Search Results (15)

Polyphosphates are biopolymers composed of phosphate monomers linked by high-energy phosphoanhydride bonds. They are present across all life domains, serving as a source of energy, metal chelators, and playing a crucial role in stress defense. In Escherichia coli, polyphosphates also function as inorganic molecular chaperones. The present study aims to investigate whether polyphosphate serves a similar chaperone function in archaea, using Saccharolobus solfataricus as a model organism. To this end, polyphosphate was extracted and quantified, the ADP/ATP ratio was determined, insoluble protein extracts were analyzed at different time points after copper exposure, and qPCR was performed to measure the expression of stress-related genes. PolyP was extracted after exposing the archaeon S. solfataricus to different copper concentrations. We determined that polyP degradation is directly correlated with metal concentration. At the minimum inhibitory concentration (MIC) of 2 mM Cu²⁺, polyP degradation stabilized 2 h after exposure and showed no recovery even after 24 h. The ADP/ATP ratio was measured and showed differences in the presence or absence of polyP. The analysis of proteins precipitated under copper stress showed a higher proportion of insoluble proteins at an elevated metal concentration. On the other hand, increased protein precipitation was detected in the absence of polyP. Gene expression analysis via qPCR was conducted to assess the expression of genes involved in chaperone and chaperonin production, copper resistance, oxidative stress response, and phosphate metabolism under prolonged copper exposure, both in the presence and absence of polyP. The results indicated an upregulation of all the chaperonins measured in the presence of polyP. Interestingly, just some of these genes were upregulated in polyP’s absence. Despite copper stress, there was no upregulation of superoxide dismutase in our conditions. These results highlight the role of polyP in the copper stress response in S. solfataricus, particularly to prevent protein precipitation, likely due to its function as an inorganic chaperone. Additionally, the observed protein precipitation could be attributable to interactions between copper and some amino acids on the protein structures rather than oxidative stress induced by copper exposure, as previously described in E. coli. Our present findings provide new insights into the protective role of polyP as an inorganic chaperone in S. solfataricus and emphasize its importance in maintaining cellular homeostasis under metal stress conditions. Full article

Encapsulated phosphotriesterase nanoreactors show their efficacy in the prophylaxis and post-exposure treatment of poisoning by paraoxon. A new enzyme nanoreactor (E-nRs) containing an evolved multiple mutant (L72C/Y97F/Y99F/W263V/I280T) of Saccharolobus solfataricus phosphotriesterase (PTE) for in vivo detoxification of organophosphorous compounds (OP) was made. A comparison of nanoreactors made of three- and di-block copolymers was carried out. Two types of morphology nanoreactors made of di-block copolymers were prepared and characterized as spherical micelles and polymersomes with sizes of 40 nm and 100 nm, respectively. The polymer concentrations were varied from 0.1 to 0.5% (w/w) and enzyme concentrations were varied from 2.5 to 12.5 μM. In vivo experiments using E-nRs of diameter 106 nm, polydispersity 0.17, zeta-potential −8.3 mV, and loading capacity 15% showed that the detoxification efficacy against paraoxon was improved: the LD₅₀ shift was 23.7xLD₅₀ for prophylaxis and 8xLD₅₀ for post-exposure treatment without behavioral alteration or functional physiological changes up to one month after injection. The pharmacokinetic profiles of i.v.-injected E-nRs made of three- and di-block copolymers were similar to the profiles of the injected free enzyme, suggesting partial enzyme encapsulation. Indeed, ELISA and Western blot analyses showed that animals developed an immune response against the enzyme. However, animals that received several injections did not develop iatrogenic symptoms. Full article

Some archaea from the genus Sulfolobus are important for bioleaching of copper, where metal resistant microorganisms are required. Biofilm generation is one of the ways microorganisms cope with some stimuli in nature, including heavy metals. The response to external factors, particularly in the biofilm form of life, is still underexplored in archaea. To explore how model thermoacidophilic archaeon Saccharolobus solfataricus faces copper stress during this lifestyle, changes in biofilms were studied using crystal violet staining, confocal fluorescence microscopy, and qPCR approaches. It was found that biofilm formation reached a maximum at 0.5 mM Cu, before starting to decrease at higher metal concentrations. The morphology of biofilms at 0.5 mM Cu was observed to be different, displaying lower thickness, different sugar patterns, and higher amounts of cells compared to standard growing conditions. Furthermore, copA, which is responsive to intracellular Cu concentration, was downregulated in biofilm cells when compared with planktonic cells exposed to the same metal concentration. The latest results suggests that cells in biofilms are less exposed to Cu than those in planktonic culture. In a PolyP-deficient strain, Cu was not able to induce biofilm formation at 0.5 mM. In summary, the findings reported here suggest that the biofilm form of life confers S. solfataricus advantages to face stress caused by Cu.Biofilm formation remains a relatively unexplored topic in archaeal research. Therefore, this knowledge in model organisms such as S. solfataricus, and how they use it to face stress, could be of great importance to engineer organisms with improved capabilities to be applied in biotechnological processes, such as bioleaching of metals. Full article

A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of this helicase/ATPase ring is associated with genetic instability and diseases such as cancer. The helicase/ATPase rings of eukaryotes and archaea consist of six minichromosome maintenance (MCM) proteins. Prior structural studies have shown that MCM rings bind one encircled strand of DNA in a spiral staircase, suggesting that the ring pulls this strand of DNA through its central pore in a hand-over-hand mechanism where the subunit at the bottom of the staircase dissociates from DNA and re-binds DNA one step above the staircase. With high-resolution cryo-EM, we show that the MCM ring of the archaeal organism Saccharolobus solfataricus binds an encircled DNA strand in two different modes with different numbers of subunits engaged to DNA, illustrating a plausible mechanism for the alternating steps of DNA dissociation and re-association that occur during DNA translocation. Full article

Superoxide dismutase (SOD) is a fundamental antioxidant enzyme that neutralises superoxide ions, one of the main reactive oxygen species (ROS). Extremophile organisms possess enzymes that offer high stability and catalytic performances under a wide range of conditions, thus representing an exceptional source of biocatalysts useful for industrial processes. In this study, SODs from the thermo-halophilic Aeropyrum pernix (SOD_Ap) and the thermo-acidophilic Saccharolobus solfataricus (SOD_Ss) were heterologously expressed in transgenic tomato cell cultures. Cell extracts enriched with SOD_Ap and SOD_Ss showed a remarkable resistance to salt and low pHs, respectively, together with optimal activity at high temperatures. Moreover, the treatment of tuna fillets with SOD_Ap-extracts induced an extension of the shelf-life of this product without resorting to the use of illicit substances. The results suggested that the recombinant plant extracts enriched with the extremozymes could find potential applications as dietary supplements in the nutrition sector or as additives in the food preservation area, representing a more natural and appealing alternative to chemical preservatives for the market. Full article

Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO₂. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, T_opt = 80 °C) and Sulfolobus acidocaldarius (Sa, T_opt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from S. solfataricus and S. acidocaldarius showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen. Full article

The outermost component of cell envelopes of most bacteria and almost all archaea comprise a protein lattice, which is termed Surface (S-)layer. The S-layer lattice constitutes a highly porous structure with regularly arranged pores in the nm-range. Some archaea thrive in extreme milieus, thus producing highly stable S-layer protein lattices that aid in protecting the organisms. In the present study, fragments of the cell envelope from the hyperthermophilic acidophilic archaeon Saccharolobus solfataricus P2 (SSO) have been isolated by two different methods and characterized. The organization of the fragments and the molecular sieving properties have been elucidated by transmission electron microscopy and by determining the retention efficiency of proteins varying in size, respectively. The porosity of the archaeal S-layer fragments was determined to be 45%. S-layer fragments of SSO showed a retention efficiency of up to 100% for proteins having a molecular mass of ≥ 66 kDa. Moreover, the extraction costs for SSO fragments have been reduced by more than 80% compared to conventional methods, which makes the use of these archaeal S-layer material economically attractive. Full article

Generation of the 3′ overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved MRE11/RAD50 proteins, play an important role in generating 3′ single-stranded DNA during archaeal HR. Little is known, however, about HerA-NurA function and activation of this fundamental and complicated DNA repair process. Herein, we analyze the functional relationship among NurA, HerA and the single-strand binding protein SSB from Saccharolubus solfataricus. We demonstrate that SSB clearly inhibits NurA endonuclease activity and its exonuclease activities also when in combination with HerA. Moreover, we show that SSB binding to DNA is greatly stimulated by the presence of either NurA or NurA/HerA. In addition, if on the one hand NurA binding is not influenced, on the other hand, HerA binding is reduced when SSB is present in the reaction. In accordance with what has been observed, we have shown that HerA helicase activity is not stimulated by SSB. These data suggest that, in archaea, the DNA end resection process is governed by the strictly combined action of NurA, HerA and SSB. Full article

Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA₄) and that Csa3a lacks self-regulating “ring nuclease” activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA₄ recognition are described, as are conformational changes in Csa3a associated with cA₄ binding. We also characterized the effect of cA₄ on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA₄-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA₄ interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA₄ interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response. Full article

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed “recoding”. In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for −1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed −1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed. Full article

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus: a GH1 β-gluco-/β-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of β-gluco-/β-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan. Full article

DNA polymerase B1 (PolB1) is a member of the B-family DNA polymerase family and is a replicative DNA polymerase in Crenarchaea. PolB1 is responsible for the DNA replication of both the leading and lagging strands in the thermophilic crenarchaeon Sulfolobus acidocaldarius. Recently, two subunits, PolB1-binding protein (PBP)1 and PBP2, were identified in Saccharolobus solfataricus. Previous in vitro studies suggested that PBP1 and PBP2 influence the core activity of apoenzyme PolB1 (apo-PolB1). PBP1 contains a C-terminal acidic tail and modulates the strand-displacement synthesis activity of PolB1 during the synthesis of Okazaki fragments. PBP2 modestly enhances the DNA polymerase activity of apo-PolB1. These subunits are present in Sulfolobales, Acidilobales, and Desulfurococcales, which belong to Crenarchaea. However, it has not been determined whether these subunits are essential for the activity of apo-PolB1. In this study, we constructed a pbp1 deletion strain in S. acidocaldarius and characterized its phenotypes. However, a pbp2 deletion strain was not obtained, indicating that PBP2 is essential for replication by holoenzyme PolB1. A pbp1 deletion strain was sensitive to various types of DNA damage and exhibited an increased mutation rate, suggesting that PBP1 contribute to the repair or tolerance of DNA damage by holoenzyme PolB1. The results of our study suggest that PBP1 is important for DNA repair by holoenzyme PolB1 in S. acidocaldarius. Full article

Polyphosphates (polyP) are polymers of orthophosphate residues linked by high-energy phosphoanhydride bonds that are important in all domains of life and function in many different processes, including biofilm development. To study the effect of polyP in archaeal biofilm formation, our previously described Sa. solfataricus polyP (−) strain and a new polyP (−) S. acidocaldarius strain generated in this report were used. These two strains lack the polymer due to the overexpression of their respective exopolyphosphatase gene (ppx). Both strains showed a reduction in biofilm formation, decreased motility on semi-solid plates and a diminished adherence to glass surfaces as seen by DAPI (4′,6-diamidino-2-phenylindole) staining using fluorescence microscopy. Even though arlB (encoding the archaellum subunit) was highly upregulated in S. acidocardarius polyP (−), no archaellated cells were observed. These results suggest that polyP might be involved in the regulation of the expression of archaellum components and their assembly, possibly by affecting energy availability, phosphorylation or other phenomena. This is the first evidence indicating polyP affects biofilm formation and other related processes in archaea. Full article

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 10⁵ M⁻¹ s⁻¹ against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut. Full article

Factors that give enzymes stability, activity, and substrate recognition result from the combination of few weak molecular interactions, which can be difficult to study through rational protein engineering approaches. We used irrational random mutagenesis and in vivo selection to test if a β-glycosidase from the thermoacidophile Saccharolobus solfataricus (Ssβ-gly) could complement an Escherichia coli strain unable to grow on lactose. The triple mutant of Ssβ-gly (S26L, P171L, and A235V) was more active than the wild type at 85 °C, inactivated at this temperature almost 300-fold quicker, and showed a 2-fold higher k_cat on galactosides. The three mutations, which were far from the active site, were analyzed to test their effect at the structural level. Improved activity on galactosides was induced by the mutations. The S26L and P171L mutations destabilized the enzyme through the removal of a hydrogen bond and increased flexibility of the peptide backbone, respectively. However, the flexibility added by S26L mutation improved the activity at T > 60 °C. This study shows that random mutagenesis and biological selection allowed the identification of residues that are critical in determining thermal activity, stability, and substrate recognition. Full article