The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase.

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively supercoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes.

Nitrate-Responsive Suppression of Dimethyl Sulfoxide Respiration in a Facultative Anaerobic Haloarchaeon, Haloferax volcanii.

Haloferax volcanii is a facultative anaerobic haloarchaeon that can grow using nitrate or dimethyl sulfoxide (DMSO) as a respiratory substrate under anaerobic conditions. Comparative transcriptome analysis of denitrifying and aerobic cells of H. volcanii indicated extensive changes in gene expression involving the activation of denitrification, suppression of DMSO respiration, and conversion of the heme biosynthetic pathway under denitrifying conditions. The anaerobic growth of H. volcanii by DMSO respiration was inhibited at nitrate concentrations of <1 mM, whereas nitrate-responsive growth inhibition was not observed in the ΔnarO mutant. A reporter assay demonstrated that the transcription of the dms operon was suppressed by nitrate. In contrast, the anaerobic growth of the ΔdmsR mutant by denitrification was little affected by the addition of DMSO. NarO has been identified as an activator of denitrification-related genes in response to anaerobic conditions, and here, we found that NarO is also involved in nitrate-responsive suppression of the dms operon. Nitrate-responsive suppression of DMSO respiration is known in several bacteria such as Escherichia coli and photosynthetic Rhodobacter species. This is the first report to show that a regulatory mechanism that suppresses DMSO respiration in response to nitrate exists not only in bacteria but also in haloarchaea. IMPORTANCE Haloferax volcanii can grow anaerobically by denitrification (nitrate respiration) or DMSO respiration. In facultative anaerobic bacteria that can grow by both nitrate respiration and DMSO respiration, nitrate respiration is preferentially induced when both nitrate and DMSO are available as the respiratory substrates. The results of transcriptome analysis, growth phenotyping, and reporter assays indicated that DMSO respiration is suppressed in response to nitrate in H. volcanii. The haloarchaeon-specific regulator NarO, which activates denitrification under anaerobic conditions, is suggested to be involved in the nitrate-responsive suppression of DMSO respiration.

CTP synthase forms cytoophidia in archaea.

CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of nucleotide CTP de novo synthesis. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes, which are termed cytoophidia. However, it is unknown whether cytoophidia exist in the third domain of life, archaea. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaea. Under stimulated emission depletion microscopy, we find that the structures of H. hispanica CTPS are elongated, similar to cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. In addition, treatment of H. hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly. Our study reveals that CTPS can form cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS.

Comparing DNA, RNA and protein levels for measuring microbial dynamics in soil microcosms amended with nitrogen fertilizer.

To what extent multi-omic techniques could reflect in situ microbial process rates remains unclear, especially for highly diverse habitats like soils. Here, we performed microcosm incubations using sandy soil from an agricultural site in Midwest USA. Microcosms amended with isotopically labeled ammonium and urea to simulate a fertilization event showed nitrification (up to 4.1 ± 0.87 µg N-NO3- g-1 dry soil d-1) and accumulation of N2O after 192 hours of incubation. Nitrification activity (NH4+ → NH2OH → NO → NO2- → NO3-) was accompanied by a 6-fold increase in relative expression of the 16S rRNA gene (RNA/DNA) between 10 and 192 hours of incubation for ammonia-oxidizing bacteria Nitrosomonas and Nitrosospira, unlike archaea and comammox bacteria, which showed stable gene expression. A strong relationship between nitrification activity and betaproteobacterial ammonia monooxygenase and nitrite oxidoreductase transcript abundances revealed that mRNA quantitatively reflected measured activity and was generally more sensitive than DNA under these conditions. Although peptides related to housekeeping proteins from nitrite-oxidizing microorganisms were detected, their abundance was not significantly correlated with activity, revealing that meta-proteomics provided only a qualitative assessment of activity. Altogether, these findings underscore the strengths and limitations of multi-omic approaches for assessing diverse microbial communities in soils and provide new insights into nitrification.

Salt-dependent regulation of archaellins in Haloarcula marismortui.

Microorganisms require a motility structure to move towards optimal growth conditions. The motility structure from archaea, the archaellum, is fundamentally different from its bacterial counterpart, the flagellum, and is assembled in a similar fashion as type IV pili. The archaellum filament consists of thousands of copies of N-terminally processed archaellin proteins. Several archaea, such as the euryarchaeon Haloarcula marismortui, encode multiple archaellins. Two archaellins of H. marismortui display differential stability under various ionic strengths. This suggests that these proteins behave as ecoparalogs and perform the same function under different environmental conditions. Here, we explored this intriguing system to study the differential regulation of these ecoparalogous archaellins by monitoring their transcription, translation, and assembly into filaments. The salt concentration of the growth medium induced differential expression of the two archaellins. In addition, this analysis indicated that archaellation in H. marismortui is majorly regulated on the level of secretion, by a still unknown mechanism. These findings indicate that in archaea, multiple encoded archaellins are not completely redundant, but in fact can display subtle functional differences, which enable cells to cope with varying environmental conditions.

Global effect of the lack of inorganic polyphosphate in the extremophilic archaeon Sulfolobus solfataricus: A proteomic approach.

Inorganic polyphosphates (polyP) are present in all living cells and several important functions have been described for them. They are involved in the response to stress conditions, such as nutrient depletion, oxidative stress and toxic metals amongst others. A recombinant strain of Sulfolobus solfataricus unable to accumulate polyP was designed by the overexpression of its endogenous ppx gene. The overall impact of the lack of polyP on this S. solfataricus polyP (-) strain was analyzed by using quantitative proteomics (isotope-coded protein label, ICPL). Stress-related proteins, such as peroxiredoxins and heat shock proteins, proteins involved in metabolism and several others were produced at higher levels in the ppx expression strain. The polyP deficient strain showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA gene coding for the P-type copper-exporting ATPase. This implies a complementary function of both copper resistance systems. These results strongly suggests that the lack of polyP makes this hyperthermophilic archaeon more sensitive to toxic conditions, such as an exposure to metals or other harmful stimuli, emphasizing the importance of this inorganic phosphate polymers in the adaptations to live in the environmental conditions in which thermoacidophilic archaea thrive. SIGNIFICANCE: Inorganic polyphosphate (polyP) are ubiquitous molecules with many functions in living organisms. Few studies related to these polymers have been made in archaea. The construction of a polyP deficient recombinant strain of Sulfolobus solfataricus allowed the study of the global changes in the proteome of this thermoacidophilic archaeon in the absence of polyP compared with the wild type strain. The results obtained using quantitative proteomics suggest an important participation of polyP in the oxidative stress response of the cells and as having a possible metabolic role in the cell, as previously described in bacteria. The polyP deficient strain also showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA, implying a complementary role of both copper resistance systems.

Inhibition of MyD88 Signaling Skews Microglia/Macrophage Polarization and Attenuates Neuronal Apoptosis in the Hippocampus After Status Epilepticus in Mice.

Inflammation is implicated in epileptogenesis. Activated microglia and macrophages (MG/MΦ) are found in the brains of patients with epilepsy-related diseases and animal models of epilepsy. It is not yet known how the MG/MΦ activation phenotype affects pathological changes in the brain after a single seizure. In this study, we had 2 main purposes: first, to characterize post-status epilepticus (SE) inflammation by tracking MG/MΦ polarization, and, second, to explore the role of an innate immune receptor adaptor protein, namely, myeloid differentiation primary response gene 88 (MyD88), in the induction of SE in a mouse model. A lithium-pilocarpine model of seizure conditions was generated in C57BL/6 mice. The intensity and distribution of MG/MΦ polarization were tracked by fluorescent immunohistochemistry and Western blotting for the polarization markers inducible nitrogen oxygenized synthase, arginase-1, CD163, and mannose receptor. We observed steadily increasing M1 MG/MΦ along with MyD88 signal upregulation after SE in the hippocampi of mice, whereas the M2 marker arginase-1 was localized mainly in astrocytes rather than in MG/MΦ. Inhibition or gene knockout of MyD88 reduced M1 MG/MΦ and gliosis although increasing M2 MG/MΦ in the hippocampi of SE mice. MyD88 inhibition also augmented glutamate transporter 1 expression and reduced N-methyl-D-aspartate receptor NR1 subunit expression in the hippocampus to protect pyramidal neurons from apoptosis. These data suggest that MG/MΦ polarization after SE impacts the pathological outcome of the hippocampus via MyD88 signaling and point to MyD88 as a potential neuroprotective target for epilepsy therapy.

Phytohormones Regulate the Expression of Nuclear Genes Encoding the Components of the Plastid Transcription Apparatus.

As multifunctional regulators of physiological processes, phytohormones play an important role in the regulation of expression of the plastid genome and chloroplast biogenesis. Hormones can directly regulate the expression of genes localized in the chloroplast genome. However, many components of the plastid transcription apparatus are encoded by nuclear rather than plastid genes. It remains obscure whether these nuclear genes are subject to hormonal regulation. This is the first study to show that phytohormones exert differential effects on the expression of nuclear genes of the transcription machinery of the Arabidopsis thaliana plastome. RT-PCR analysis showed that the level of transcripts of the majority of studied genes was activated by trans-zeatin but decreased under the influence of ABA, methyl jasmonate, and salicylic acid, whereas ethylene had no significant effect, and the effects of brassinolide depended on the illumination conditions. The results of this study indicate that the hormonal regulation of the plastome expression can be mediated by differential regulation of the nuclear genes encoding plastid transcription machinery components.

Formate hydrogenlyase and formate secretion ameliorate H2 inhibition in the hyperthermophilic archaeon Thermococcus paralvinellae.

Some hyperthermophilic heterotrophs in the genus Thermococcus produce H2 in the absence of S° and have up to seven hydrogenases, but their combined physiological roles are unclear. Here, we show which hydrogenases in Thermococcus paralvinellae are affected by added H2 during growth without S°. Growth rates and steady-state cell concentrations decreased while formate production rates increased when T. paralvinallae was grown in a chemostat with 65 µM of added H2(aq) . Differential gene expression analysis using RNA-Seq showed consistent expression of six hydrogenase operons with and without added H2 . In contrast, expression of the formate hydrogenlyase 1 (fhl1) operon increased with added H2 . Flux balance analysis showed H2 oxidation and formate production using FHL became an alternate route for electron disposal during H2 inhibition with a concomitant increase in growth rate relative to cells without FHL. T. paralvinellae also grew on formate with an increase in H2 production rate relative to growth on maltose or tryptone. Growth on formate increased fhl1 expression but decreased expression of all other hydrogenases. Therefore, Thermococcus that possess fhl1 have a competitive advantage over other Thermococcus species in hot subsurface environments where organic substrates are present, S° is absent and slow H2 efflux causes growth inhibition.

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment.

First description of small proteins encoded by spRNAs in Methanosarcina mazei strain Gö1.

In Methanosarcina mazei several small RNAs have been identified containing a small putative open reading frame (sORF) and thus classified as spRNAs. Here, we report on the first detection of three small proteins in M. mazei encoded by spRNAs using LC-MS/MS analysis of total protein extracts of cells grown under various stress conditions. Each spRNA shows high conservation in Methanosarcina species with regard to the sORF and the flanking non-coding RNA regions, moreover the predicted RNA structures are as well highly conserved. Characterizing the respective transcript levels in response to several stress conditions by northern blots demonstrated an enormous decrease of spRNA36 and spRNA44 during stationary growth (to less than 5%), and a significant increase of spRNA36 (2.5-fold) in response to nitrogen limitation. spRNA41, however, was only detected by RNA-Seq approaches. Quantification of the small proteins by LC-MS/MS using synthetic stable isotope labeled oligopeptides as standards indicated that the concentration of oligopetide36 and 41 in mid exponential phase is induced under nitrogen limitation, which in case of oligopeptide36 is in accordance with its transcript level. The relative amount of the three oligopeptides did not change upon entering stationary growth phase, even though the transcript levels decreased dramatically. Additional production of the oligopeptides in M. mazei did not result in any evident phenotype under standard or nitrogen limiting growth conditions. However, overall the transcript levels of several genes involved in carbon metabolism or in heat shock response were reduced 2-3 fold due to the overproduction, though no sORF specific change was observed. Based on our findings we hypothesize that oligopeptide36 might have a regulatory function in nitrogen metabolism by modulating the activity of a yet unknown target protein involved in the central nitrogen metabolism.

The Methanosarcina acetivorans thioredoxin system activates DNA binding of the redox-sensitive transcriptional regulator MsvR.

The production of biogas (methane) by an anaerobic digestion is an important facet to renewable energy, but is subject to instability due to the sensitivity of strictly anaerobic methanogenic archaea (methanogens) to environmental perturbations, such as oxygen. An understanding of the oxidant-sensing mechanisms used by methanogens may lead to the development of more oxidant tolerant (i.e., stable) methanogen strains. MsvR is a redox-sensitive transcriptional regulator that is found exclusively in methanogens. We show here that oxidation of MsvR from Methanosarcina acetivorans (MaMsvR) with hydrogen peroxide oxidizes cysteine thiols, which inactivates MaMsvR binding to its own promoter (P(msvR)). Incubation of oxidized MaMsvR with the M. acetivorans thioredoxin system (NADPH, MaTrxR, and MaTrx7) results in reduction of the cysteines back to thiols and activation of P msvR binding. These data confirm that cysteines are critical for the thiol-disulfide regulation of P(msvR) binding by MaMsvR and support a role for the M. acetivorans thioredoxin system in the in vivo activation of MaMsvR. The results support the feasibility of using MaMsvR and P(msvR), along with the Methanosarcina genetic system, to design methanogen strains with oxidant-regulated gene expression systems, which may aid in stabilizing anaerobic digestion.

A novel CO-responsive transcriptional regulator and enhanced H2 production by an engineered Thermococcus onnurineus NA1 strain.

Genome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism in Thermococcus onnurineus NA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes of Thermococcus species and "Candidatus Korarchaeum cryptofilum" OPF8. In-frame deletion of either corQ or corR caused a severe impairment in CO-dependent growth and H2 production. When corQ and corR deletion mutants were complemented by introducing the corQR genes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integrated corQR (ΔCorR/corQR(↑)) compared with those in the wild-type strain. In addition, the ΔCorR/corQR(↑) strain exhibited a much higher H2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2 production rate (191.9 mmol liter(-1) h(-1)) and the specific H2 production rate (249.6 mmol g(-1) h(-1)) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that the corQR genes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2 production.

Assessment of the sensitivity of three North American fish species to disruptors of steroidogenesis using in vitro tissue explants.

There is concern regarding exposure of aquatic organisms to chemicals that interfere with the endocrine system. One critical mechanism of endocrine disruption is impairment of steroidogenesis that can lead to altered hormone levels, altered or delayed sexual development, and ultimately reproductive failure. With the current large gap in knowledge and a high degree of uncertainty regarding the sensitivity of fishes native to northern ecosystems to endocrine disrupting chemicals (EDCs), the aim of this study was to develop an in vitro gonadal explant assay enabling the assessment of EDCs on sex-steroid production in wild fish species native to North America. Northern pike (Esox lucius), walleye (Sander vitreus), and white sucker (Catostomus commeroni) were sampled from a reference location in Lake Diefenbaker, Saskatchewan, Canada, at spawn and multiple post-spawn time points. Gonads were excised and immediately exposed for 24h to a model inducer (forskolin) or inhibitor (prochloraz) of steroidogenesis in L-15 supplemented media. Furthermore, seasonal profiles of plasma 11-ketotestosterone (11-KT) and 17-ß estradiol (E2) concentrations were characterized. Enzyme-linked immunosorbent assays were used to quantify hormone concentrations in plasma and media. The seasonal profile of plasma hormones was significantly correlated with basal in vitro hormone production. Gonad tissue exposed to forskolin showed a concentration-dependent increase in E2 and a general increase in 11-KT. Gonad tissue exposed to prochloraz resulted in a decrease of concentrations of 11-KT and E2. These results illustrated that gonadal tissue is undergoing steroidogenesis in an in vitro setting that is comparable to in vivo hormone profiles, and which is responsive to chemical exposure in a concentration-dependent manner. The seasonal time point during which gonad explants were excised and exposed had an impact on the potency and magnitude of responses, resulting in a seasonal effect on sensitivity. Male and female white sucker showed greatest sensitivity to forskolin, while male and female walleye showed greatest sensitivity to prochloraz. Also, gonad explants from these species were found to have greater sensitivity than responses previously reported for in vitro explants of other fish species such as the fathead minnow (Pimephales promelas), and stable cell lines currently used as screening applications to detect chemicals that might disrupt the endocrine system. Therefore, current approaches that use stable cell lines or tissue explants from standardized small bodied laboratory species might not be protective of some wild fish species. Future research is required that investigates whether this in vitro gonadal explant assay is predictive of in vivo effects in wild species of fishes.

Basal and benzo[a]pyrene-induced expression profile of phase I and II enzymes and ABC transporter mRNA in the early life stage of Chinese rare minnows (Gobiocypris rarus).

ATP-binding cassette (ABC) transporters together with phase I and II detoxification enzymes have been considered as included in a cellular detoxification system. Previous studies have highlighted the involvement of aryl hydrocarbon receptor (AHR) and Cyp1a in PAH-induced embryo toxicity. However, the response of other xenobiotic enzymes/transporters in PAH-mediated embryo toxicity is not fully characterized. In the present study, rare minnow embryos were exposed to 10 and 100µg/L BaP within 4h post-fertilization (hpf) up to 168 hpf. RNA was extracted at 24, 48, 96, and 168 hpf. The basal and BaP-induced expression of phase I enzyme genes (cyp1a, 1b1, and 1c1), phase II enzyme gene (gstm and ugt1a), and ABC transporter genes (abcb1, abcc1, abcc2, and abcg2) mRNA was determined using real-time PCR. Severe developmental defects (e.g., spinal deformities, pericardial and yolk-sac edema) were observed in the BaP treated groups. The basal expression showed that gstm was most strongly expressed, followed by abcb1, ugt1a, and abcc2, whereas cyp1a, 1b1, 1c1, and abcg2 showed weak expression. BaP significantly induced the mRNA expression of three CYP1s (cyp1a, 1b1, and 1c1) (p<0.05) and the ABC transporters (abcc1, abcc2, and abcg2) in a dose-dependent manner. However, the mRNA expression of Phase II enzymes (gstm, ugt1a) for the BaP treatments showed no significant difference with that of the controls. Furthermore, distinct induced patterns of these genes were observed during different exposure periods. Simultaneous up-regulation of the cyp and ABC transporter gene transcripts suggests that a possible involvement and cooperation in the detoxification process could provide protection against the BaP toxicity of rare minnows at the early life stage.

DNA double strand break end-processing and RecA induce RecN expression levels in Bacillus subtilis.

Bacillus subtilis cells respond to double strand breaks (DSBs) with an ordered recruitment of repair proteins to the site lesion, being RecN one of the first responders. In B. subtilis, one of the responses to DSBs is to increase RecN expression rather than modifying its turnover rate. End-processing activities and the RecA protein itself contribute to increase RecN levels after DNA DSBs. RecO is required for RecA filament formation and full SOS induction, but its absence did not significantly affect RecN expression. Neither the absence of LexA nor the phosphorylation state of RecA or SsbA significantly affect RecN expression levels. These findings identify two major mechanisms (SOS and DSB response) used to respond to DSBs, with LexA required for one of them (SOS response). The DSB response, which requires end-processing and RecA or short RecO-independent RecA filaments, highlights the importance of guarding genome stability by modulating the DNA damage responses.

Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1)) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

Uranium extremophily is an adaptive, rather than intrinsic, feature for extremely thermoacidophilic Metallosphaera species.

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 (U). Metallosphaera prunae, isolated from a smoldering heap on a uranium mine in Thüringen, Germany, could be viewed as a "spontaneous mutant" of Metallosphaera sedula, an isolate from Pisciarelli Solfatara near Naples. Metallosphaera prunae tolerated triuranium octaoxide (U(3)O(8)) and soluble uranium [U(VI)] to a much greater extent than M. sedula. Within 15 min following exposure to "U(VI) shock," M. sedula, and not M. prunae, exhibited transcriptomic features associated with severe stress response. Furthermore, within 15 min post-U(VI) shock, M. prunae, and not M. sedula, showed evidence of substantial degradation of cellular RNA, suggesting that transcriptional and translational processes were aborted as a dynamic mechanism for resisting U toxicity; by 60 min 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 U(3)O(8) to soluble U(VI) for bioenergetic purposes, a chemolithoautotrophic feature not previously reported. M. prunae, however, did not solubilize solid U(3)O(8) 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.

A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus.

Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6×His tag, as shown with pSeSD-N-lacS. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon Sulfolobus can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site.

Harmaline-resistant mutant of Methanothermobacter thermautotrophicus with a lesion in Na(+)/H(+) antiport.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the Na(+)/H(+) antiporter inhibitor harmaline was isolated. The Na(+)/H(+) exchange activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Na(+)/H(+) antiport activity of wild-type cells grown in the high Na(+) concentration (125 mmol/l) was significantly increased as compared to the cells grown under low Na(+) concentration (6.25 mmol/l) conditions. In contrast, harmaline resistant mutant showed almost the same Na(+)/H(+) antiport activity under both these conditions. While harmaline profoundly inhibited methanogenesis in the wild-type, increased methanogenesis was observed both in the presence and absence of harmaline in the mutant strain. ATP synthesis driven by methanogenic electron transport was significantly enhanced in the mutant cells. The experimental data revealed the differential expression of A flavoprotein and molybdenum-containing formylmethanofuran dehydrogenase 1 subunit C in harmaline-resistant mutant. The overexpression of these proteins might contribute to harmaline resistance. Taken together the results indicate that harmaline resistance in this mutant has arisen as a consequence of mutation(s) in antiporter gene(s) or protein(s) linked to antiporter activity. Moreover this work provides the evidence that Na(+)/H(+) exchanger deficiency in harmaline-resistant mutant can induce overexpression of several proteins participating in methanogenesis.