Molecular Hydrogen Impact on Chalcopyrite Bioleaching by the Extremely Thermoacidophilic Archaeon Metallosphaera sedula
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ABSTRACT:
ORGANISM(S): Metallosphaera sedula
SUBMITTER: Kathryne Auernik
PROVIDER: E-GEOD-20554 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:(from abstract): Iron oxidation is a desirable trait of biomining microorganisms, although the mechanism is not well-understood in extreme thermoacidophiles. The complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ~2300 ORFs) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins (in)directly involved with bioleaching. As expected, the M. sedula genome encodes genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, could also be identified. These iron-oxidizing components are missing from genomes of non-leaching Sulfolobales like Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole genome transcriptional response analysis showed that 88 ORFs were up-regulated 2-fold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized. Dye flip of Mse cells includes two samples, yeast exact (YE) and yeast extract + ferrous sulfate (YEF). The first slide, Y3F5, has YE RNA labeled with cy3 and YEF RNA labeled with cy5, while the second slide, F3Y5, has YEF RNA labeled with cy3 and YE RNA labeled with cy5. YE serves as the reference condition, with the expectation that ORFs involved with Fe2+ oxidation (and SO4 metabolism) will be upregulated on YEF (log2 fold change of YE-YEF < -1).
2008-05-02 | E-GEOD-11296 | biostudies-arrayexpress
Project description:To study mixotrophy, it is desirable to have an organism capable of growth in the presence and absence of both organic and inorganic carbon sources, as well as organic and inorganic energy sources. Metallosphaera sedula is an extremely thermoacidophilic archaeon which has been shown to grow in the presence of inorganic carbon and energy source supplements (autotrophy), organic carbon and energy source supplements (heterotrophy), and in the presence of organic carbon and inorganic energy source supplements. The recent elucidation of M. sedulaâ??s inorganic carbon fixation cycle and its genome sequence further facilitate its use in mixotrophic studies. In this study, we grow M. sedula heterotrophically in the presence of organic carbon and energy sources (0.1% tryptone), autotrophically in the presence of inorganic carbon and energy sources (H2 + CO2), and â??mixotrophicallyâ?? in the presence of both organic and inorganic carbon and energy sources (0.1% tryptone + H2 + CO2 ) to characterize the nature of mixotrophy exhibited. Two 3 slide loops joined at equivalent conditions (8 slides total) for Mse cells includes 3 conditions tested in duplicate (biological repeats): heterotrophy (H1 and H2), autotrophy (A1 and A2), and mixotrophy (M1 and M2). Half of an RNA sample for one condition was labeled with Cy3 while the other half was labeled with Cy5. The two differently labeled samples were run on different slides. Each probe is spotted on each slide 5 times (5 replicates; spot intensities for all replicates on slide provided in associated raw data file).
2010-06-20 | E-GEOD-14978 | biostudies-arrayexpress
Project description:Abstract: The crenarchaeal order Sulfolobales collectively contains at least five major terminal oxidase complexes. Based on genome sequence information, all five complexes are found only in Metallosphaera sedula and Sulfolobus tokodaii, the two sequenced Sulfolobales capable of iron oxidization. While specific respiratory complexes in certain Sulfolobales have been characterized previously as proton pumps for maintaining intracellular pH and generating proton motive force (pmf), their contribution to sulfur and iron biooxidation has not been considered. For M. sedula growing in the presence of ferrous iron and reduced inorganic sulfur compounds (RISCs), global transcriptional analysis was used to track the response of specific genes associated with these complexes, as well as other known and putative respiratory electron transport chain elements. ORFs from all five terminal oxidase or bc1-like complexes were stimulated on one or more conditions tested. Components of the fox (Msed0467-0489) and soxNL-cbsABA (Msed0500-0505) terminal/quinol oxidase clusters were triggered by ferrous iron, while the soxABCDD' terminal oxidase cluster (Msed0285-0291) were induced by tetrathionate and S°. Chemolithotrophic electron transport elements, including a putative tetrathionate hydrolase (Msed0804), a novel polysulfide/sulfur/DMSO reductase-like complex (Msed0812-0818), and a novel heterodisulfide reductase-like complex (Msed1542-1550), were also stimulated by RISCs. Furthermore, several hypothetical proteins were found to have strong responses to ferrous iron or RISCs, suggesting additional candidates in iron or sulfur oxidation-related pathways. From this analysis, a comprehensive model for electron transport in M. sedula could be proposed as the basis for examining specific details of iron and sulfur oxidation in this bioleaching archaeon. 5-slide loop of Mse cells includes 5 conditions tested: yeast exact (Y), yeast extract + ferrous sulfate (YFS), yeast extract + potassium sulfate (YKS), yeast extract + potassium tetrathionate (YKT), and yeast extract + elemental sulfur (YS). Half of an RNA sample for one condition was labeled with Cy3 while the other half was labeled with Cy5. The two differently labeled samples were run on different slides. Each probe is spotted on each slide 5 times (5 replicates; spot intensities for all replicates on slide provided in associated raw data file).
2008-10-22 | E-GEOD-12044 | biostudies-arrayexpress
Project description:Metallosphaera sedula is an extremely thermoacidophilic archaeon that grows heterotrophically on peptides, and chemolithoautotrophically on hydrogen, sulfur, or reduced metals as energy sources. During autotrophic growth, carbon dioxide is incorporated into cellular carbon via the 3-hydroxypropionate /4-hydroxybutyrate cycle (3HP/4HB). To date, all of the steps in the pathway have been connected to enzymes encoded in specific ORFs, except for the one responsible for ligation of coenzyme A (CoA) to 4-hydroxybutyrate (4HB). While several candidates for this step have been identified through bioinformatic analysis of the M. sedula genome, none have been shown to catalyze this biotransformation. Transcriptomic analysis of cells grown under strict H2-CO2 autotrophy was used elucidate additional candidate genes involved in carbon fixation and identify the genes which encode for 4HB-CoA synthetase. Three slide loop for Mse cells includes 3 conditions tested in duplicate (biological repeats from tandem fermentors): autotrophic carbon limited (ACL), autotrophic carbon rich (ACR), and heterotrophic (HTR). Half of an RNA sample for one condition was labeled with Cy3 while the other half was labeled with Cy5. The two differently labeled samples were run on different slides. Each probe is spotted on each slide 5 times (5 replicates; spot intensities for all replicates on slide provided in associated raw data file).
2013-03-05 | E-GEOD-39944 | biostudies-arrayexpress
Project description:Thermoacidophilic archaea are found in heavy metal-rich environments and, in some cases, these microorganisms are causative agents of metal mobilization through cellular processes related to their bioenergetics. Given the nature of their habitats, these microorganisms must deal with the potentially toxic effect of heavy metals. Here, we show that two thermoacidophilic Metallosphaera species with nearly identical (99.99%) genomes differed significantly in their sensitivity and reactivity to uranium. M. prunae, isolated from a smoldering heap on a uranium mine in Thuringen, Germany, could be viewed as a M-bM-^@M-^\spontaneous mutantM-bM-^@M-^] of M. sedula, an isolate from Pisciarelli solfatara near Naples, Italy. M. prunae tolerated U3O8 and U(VI) to a much greater extent than M. sedula. Within 15 minutes following exposure to M-bM-^@M-^\U(VI) shockM-bM-^@M-^], M. sedula, and not M. prunae, exhibited transcriptomic features associated with severe stress response. Furthermore, within 15 minutes post-U(VI) shock, M. prunae, and not M. sedula, showed evidence of substantial degradation of cellular RNA. This suggested that transcriptional and translational processes were aborted as a dynamic mechanism for resisting U toxicity; by 60 minutes post-U(VI) shock, RNA integrity in M. prunae recovered, and known modes for heavy metal resistance were activated. In addition, M. sedula rapidly oxidized solid U3O8 to soluble U(VI) for bioenergetic purposes, a chemolithoautotrophic feature not previously reported. M. prunae, however, did not solubilize solid U3O8 to any significant extent, thereby not exacerbating U(VI) toxicity. These results point to uranium extremophily as an adaptive, rather than intrinsic, feature for Metallosphaera species, driven by environmental factors. The study comprises 9 Samples, described in detail below. MprAU_MseAU: Transcriptional analysis of the response of Metallosphaera prunae (Mpr) and Metallosphaera sedula(Mse) to chemolithoautotrophic conditions (0.1 wt% Uranium octaoxide with CO2 supplementation in headspace). This experiment was done to identify the key terminal oxidases which responded to a Uranium oxide while doing inter-species comparison between Mpr and Mse. Transcriptional response of the terminal oxidase clusters proved that certain key genes play a role in the vastly different physiologies of these two species. MprN_MprU60: Transcriptional analysis of the response of Metallosphaera prunae (Mpr) to 60 min of Uranium shock. This experiment was done to analyze the differential transcription of Mpr cells challenged with 1 mM uranyl acetate shock (U shock) compared to normal growth. The Uranium cultures were harvested 60 min after the shock. MprN_MseN: Differential transcription of Metallosphaera species under normal growth conditions. This experiment was done to analyze the differential transcription of Mpr cells compared with Mse cells at mid log phase. MprN_MprU3h: Transcriptional response of Metallosphaera prunae (Mpr) to 3h of Uranium shock compared to normal growth. This experiment was done to analyze the differential transcription of Mpr cells challenged with 1 mM uranyl acetate shock (U shock) . The Uranium cultures were harvested 3 h after the shock. MseN_MseU15: Transcriptional response of Metallosphaera sedula (Mse) to 15 min of Uranium shock. This experiment was done to analyze the differential transcription of Mse cells challenged with 1 mM uranyl acetate shock (U shock) compared to normal growth. The Uranium cultures were harvested 15 min after the shock. MseN_MseU60: Transcriptional response of Metallosphaera sedula to 60 min of Uranium shock. Mse cells were grown upto mid log phase after which the cells were subjected to U shock and harvested 60 min later. Biological repeats were done for both experimental conditions. MseN_MseU3h: Transcriptional response of Metallosphaera sedula (Mse) to 3h of Uranium shock compared to normal growth. This experiment was done to analyze the differential transcription of Mse cells challenged with 1 mM uranyl acetate shock (U shock) . The Uranium cultures were harvested 3 h after the shock. MseU15_MseU60: Transcriptional response of Metallosphaera sedula to 15 min of Uranium shock compared with 60 min of Uranium shock. This experiment was done to analyze the differential transcription of Mse cells challenged with 1 mM uranyl acetate shock (U shock) . The Uranium cultures were harvested 15 min and 60 min after the shock. MprU3h_MseU3h: Differential transcription of Metallosphaera cells under Uranium shock. This experiment was done to analyze the differential transcription of Metallosphaera sedula (Mse) and Metallosphaera prunae (Mpr) challenged with 1 mM uranyl acetate.
2012-09-25 | E-GEOD-40796 | biostudies-arrayexpress
Project description:Phylogenetic, microbiological and comparative genomic analysis was used to examine the diversity among members of the genus Caldicellulosiruptor with an eye towards the capacity of these extremely thermophilic bacteria for degrading the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii (formerly Anaerocellum thermophilum), C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA phylogeny and cross-species DNA-DNA hybridization to a whole genome C. saccharolyticus oligonucleotide microarray. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid pre-treated switchgrass, only C. saccharolyticus, C. besci, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman No. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that species capable of crystalline cellulose hydrolysis also had diverse secretome fingerprints. The two-dimensional secretome of C. saccharolyticus revealed a prominent S-layer protein that appears to be also indicative of highly cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interaction. These growth physiology results were also linked to glycoside hydrolase and carbohydrate-binding module inventories for the seven bacteria, deduced from draft genome sequence information. These preliminary inventories indicated that the absence of a single glycoside hydrolase family and carbohydrate binding motif family appear to be responsible for some Caldicellulosiruptor species’ diminished cellulolytic capabilities. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and is well suited for biomass deconstruction applications. Six dye-flip experiments were conducted using C. saccharolyticus genomic DNA as the reference in each dye-flip, and one of six different Caldicellulosiruptor spp. as a tester in each dye-flip
2010-11-29 | E-GEOD-23606 | biostudies-arrayexpress
Project description:Thermomacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, a M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supra-normal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to up-regulation of 55 genes. Genome re-sequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism or transport. These mutations included 7 non-synonymous substitutions, 4 insertions and 1 deletion. One of the insertion mutations mapped to pseudogene, Msed_1517, and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that include the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula was naturally lacking this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low affinity high velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated spontaneous arsenate resistant mutants derived from CuR1 all underwent mutation in pitA and non-selectively became copper resistant. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. The study comprises 5 samples, described in detail below. WT_CuR1: Differential transcriptional response of Metallosphaera sedula DSM 5348, WT, to the supra-normal copper resistant spontaneous Metallosphaera sedula mutant, CuR1 under normal growth conditions. This experiment was done to analyze the differential transcription of WT cells compared with CuR1 cells at mid log phase. WT-15_CuR1-15: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 15 minutes post copper challenge. The copper cultures were harvested 15 minutes after the shock. WT-60_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 60 minutes post copper challenge. The copper cultures were harvested 60 minutes after the shock. WT-15_WT-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively. CuR1-15_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula CuR1 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively.
2014-07-10 | E-GEOD-59253 | biostudies-arrayexpress
Project description:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, Gram-positive anaerobe, which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO2 and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to co-utilize glucose and xylose, make this bacterium an attractive candidate for microbial bioenergy production. Glycolytic pathways and an ABC-type sugar transporter were significantly up-regulated during growth on glucose and xylose, indicating that C. saccharolyticus co-ferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks represents a highly desirable feature of a lignocellulose-utilizing, biofuel-producing bacterium. Keywords: substrate response C. saccharolyticus was subcultured (overnight) 3 times on the substrate of interest in modified DSMZ 640 medium before inoculating a pH-controlled (pH = 7) 1-liter fermentor containing 4 gram substrate per liter. Cells were grown at 70 °C until mid-logarithmic phase (~OD660 = 0.3-0.4) and harvested by centrifugation and rapid cooling to 4 °C and stored at -80 °C. To elucidate the central carbon metabolic pathways and their regulation, transcriptome analysis was performed after growth on glucose, xylose and a 1:1 mixture of both substrates. L-Rhamnose, which was likely to follow another pathway, was used as a reference substrate.
2008-04-15 | E-GEOD-11153 | biostudies-arrayexpress
Project description:The co-utilization of hexoses and pentoses derived from lignocellulose is an attractive trait for in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H2-producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus growing on oligosaccharides (composed of glucose and xylose) compared to monosaccharides. Based on the whole-genome transcriptional response analysis and comparative genomics, carbohydrate specificities for transport systems could be proposed for a number of the 24 putative carbohydrate ATP-binding cassette (ABC) transporters in the C. saccharolyticus genome. Transcriptome analysis showed different transporters were utilized for uptake of oligosaccharides than those used for monosaccharides uptake. No evidence for catabolite repression was noted for either growth on multi-sugar mixtures or in the corresponding transcriptomes. C. saccharolyticus was subcultured (overnight) seven times on the substrate of interest in modified DSMZ 640 medium before inoculating a 1-liter batch containing 0.5 gram substrate per liter. Cells were grown at 70 °C until mid-logarithmic phase (3-5*107) and harvested by rapid cooling to 4 °C and centrifugation and then stored at -80 °C. To elucidate the transporters plus the central carbon metabolic pathways and their regulation utilized on the different sugars, transcriptome analysis was performed after growth on xylan (oat spelt), xyloglucan, xylogluco-oligosaccharides, glucose and xylose.
2010-05-16 | E-GEOD-17427 | biostudies-arrayexpress
Project description:The co-utilization of hexoses and pentoses derived from lignocellulose is an attractive trait for in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H2-producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus (Csac) growing on individual monosaccharides (arabinose, fructose, galactose, glucose, mannose and xylose), mixtures of these sugars. Based on the whole-genome transcriptional response analysis and comparative genomics, carbohydrate specificities for transport systems could be proposed for most of the 24 putative carbohydrate ATP-binding cassette (ABC) transporters in the C. saccharolyticus genome. Transcriptome contrasts for monosacchride growth showed that minimal changes were observed in some cases (e.g., 31 ORFs changed ï³ 2-fold for glucose/galactose) while substantial changes occurred for cases involving mannose (e.g., 363 ORFs ï³ 2-fold for glucose/mannose). No evidence for catabolite repression was noted for either growth on multi-sugar mixtures or in the corresponding transcriptomes. C. saccharolyticus was subcultured (overnight) seven times on the substrate of interest in modified DSMZ 640 medium before inoculating a 1-liter batch containing 0.5 gram substrate per liter. Cells were grown at 70 °C until mid-logarithmic phase (3-5*107) and harvested by rapid cooling to 4 °C and centrifugation and then stored at -80 °C. To elucidate the transporters plus the central carbon metabolic pathways and their regulation utilized on the different sugars, transcriptome analysis was performed after growth on arabinose, fructose, galactose, glucose, mannose, xylose and a mixture of all six substrates.
2010-05-16 | E-GEOD-17425 | biostudies-arrayexpress