Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

M. sedula transcriptional response to ferrous iron and RISC supplements

ABSTRACT: 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).

ORGANISM(S): Metallosphaera sedula

SUBMITTER: Kathryne Auernik

PROVIDER: E-GEOD-12044 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Identification of components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula through iron and sulfur compound oxidation transcriptomes.

Auernik Kathryne S KS Kelly Robert M RM

Applied and environmental microbiology 20081017 24

The crenarchaeal order Sulfolobales collectively contain 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, their contribution to sulfur and ...[more]

PMID: 18931292

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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).

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.

Project description:Arsenic metalloid is a double-edge sword. On the one hand it is a very toxic and powerful carcinogen, and on the other it has been successfully used in the treatment of acute promyelocytic leukemia. In order to prevent the deleterious effects caused by arsenic compounds, almost all living organisms have developed mechanisms to eliminate it. In this study genome-wide response of S. cerevisiae to arsenic shows that this metal interferes with genes involved in the iron homeostasis including those encoding proteins that function in iron uptake, incorporation into Fe–S clusters, and more. In addition our data indicate that Yap1 transcriptionally controls the iron homeostasis regulator AFT2 as well as its direct target, MRS4. Most importantly in response to arsenate exposure Yap1 strongly regulates the expression of several genes involved in the Fe-S proteins biosynthesis, namely NBP35 and YFH1. Interestingly mRNA levels encoding Fet3, Ferro-O2-oxidoreductase required for high-affinity iron uptake, are drastically destabilized upon arsenic exposure. Such destabilization is due to the 5’ to 3’ exonuclease Xrn1 localized in the P Bodies. Moreover FET3 mRNA decay is not mediated by Cth2 and is independent on the formation of ROS responsible for the toxic effects of arsenic compounds. Strikingly, in presence of arsenate fet3 mutant shows resistance over the wild-type which leads us to suggest that Fet3 has a role in arsenic toxicity. Unexpectedly arsenic treatment seems to activate the non-reductive iron uptake in order to maintain the cellular iron homeostasis. Furthermore our genetic, biochemical, and physiological analysis demonstrate that aft1 mutant is sensitive to arsenic compounds and such phenotype is reversible upon addition of iron. We also show that arsenic exposure induces iron deficiency in aft1 mutant. In conclusion this work shows for the first time that arsenic, a chemotherapy drug used to treat a specific type of acute promyelocytic leukemia (APL), disrupts iron homeostasis and our results suggest that this disruption is independent on ROS generation. Finally we provide preliminary data confirming that such disruption also takes place in mammalian cells, an observation that can be very relevant in term of clinical applications. yap1yap8 mutant cells independently transformed with pRS416 and YcpLac111, YcpLac111-YAP1, or pRS416-YAP8 were grown in triplicates in SC-URA-LEU containing 2mM of AsV until exponential growth phase, and RNA was extracted, labeled, and hybridized to Affymetrix Yeast Genome S98 arrays.

Dataset Information

M. sedula transcriptional response to ferrous iron and RISC supplements

Publications

Identification of components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula through iron and sulfur compound oxidation transcriptomes.

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