these strains commonly display antimicrobial resistance and can persist despite cleaning protocols. Thus, this work aimed to isolate S. Infantis strains from a poultry meat farm in Santiago, Chile and to characterize genetic variations present in them. We determined their genomic and phenotypic profiles at different points along the production line. The results indicate that the strains encompass 853 polymorphic sites (core-SNPs) with isolates differing from one another by 0–347 core SNPs, suggesting variation among them, however, we found discrete correlations with the source of the sample in the production line. Furthermore, the pan-genome was composed of 4854 total gene clusters of which 2618 (53.9%) corresponds to the core-genome and only 181 (3.7%) are unique genes (those present in one particular strain). This preliminary analysis will enrich the surveillance of Salmonella, yet further studies are required to assess their evolution and phylogeny.

Relatively little is known about the microbial communities present in natural environments that meet physico-chemical conditions for the development of potential leaching microorganisms such as thermal ecosystems from the Chilean Altiplano. Thermophilic leaching enrichments were obtained and identified from a high altitude solfataric pound in Lirima hot springs in the Chilean Altiplano. This ecosystem is characterized by hot underground freshwaters, enriched in sulfur compounds showing pH from neutral to acidic. Microbial diversity has been scarcely explored here, and preliminary results demonstrate that hydrothermal pounds are represented by thermophilic anaerobic and acidophilic taxa. Thermophilic leaching cultures in shake flasks were obtained using ferrous iron and pyrite as energy source. The presence of Bacteria and Archaea in oxidizing enrichments was determined by PCR amplification of 16S rRNA genes. A preliminary analysis of microbial diversity using massive sequencing revealed that Bacteria were more abundant than Archaea in both enrichments. Specifically for the iron-oxidizing culture, the majority of the sequences clustered within the Proteobacteria phylum (79%). Among Proteobacteria, the proportion of Betaproteobacteria (42.2%) and Gammaproteobacteria (21.27%) was much higher than that of Alphaproteobacteria (15.5%). Within the Betaproteobacteria class, the most frequent genus was Leptothrix-like. Similar results were obtained for the pyrite oxidizing culture. Interestingly, this study shows the presence of microorganisms close to the Leptothrix genus under low pH conditions (1.7-2.8) and their capacity to grow at high temperatures with ferrous iron or pyrite as sole energy source

The objective of this study was to compare the public perception in desert and rainy regions of Chile regarding the reuse of treated sewage water. The methodology of this study consisted of applying a survey to the communities of San Pedro de Atacama (desert region) and Hualqui (rainy region) to identify attitudes about the reuse of sewage water. The survey was applied directly to men and women, 18 to 90 years old, who were living in the studied communities. The results indicate that inhabitants of San Pedro de Atacama (desert region) were aware of the state of their water resources, with 86% being aware that there are water shortages during some part of the year. In contrast, only 55% of residents in Hualqui (rainy region) were aware of water shortages. With respect of the reuse of treated sewage water, 47% of respondents in San Pedro de Atacama understood the concept, as compared to 27% in Hualqui. There was more acceptance of using treated sewage water for non-potable purposes than as drinking water.

Lead (Pb) dust exposure can have detrimental environmental and human health effects. Improperly enclosed stockpiles of Pb concentrates can cause dust emissions, subsequent pollution of the soil and environmental risk. The aim of this work was to study Pb form, distribution and immobilization (by using eggshell and seashell) in an industrial arid soil near a storage area of Pb mineral concentrates in northern Chile. High amounts of sulfur (S; 9900 mg kg−1) and Pb (6530 mg kg−1) were found in the polluted soil. The energy-dispersive X-ray spectroscopy analysis revealed a lead sulfide (PbS: galena). Metallic Pb particles, which were between 41 and 46 µm, were identified in the soil. After eggshell and seashell (20%) were applied, the soil pH increased from 6.0 to 7.84 and 8.07, respectively. In the studied soil, the leaching test showed a 59 mg L−1 average Pb extractable concentration. After 240 days, extractable Pb by toxicity characteristics leaching procedure decreased to 4.79 mg L−1 (93.3%) with the application of seashell at 20% compared with a decrease of 33.33 mg L−1 (53.6%) using eggshell. Pb in the polluted soil was mainly found in the exchangeable fraction (66%), followed by the reducible (24%), residual (7%) and oxidizable (6%) fractions. According to the risk assessment code, the contaminated soil before treatment was classified as very high risk. Adding eggshell (20%) and seashell (20%) decreased the exchangeable fractions to 39 and 35%, respectively. Applying these liming materials achieved Pb immobilization in the soil, but the soil remained in the high environmental risk category. We conclude that the application of seashell waste, resulting from high aquaculture activity, opens an interesting window to the treatment of contaminated arid soils.

Indexación: Web of Science; Scopus. The Atacama Desert hosts diverse ecosystems including salt flats and shallow Andean lakes. Several heavy metals are found in the Atacama Desert, and microorganisms growing in this environment show varying levels of resistance/tolerance to copper, tellurium, and arsenic, among others. Herein, we report the genome sequence and comparative genomic analysis of a new Exiguobacterium strain, sp. SH31, isolated from an altiplanic shallow athalassohaline lake. Exiguobacterium sp. SH31 belongs to the phylogenetic Group II and its closest relative is Exiguobacterium sp. S17, isolated from the Argentinian Altiplano (95% average nucleotide identity). Strain SH31 encodes a wide repertoire of proteins required for cadmium, copper, mercury, tellurium, chromium, and arsenic resistance. Of the 34 Exiguobacterium genomes that were inspected, only isolates SH31 and S17 encode the arsenic efflux pump Acr3. Strain SH31 was able to grow in up to 10 mM arsenite and 100 mM arsenate, indicating that it is arsenic resistant. Further, expression of the ars operon and acr3 was strongly induced in response to both toxics, suggesting that the arsenic efflux pump Acr3 mediates arsenic resistance in Exiguobacterium sp. SH31. http://journal.frontiersin.org/article/10.3389/fmicb.2017.00456/full

The occurrence of microorganisms from the Vibrio genus in saline lakes from northern Chile had been evidenced using Numerical Taxonomy decades before and, more recently, by phylogenetic analyses of environmental samples and isolates. Most of the knowledge about this genus came from marine isolates and showed temperature and salinity to be integral agents in shaping the niche of the Vibrio populations. The stress tolerance phenotypes of Vibrio sp. Teb5a1 isolated from Salar de Atacama was investigated. It was able to grow without NaCl and tolerated up to 100 g/L of the salt. Furthermore, it grew between 17° and 49°C (optimum 30°C) in the absence of NaCl, and the range was expanded into cold temperature (4-49°C) in the presence of the salt. Other additional adaptive strategies were observed in response to the osmotic stress: pigment production, identified as the known antibacterial prodigiosin, swimming and swarming motility and synthesis of a polar flagellum. It is possible to infer that environmental congruence might explain the cellular phenotypes observed in Vibrio sp. considering that coupling between temperature and salinity tolerance, the production of antibacterial agents at higher temperatures, flagellation and motility increase the chance of Vibrio sp. to survive in salty environments with high daily temperature swings and UV radiation.

Mesophilic iron and sulfur-oxidizing acidophiles are commonly used for the extraction of base metals from low-grade sulfide ores in some copper Chilean mines. However, relatively little is known about their activities in cold environments. Some natural ecosystems present in the Andes Mountains, such as the Chilean Altiplano, meet environmental conditions for the growth of psychrotolerant leaching microorganisms. In this work, we obtained enrichment cultures of iron- and sulfur-oxidizing microorganisms from an acid river in the Chilean Altiplano. Molecular identification was performed using PCR products of bacterial 16S rRNA clone libraries, and the sequences analysis revealed the presence of a microorganism related to the recently described psychrotolerantAcidithiobacillus ferrivorans. TheAcidithiobacillusstrain was able to grow at temperatures ranging between 4 and 30°C, and pH values ranged between 1.7 and 2.5. According to the energy sources, this microorganism was able to grow using ferric iron, sulfur, thiosulfate and tetrathionate. Optimal growth was observed in presence of ferric ion, where the culture reached a potential redox value of 600 mV and a cellular number of 3×107cells/mL. Molecular analysis of variants of gene encoding for rusticyanin showed thatrusBgene was amplified fromA. ferrivoransstrain and no PCR product was obtained for therusAgene. Our description is consistent with data previously reported forA. ferrivoransstrains. Finally, results of this study highlight the importance and potential of novel native bacterial species proficient in mineral oxidation under low-temperature conditions.

Two cell lines derived from a single Trypanosoma cruzi clone by long-term passaging generated a highly virulent (C8C3hvir) and a low virulent (C8C3lvir) cell line. The C8C3hvir cell line was highly infective and lethal to Balb/c mice, and the C8C3lvir cell line was three- to five-fold less infective to mouse cardiomyocytes than C8C3hvir. The highly virulent T. cruzi cell line abundantly expressed the major cysteine proteinase cruzipain (Czp), complement regulatory protein (CRP) and trans-sialidase (TS), all of which are known to act as virulence factors in this parasite. The in vitro invasion capacity and in vivo Balb/c mouse infectiveness of the highly virulent strain was strongly reduced by pre-treatment with antisense oligonucleotides targeting TS or CRP or with E64d. Based on these results, we conclude that decreased levels of TS, CRP and Czp expression could contribute to loss of T. cruzi trypomastigote virulence.

Summary The bioleaching of metal sulfide has developed into a very important industrial process and understanding the microbial dynamic is key to advancing commer- cial bioleaching operations. Here we report the first quantitative description of the dynamic of active com- munities in an industrial bioleaching heap. Acidithio- bacillus ferrooxidans was the most abundant during the first part of the leaching cycle, while the abun- dance of Leptospirillum ferriphilum and Ferroplasma acidiphilum increased with age of the heap. Acidithio- bacillus thiooxidans kept constant throughout the leaching cycle, and Firmicutes group showed a low and a patchy distribution in the heap. The Acidiphilium-like bacteria reached their highest abun- dance corresponding to the amount of autotrophs. The active microorganisms in the leaching system were determined using two RNA-based sensitive techniques. In most cases, the 16S rRNA copy numbers of At. ferrooxidans, L. ferriphilum, At. thiooxidans and F. acidiphilum, was concomitant with the DNA copy numbers, whereas Acidiphilium-like bacteria and some Firmicutes members did not show a clear correlation between 16S rRNA accumulation and DNA copy numbers. However, the prokaryotic acidophile microarray (PAM) analysis showed active members of Alphaproteobacteria in all samples and of Sulfobacillus genus in older ones. Also, new active groups such as Actinobacteria and Acidobacterium genus were detected by PAM. The results suggest that changes during the leaching cycle in chemical and physical conditions, such as pH and Fe 3+ /Fe 2+ ion rate, are primary factors shaping the microbial dynamic in the heap.

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.

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.

During the past few decades the microbial bioleaching of metallic sulphide ores has become a very important process utilized by the mining industry. The biodiversity studies of industrial systems have revealed the occurrence of acidophilic microorganisms, forming dynamic communities that include bacteria and archaea. A combination of molecular and cultivation-based approaches has been used during the leaching cycle to understand the key microorganisms present and their function in the Escondida. Sulphide Leach Plant. The global genomic expression in solution samples has been analyzed by random arbitrary primed polymerase chain reaction (RAP-PCR), to pinpoint those microorganisms with active metabolism in the communities present in regions of the heap leach operation of different ages. Four random primers were used and one hundred and twenty RAP-PCR products were cloned, sequenced and compared to databases using the BLAST search algorithm. Approximately 48% of the RAP-PCR products were 16S and 23S rRNA genes, and many of these results are consistent with community analysis carried out on the same heap using 16S rRNA gene libraries and real-time PCR. Interestingly, different microorganisms to those previously detected by 16S rRNA PCR-DGGE, represented by sequences distantly related to the archaea Picrophilus torridus, and Thermoplasma acidophilum sequences, were detected using RNA analysis. Eight different RAP-PCR products shared similarity with functional genes, and sequence analysis revealed the expression of genes associated with transcription (RNA polymerase) and translation (elongation factor and ribosomal protein) components, metabolic function (2-methylisocitrate synthase and pyruvate dehydrogenase), and electron transport (cytochrome). Other genes such as mercuric reductase and ABC transporter ATP-binding proteins were also detected. However, 30% of RAP-PCR products showed no similarity with any sequences currently in the database. This report represents the first transcriptomic study of the microbial community inhabiting an industrial bioleaching system. The data suggest that the functional spectrum of expressed genes was consistent with genomic and proteomic reports from pure cultures and environmental AMD samples containing microorganisms involved in bioleaching processes. Both studies revealed the expression of genes mainly related to energetic metabolisms and stress responses. (C) 2008 Published by Elsevier B.V.

Microbial heap bioleaching is being used as an industrial process to recover copper from low grade ores. It is known that a consortium of different microorganisms participates in this process. Therefore identification and quantification of communities inhabiting heap bioleaching operations is a key step for understanding the dynamics and role of these microorganisms in the process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in this process. To study the microbial population inhabiting a low-grade copper sulphide ore bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences retrieved from genetic libraries showed that the community is mainly composed by microoganisms related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma. Specific primers for real-time PCR determination were designed and tested to amplify each of the sequences obtained by cloning. Standard curves for real time PCR were performed using plasmid DNA from selected clones. This methodology is actually being used to monitor relevant microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.

During the last decades, microbial bioleaching of metallic sulphide ores has become a very important process in the mining industry. The study of biodiversity in those systems has revealed the occurrence of several acidophilic organisms, forming a dynamic community including Bacteria and Archaea. Despite the importance of the heap bioleaching process, the metabolic behaviour of a microbial community in bioleaching systems remains unknown. The role played by members of the community inhabiting a bioheap environment has been estimated by the phenotypic characteristics of pure cultures, such as iron oxidation, sulphur oxidation, resistance to high ion concentration and metal tolerance, among others. The global genomic expression of the community inhabiting an industrial bioheap of low-grade copper sulphide ore at Escondida mine in Chile was investigated by random arbitrary primed polymerase chain reaction (RAP-PCR). Three random primers were used and twenty differentially expressed bands were cloned and sequenced. Several sequences were related to 16S rRNA of members of the microbial community; two sequences were related to the α subunit of pyruvate dehydrogenase from Acidithiobacillus ferrooxidans. The expression levels of the pyruvate dehydrogenase gene in samples from an industrial bioleaching operation at different stages were checked by quantitative real-time PCR.

The sulfur oxygenase reductase (Sor) catalyzes the oxygen dependent disproportionation of elemental sulfur, producing sulfite, thiosulfate and sulfide. Being considered an "archaeal like" enzyme, it is also encoded in the genomes of some acidophilic leaching bacteria such as Acidithiobacillus caldus, Acidithiobacillus thiooxidans, Acidithiobacillus ferrivorans and Sulfobacillus thermosulfidooxidans, among others. We measured Sor activity in crude extracts from Sb. thermosulfidooxidans DSM 9293(T). The optimum temperature for its oxygenase activity was achieved at 75 °C, confirming the "thermophilic" nature of this enzyme. Additionally, a search for genes probably involved in sulfur metabolism in the genome sequence of Sb. thermosulfidooxidans DSM 9293(T) was done. Interestingly, no sox genes were found. Two sor genes, a complete heterodisulfidereductase (hdr) gene cluster, three tetrathionate hydrolase (tth) genes, three sulfide quinonereductase (sqr), as well as the doxD component of a thiosulfate quinonereductase (tqo) were found. Seven At. caldus strains were tested for Sor activity, which was not detected in any of them. We provide evidence that an earlier reported Sor activity from At. caldus S1 and S2 strains most likely was due to the presence of a Sulfobacillus contaminant.

It has been postulated that inorganic polyphosphate (polyP) and transport of metal–phosphate complexes could participate in heavy metal tolerance in some bacteria. To study if such a system exists in archaea, the presence of polyP was determined by the electron energy loss spectroscopy (EELS) procedure and quantified by using specific enzymic methods inSulfolobus acidocaldarius,Sulfolobus metallicusandSulfolobus solfataricus. All three micro-organisms synthesized polyP during growth, but onlyS. metallicusgreatly accumulated polyP granules. The differences in the capacity to accumulate polyP between these archaea may reflect adaptive responses to their natural environment. Thus,S. metallicuscould grow in and tolerate up to 200 mM copper sulfate, with a concomitant decrease in its polyP levels with increasing copper concentrations. On the other hand,S. solfataricuscould not grow in or tolerate more than 1–5 mM copper sulfate, most likely due to its low levels of polyP. ShiftingS. metallicuscells to copper sulfate concentrations up to 100 mM led to a rapid increase in their exopolyphosphatase (PPX) activity which was concomitant in time with a decrease in their polyP levels and a stimulation of phosphate efflux. Furthermore, copper in the range of 10 μM greatly stimulated PPX activity in cell-free extracts fromS. metallicus. The results strongly suggest that a metal tolerance mechanism mediated through polyP is functional in members of the genusSulfolobus. This ability to accumulate and hydrolyse polyP may play an important role not only in the survival of these micro-organisms in sulfidic mineral environments containing high toxic metals concentrations, but also in their applications in biomining.

Microorganisms are used to aid the extraction of valuable metals from low-grade sulfide ores in mines worldwide, but relatively little is known about this process in cold environments. This study comprises a preliminary analysis of the bacterial diversity of the polyextremophilic acid River Aroma located in the Chilean Altiplano, and revealed that Betaproteobacteria was the most dominant bacterial group (Gallionella-like and Thiobacillus-like). Taxa characteristic of leaching environments, such Acidithiobacillus and Leptospirillum, were detected at low abundances. Also, bacteria not associated with extremely acidic, metal-rich environments were found. After enrichment in iron- and sulfur-oxidizing media, we isolated and identified a novel psychrotolerant Acidithiobacillus ferrivorans strain ACH. This strain can grow using ferrous iron, sulfur, thiosulfate, tetrathionate and pyrite, as energy sources. Optimal growth was observed in the presence of pyrite, where cultures reached a cell number of 6.5 · 10(7) cells mL(-1). Planktonic cells grown with pyrite showed the presence of extracellular polymeric substances (10 °C and 28 °C), and a high density of cells attached to pyrite grains were observed at 10 °C by electron microscopy. The attachment of cells to pyrite coupons and the presence of capsular polysaccharides were visualized by using epifluorescence microscopy, through nucleic acid and lectin staining with Syto(®)9 and TRITC-Con A, respectively. Interestingly, we observed high cell adhesion including the formation of microcolonies within 21 days of incubation at 4 °C, which was correlated with a clear induction of capsular polysaccharides production. Our data suggests that attachment to pyrite is not temperature-dependent in At. ferrivorans ACH. The results of this study highlight the potential of this novel psychrotolerant strain in oxidation and attachment to minerals under low-temperature conditions.

Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya , Archaea , and several bacteria. To further study the metabolism of polyP in Archaea , we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162–5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.

Summary The bioleaching of metal sulfide has developed into a very important industrial process and understanding the microbial dynamic is key to advancing commercial bioleaching operations. Here we report the first quantitative description of the dynamic of active communities in an industrial bioleaching heap. Acidithiobacillus ferrooxidans was the most abundant during the first part of the leaching cycle, while the abundance of Leptospirillum ferriphilum and Ferroplasma acidiphilum increased with age of the heap. Acidithiobacillus thiooxidans kept constant throughout the leaching cycle, and Firmicutes group showed a low and a patchy distribution in the heap. The Acidiphilium‐like bacteria reached their highest abundance corresponding to the amount of autotrophs. The active microorganisms in the leaching system were determined using two RNA‐based sensitive techniques. In most cases, the 16S rRNA copy numbers of At. ferrooxidans, L. ferriphilum, At. thiooxidans and F. acidiphilum, was concomitant with the DNA copy numbers, whereas Acidiphilium‐like bacteria and some Firmicutes members did not show a clear correlation between 16S rRNA accumulation and DNA copy numbers. However, the prokaryotic acidophile microarray (PAM) analysis showed active members of Alphaproteobacteria in all samples and of Sulfobacillus genus in older ones. Also, new active groups such as Actinobacteria and Acidobacterium genus were detected by PAM. The results suggest that changes during the leaching cycle in chemical and physical conditions, such as pH and Fe3+/Fe2+ ion rate, are primary factors shaping the microbial dynamic in the heap.

Analyses of clone libraries from water and sediments of different sites from Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano, revealed the presence of five unique clusters of uncultured Archaea that have not been previously reported or specifically assigned. These sequences were distantly related (83-96% sequence identity) to a limited number of other clone sequences and revealed no identity to cultured Archaea. The abundance of Archaea and Bacteria was estimated using qPCR and community composition was examined through the construction of clone libraries of archaeal 16S rRNA gene. Archaea were found to be dominant over Bacteria in sediments from two saline sites (sites H4: 6.31 x 10(4) and site H6: 1.37 x 10(4) microS cm(-1)) and in one of the water samples (freshwater from site H0: 607 muS cm(-1)). Euryarchaeotal sequences were more abundant than crenarchaeotal sequences. Many of the clone sequences (52%) were similar to uncultured archaeal groups found in marine ecosystems having identity values between 99% and 97%. A major fraction of the sequences (40%) were members of Methanobacteria, while others were included in the Marine Benthic Groups B and D, the Miscellaneous Crenarchaeotic Group, the Terrestrial Miscellaneous Euryarchaeotal Group, Marine Group I and Halobacteria. The presence of uncultured archaeal groups in Salar de Huasco extends their known distribution in inland waters, providing new clues about their possible function in the environment.