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Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.

Parvovirus B19 (B19V) is pathogenic to humans and causes various human diseases. However, no antiviral agents or vaccines currently exist for the treatment or prevention of B19V infection. Therefore, developing sensitive and specific methods for B19V infection diagnosis is essential for accurate diagnoses. Previously, a Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas12a (cpf1)-based electrochemical biosensor (E-CRISPR) with a picomole sensitivity for B19V detection was established. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the nonstructural protein 1 (NS1) region of the B19V viral genome (abbreviated B19-NS1 PAND). Benefiting from independent protospacer adjacent motif (PAM) sequences, PfAgo can recognize their target with guide DNA (gDNA) that is easy to design and synthesize at a low cost. In contrast to E-CRISPR, without preamplification with Polymerase Chain Reaction (PCR), the Minimum Detectable Concentration (MDC) of three guide- or single guide-mediated B19-NS1 PAND was about 4 nM, approximately 6-fold more than E-CRISPR. However, when introducing an amplification step, the MDC can be dramatically decreased to the aM level (54 aM). In addition, the diagnostic results from clinical samples with B19-NS1 PAND revealed 100% consistency with PCR assays and subsequent Sanger sequencing tests, which may assist in molecular testing for clinical diagnosis and epidemiological investigations of B19V.

Metalloprotein diversity Metalloproteins are key components in many biological processes, including respiration, photosynthesis and drug metabolism. The presence of a metal in a protein is often not apparent until the molecule is fully characterized. For this reason, and because of the diversity of metal coordination sites, it is not yet possible to use genome sequences to predict the types of metal an organism utilizes from its environment, or to determine the composition of the organism's metalloproteins. Cvetkovic et al . have therefore taken an alternative route, using conventional liquid chromatography to identify the metals in an organism — the hyperthermophilic marine archaeon Pyrococcus furiosus — and proteomics to examine the metalloproteins. Of the 343 metal peaks in chromatography fractions, 158 did not match any known or predicted metalloprotein, some of them containing metals that the organism was not previously known to utilize. This work suggests that metalloproteomes are more extensive and diverse than previously thought. Metalloproteins are important in many biological processes, including respiration, photosynthesis and drug metabolism. Using genome sequences to predict the numbers and types of metal an organism uses is currently very challenging. These authors used a proteomics approach to identify and characterize a large number of a microorganism's metalloproteins on a genome-wide scale, and successfully separated and identified its cytoplasmic metalloproteins. Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability 1 , 2 . Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized 2 , 3 , 4 . We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism ( Pyrococcus furiosus ). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms ( Escherichia coli and Sulfolobus solfataricus ) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.

The anaerobic archaeon Pyrococcus furiosus grows by fermenting carbohydrates producing H ₂, CO ₂, and acetate. We show here that it is surprisingly tolerant to oxygen, growing well in the presence of 8% (vol/vol) O ₂. Although cell growth and acetate production were not significantly affected by O ₂, H ₂ production was reduced by 50% (using 8% O ₂). The amount of H ₂ produced decreased in a linear manner with increasing concentrations of O ₂ over the range 2–12% (vol/vol), and for each mole of O ₂ consumed, the amount of H ₂ produced decreased by approximately 2 mol. The recycling of H ₂ by the two cytoplasmic hydrogenases appeared not to play a role in O ₂ resistance because a mutant strain lacking both enzymes was not more sensitive to O ₂ than the parent strain. Decreased H ₂ production was also not due to inactivation of the H ₂-producing, ferredoxin-dependent membrane-bound hydrogenase because its activity was unaffected by O ₂ exposure. Electrons from carbohydrate oxidation must therefore be diverted to relieve O ₂ stress at the level of reduced ferredoxin before H ₂ production. Deletion strains lacking superoxide reductase (SOR) and putative flavodiiron protein A showed increased sensitivity to O ₂, indicating that these enzymes play primary roles in resisting O ₂. However, a mutant strain lacking the proposed electron donor to SOR, rubredoxin, was unaffected in response to O ₂. Hence, electrons from sugar oxidation normally used to produce H ₂ are diverted to O ₂ detoxification by SOR and putative flavodiiron protein A, but the electron flow pathway from ferredoxin does not necessarily involve rubredoxin.|

Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2′- O -ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding. RNAs undergo many types of post-transcriptional modification, including methylation of ribosomal RNAs; here the structure of the archaeal box C/D ribonucleoprotein complex bound to substrate RNA is determined, showing that the two methylation guide sequences exist in different contexts and revealing sequential regulation of methylation at the two sites. Sequential methylation of the box C/D enzyme RNAs undergo many types of post-transcriptional modification. One of these is methylation of the 2′- O -ribose of rRNAs. The modifying enzyme, the box C/D ribonucleoprotein complex, identifies its targets using guide RNAs that recognize two methylation sites. Teresa Carlomagno and colleagues have solved the structure of the 390 kDa archaeal complex bound to substrate RNA. This work shows that the two methylation guide sequences exist in different contexts, a situation that facilitates sequential methylation at the two sites. The authors speculate that this regulated process may influence RNA folding as well.

Facing rapid fluctuations in their natural environment, extremophiles, like the hyperthermophilic archaeon Pyrococcus furiosus , exhibit remarkable adaptability to extreme conditions. However, our understanding of their dynamic cellular responses remains limited. This study integrates RNA-sequencing and mass spectrometry data, thereby elucidating transcriptomic and proteomic responses to heat and cold shock stress in P. furiosus . Our results reveal rapid and dynamic changes in gene and protein expression following these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, targeting a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but sustained response. Intriguingly, cold shock at 4°C elicits distinct short- and long-term responses at both RNA and protein levels. Cluster analysis identified gene sets with either congruent or contrasting trends in RNA and protein changes, representing well-separated arCOG groups tailored to their individual cellular responses. Particularly, upregulation of ribosomal proteins and significant enrichment of 5′-leadered sequences in cold-shock responsive genes suggest that translation regulation is important during cold shock adaption. Further investigating transcriptomic features, we reveal that thermal stress genes are equipped with basal sequence elements, such as strong promoter and poly(U)-terminators, facilitating a regulated response of the respective transcription units. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and providing valuable insights into the molecular adaptations that facilitate life in extreme environments. Extreme environments provide unique challenges for life, and the study of extremophiles can shed light on the mechanisms of adaptation to such conditions. Pyrococcus furiosus , a hyperthermophilic archaeon, is a model organism for studying thermal stress response mechanisms. In this study, we used an integrated analysis of RNA-sequencing and mass spectrometry data to investigate the transcriptomic and proteomic responses of P. furiosus to heat and cold shock stress and recovery. Our results reveal the rapid and dynamic changes in gene and protein expression patterns associated with these stress responses, as well as the coordinated regulation of different gene sets in response to different stressors. These findings provide valuable insights into the molecular adaptations that facilitate life in extreme environments and advance our understanding of stress response mechanisms in hyperthermophilic archaea.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, induces severe vomiting and acute watery diarrhea in unweaned piglets. The pig industry has suffered tremendous financial losses due to the high mortality rate of piglets caused by PEDV. Consequently, a simple and rapid on-site diagnostic technology is crucial for preventing and controlling PEDV. This study established a detection method for PEDV using recombinase-aided amplification (RAA) and Pyrococcus furiosus Argonaute (PfAgo), which can detect 100 copies of PEDV without cross-reactivity with other pathogens. The entire reaction of RAA and PfAgo to detect PEDV does not require sophisticated instruments, and the reaction results can be observed with the naked eye. Overall, this integrated RAA-PfAgo cleavage assay is a practical tool for accurately and quickly detecting PEDV. Key points • PfAgo has the potential to serve as a viable molecular diagnostic tool for the detection and diagnosis of viral genomes • The RAA-PfAgo detection technique has a remarkable level of sensitivity and specificity • The RAA-PfAgo detection system can identify PEDV without needing advanced equipment

The development of tungsten biochemistry is sketched from the viewpoint of personal participation. Following its identification as a bio-element, a catalogue of genes, enzymes, and reactions was built up. EPR spectroscopic monitoring of redox states was, and remains, a prominent tool in attempts to understand tungstopterin-based catalysis. A paucity of pre-steady-state data remains a hindrance to overcome to this day. Tungstate transport systems have been characterized and found to be very specific for W over Mo. Additional selectivity is presented by the biosynthetic machinery for tungstopterin enzymes. Metallomics analysis of hyperthermophilic archaeon Pyrococcus furiosus indicates a comprehensive inventory of tungsten proteins.

Key Points The MRE11 complex, consisting of meiotic recombination 11 (MRE11), RAD50 and Nijmegen breakage syndrome 1 (NBS1; also known as nibrin), is of key importance in the DNA damage response to DNA double-strand breaks, and therefore regulates genome stability. The MRE11 complex regulates repair of DNA double-strand breaks in several contexts, including replication, telomere homeostasis, meiosis, apoptosis and immune system development. Structural studies of the MRE11 complex have provided insights into the distinct roles that different components in this complex have. The globular domain of the MRE11 complex is important for DNA binding, the RAD50 hook domain mediates complex homodimerization and NBS1 mediates regulation of the complex. Mouse models for different alleles of the MRE11 complex have been developed on the basis of these structural studies, and show clinical features of the human syndromes that the MRE11 complex has been linked to. The MRE11 complex mediates repair of DNA double-strand breaks and is essential for genome stability. Structural studies and mouse models are increasing our understanding of how the different components of this complex together coordinate the damage response. The maintenance of genome stability depends on the DNA damage response (DDR), which is a functional network comprising signal transduction, cell cycle regulation and DNA repair. The metabolism of DNA double-strand breaks governed by the DDR is important for preventing genomic alterations and sporadic cancers, and hereditary defects in this response cause debilitating human pathologies, including developmental defects and cancer. The MRE11 complex, composed of the meiotic recombination 11 (MRE11), RAD50 and Nijmegen breakage syndrome 1 (NBS1; also known as nibrin) proteins is central to the DDR, and recent insights into its structure and function have been gained from in vitro structural analysis and studies of animal models in which the DDR response is deficient.

Protein glycosylation is a critical protein modification. In biogenic membranes of eukaryotes and archaea, these reactions require activated mannose in the form of the lipid conjugate dolichylphosphate mannose (Dol- P -Man). The membrane protein dolichylphosphate mannose synthase (DPMS) catalyzes the reaction whereby mannose is transferred from GDP-mannose to the dolichol carrier Dol- P , to yield Dol- P -Man. Failure to produce or utilize Dol- P -Man compromises organism viability, and in humans, several mutations in the human dpm1 gene lead to congenital disorders of glycosylation (CDG). Here, we report three high-resolution crystal structures of archaeal DPMS from Pyrococcus furiosus , in complex with nucleotide, donor, and glycolipid product. The structures offer snapshots along the catalytic cycle, and reveal how lipid binding couples to movements of interface helices, metal binding, and acceptor loop dynamics to control critical events leading to Dol- P -Man synthesis. The structures also rationalize the loss of dolichylphosphate mannose synthase function in dpm1 -associated CDG. The generation of glycolipid dolichylphosphate mannose (Dol- P -Man) is a critical step for protein glycosylation and GPI anchor synthesis. Here the authors report the structure of dolichylphosphate mannose synthase in complex with bound nucleotide and donor to provide insight into the mechanism of Dol- P -Man synthesis.