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Toxin-antitoxin (TA) systems are important for stress adaptation in prokaryotes, including persistence, antibiotic resistance, pathogenicity, and biofilm formation. Toxins can cause cell death, reversible growth stasis, and direct inhibition of crucial cellular processes through various mechanisms, while antitoxins neutralize the effects of toxins. In bacteria, these systems have been studied in detail, whereas their function in archaea remains elusive. During heat stress, the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibited an increase in the expression of several bicistronic type II vapBC TA systems, with the highest expression observed in the vapBC4 system. In the current study, we performed a comprehensive biochemical characterization of the VapBC4 TA system, establishing it as a bonafide type II toxin-antitoxin system. The VapC4 toxin is shown to have high-temperature catalyzed RNase activity specific for mRNA and rRNA, while the VapB4 antitoxin inhibits the toxic activity of VapC4 by interacting with it. VapC4 toxin expression led to heat-induced persister-like cell formation, allowing the cell to cope with the stress. Furthermore, this study explored the impact of vapBC4 deletion on biofilm formation, whereby deletion of vapC4 led to increased biofilm formation, suggesting its role in regulating biofilm formation. Thus, during heat stress, the liberated VapC4 toxin in cells could potentially signal a preference for persister cell formation over biofilm growth. Thus, our findings shed light on the diverse roles of the VapC4 toxin in inhibiting translation, inducing persister cell formation, and regulating biofilm formation in S. acidocaldarius , enhancing our understanding of TA systems in archaea., Importance: This research enhances our knowledge of toxin-antitoxin (TA) systems in archaea, specifically in the thermoacidophilic archaeon Sulfolobus acidocaldarius . TA systems are widespread in both bacterial and archaeal genomes, indicating their evolutionary importance. However, their exact functions in archaeal cellular physiology are still not well understood. This study sheds light on the complex roles of TA systems and their critical involvement in archaeal stress adaptation, including persistence and biofilm formation. By focusing on S. acidocaldarius , which lives in habitats with fluctuating temperatures that can reach up to 90°C, the study reveals the unique challenges and survival mechanisms of this organism. The detailed biochemical analysis of the VapBC4 TA system, and its crucial role during heat stress, provides insights into how extremophiles can survive in harsh conditions. The findings of this study show the various functions of the VapC4 toxin, including inhibiting translation, inducing persister-like cell formation, and regulating biofilm formation. This knowledge improves our understanding of TA systems in thermoacidophiles and has broader implications for understanding how microorganisms adapt to extreme environments., Competing Interests: The authors declare no conflict of interest.

Archaea produce various protein filaments with specialised functions. While some archaea produce only one type of filament, the archaeal model species Sulfolobus acidocaldarius generates four. These include rotary swimming propellers analogous to bacterial flagella (archaella), pili for twitching motility (Aap), adhesive fibres (threads), and filaments facilitating homologous recombination upon UV stress (UV pili). Here, we use cryo-electron microscopy to describe the structure of the S. acidocaldarius archaellum at 2.0 Å resolution, and update the structures of the thread and the Aap pilus at 2.7 Å and 2.6 Å resolution, respectively. We define features unique to archaella of the order Sulfolobales and compare their structure to those of Aap and threads in the context of the S-layer. We define distinct N-glycan patterns in the three filaments and identify a putative O-glycosylation site in the thread. Finally, we ascertain whether N-glycan truncation leads to structural changes in archaella and Aap., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

External stress disrupts the balance of protein homeostasis, necessitating the involvement of heat shock proteins (Hsps) in restoring equilibrium and ensuring cellular survival. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, lacks the conventional Hsp100, Hsp90, and Hsp70, relying solely on a single ATP-dependent Group II chaperonin, Hsp60, comprising three distinct subunits (α, β, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αβγ, Hsp60αβ, and Hsp60β, at temperatures of 60 °C, 75 °C, and 90 °C, respectively. This study delves into the intricacies of Hsp60 complexes in S. acidocaldarius, uncovering their ability to form oligomeric structures in the presence of ATP. The recognition of substrates by Hsp60 involves hydrophobic interactions, and the subsequent refolding process occurs in an ATP-dependent manner through charge-driven interactions. Furthermore, the Hsp60β homo-oligomeric complex can protect the archaeal and eukaryotic membrane from stress-induced damage. Hsp60 demonstrates nested cooperativity in ATP hydrolysis activity, where MWC-type cooperativity is nested within KNF-type cooperativity. Remarkably, during ATP hydrolysis, Hsp60β, and Hsp60αβ complexes exhibit a mosaic behavior, aligning with characteristics observed in both Group I and Group II chaperonins, adding a layer of complexity to their functionality., (© 2024 Federation of European Biochemical Societies.)

Amongst the major types of archaeal filaments, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus system (Ups) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Ups in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius. We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility., (© 2024. The Author(s).)

Type IV pili are filamentous appendages found in most bacteria and archaea, where they can support functions such as surface adhesion, DNA uptake, aggregation, and motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess PilT homologs, it was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility. Here, we use live-cell imaging, automated cell tracking, fluorescence imaging, and genetic manipulation to show that the hyperthermophilic archaeon Sulfolobus acidocaldarius exhibits twitching motility, driven by retractable adhesion (Aap) pili, under physiologically relevant conditions (75 °C, pH 2). Aap pili are thus capable of retraction in the absence of a PilT homolog, suggesting that the ancestral type IV pili in the last universal common ancestor (LUCA) were capable of retraction., (© 2024. The Author(s).)

Importance: GPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea, the mechanism by which these GTPases mediate their function is unknown. Our study on an archaeal representative from Sulfolobus acidocaldarius showed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement of Sa GPN for motility appears to be due to its large footprint on the archaeal proteome., Competing Interests: The authors declare no conflict of interest.

Sulfolobus acidocaldarius, a thermoacidophilic crenarchaeon, frequently encounters temperature fluctuations, oxidative stress, and nutrient limitations in its environment. Here, we employed a high-throughput transcriptomic analysis to examine how the gene expression of S. acidocaldarius changes when exposed to high temperatures (92 °C). The data obtained was subsequently validated using quantitative reverse transcription-PCR (qRT-PCR) analysis. Our particular focus was on genes that are involved in the heat shock response, type-II Toxin-Antitoxin systems, and putative transcription factors. To investigate how S. acidocaldarius adapts to multiple stressors, we assessed the expression of these selected genes under oxidative and nutrient stresses using qRT-PCR analysis. The results demonstrated that the gene thβ encoding the β subunit of the thermosome, as well as hsp14 and hsp20, play crucial roles in the majority of stress conditions. Furthermore, we observed overexpression of at least eight different TA pairs belonging to the type II TA systems under all stress conditions. Additionally, four common transcription factors: FadR, TFEβ, CRISPR loci binding protein, and HTH family protein were consistently overexpressed across all stress conditions, indicating their significant role in managing stress. Overall, this work provides the first insight into molecular players involved in the cross-stress adaptation of S. acidocaldarius., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Importance: Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius , which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism's lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response., Competing Interests: The authors declare no conflict of interest.

Cellular membranes are essential for compartmentalization, maintenance of permeability, and fluidity in all three domains of life. Archaea belong to the third domain of life and have a distinct phospholipid composition. Membrane lipids of archaea are ether-linked molecules, specifically bilayer-forming dialkyl glycerol diethers (DGDs) and monolayer-forming glycerol dialkyl glycerol tetraethers (GDGTs). The antifungal allylamine terbinafine has been proposed as an inhibitor of GDGT biosynthesis in archaea based on radiolabel incorporation studies. The exact target(s) and mechanism of action of terbinafine in archaea remain elusive. Sulfolobus acidocaldarius is a strictly aerobic crenarchaeon thriving in a thermoacidophilic environment, and its membrane is dominated by GDGTs. Here, we comprehensively analyzed the lipidome and transcriptome of S. acidocaldarius in the presence of terbinafine. Depletion of GDGTs and the accompanying accumulation of DGDs upon treatment with terbinafine were growth phase-dependent. Additionally, a major shift in the saturation of caldariellaquinones was observed, which resulted in the accumulation of unsaturated molecules. Transcriptomic data indicated that terbinafine has a multitude of effects, including significant differential expression of genes in the respiratory complex, motility, cell envelope, fatty acid metabolism, and GDGT cyclization. Combined, these findings suggest that the response of S. acidocaldarius to terbinafine inhibition involves respiratory stress and the differential expression of genes involved in isoprenoid biosynthesis and saturation.

The saccharification of cellulose and hemicellulose is essential for utilizing lignocellulosic biomass as a biofuel. While cellulose is composed of glucose only, hemicelluloses are composed of diverse sugars such as xylose, arabinose, glucose, and galactose. Sulfolobus acidocaldarius is a good potential candidate for biofuel production using hemicellulose as this archaeon simultaneously utilizes various sugars. However, S. acidocaldarius has to be manipulated because the enzyme that breaks down hemicellulose is not present in this species. Here, we engineered S. acidocaldarius to utilize xylan as a carbon source by introducing xylanase and β-xylosidase. Heterologous expression of β-xylosidase enhanced the organism's degradability and utilization of xylooligosaccharides (XOS), but the mutant still failed to grow when xylan was provided as a carbon source. S. acidocaldarius exhibited the ability to degrade xylan into XOS when xylanase was introduced, but no further degradation proceeded after this sole reaction. Following cell growth and enzyme reaction, S. acidocaldarius successfully utilized xylan in the synergy between xylanase and β-xylosidase.

Apurinic/apyrimidic (AP) sites are severe DNA damages and strongly block DNA extension by major DNA polymerases. Y-family DNA polymerases possess a strong ability to bypass AP sites and continue the DNA synthesis reaction, which is called translesion synthesis (TLS) activity. To investigate the effect of the molecular structure of the AP site on the TLS efficiency of Dbh, a Y-family DNA polymerase from Sulfolobus acidocaldarius , a series of different AP site analogues (various spacers) are used to characterize the bypass efficiency. We find that not only the molecular structure and atomic composition but also the number and position of AP site analogues determine the TLS efficiency of Dbh. Increasing the spacer length decreases TLS activity. The TLS efficiency also decreases when more than one spacer exists on the DNA template. The position of the AP site analogues is also an important factor for TLS. When the spacer is opposite to the first incorporated dNTPs, the TLS efficiency is the lowest, suggesting that AP sites are largely harmful for the formation of hydrogen bonds. These results deepen our understanding of the TLS activity of Y-family DNA polymerases and provide a biochemical basis for elucidating the TLS mechanism in Sulfolobus acidocaldarius cells.

In archaeal microorganisms, the compaction and organization of the chromosome into a dynamic but condensed structure is mediated by diverse chromatin-organizing proteins in a lineage-specific manner. While many archaea employ eukaryotic-type histones for nucleoid organization, this is not the case for the crenarchaeal model species Sulfolobus acidocaldarius and related species in Sulfolobales, in which the organization appears to be mostly reliant on the action of small basic DNA-binding proteins. There is still a lack of a full understanding of the involved proteins and their functioning. Here, a combination of in vitro and in vivo methodologies is used to study the DNA-binding properties of Sul12a, an uncharacterized small basic protein conserved in several Sulfolobales species displaying a winged helix-turn-helix structural motif and annotated as a transcription factor. Genome-wide chromatin immunoprecipitation and target-specific electrophoretic mobility shift assays demonstrate that Sul12a of S. acidocaldarius interacts with DNA in a non-sequence specific manner, while atomic force microscopy imaging of Sul12a-DNA complexes indicate that the protein induces structural effects on the DNA template. Based on these results, and a contrario to its initial annotation, it can be concluded that Sul12a is a novel chromatin-organizing protein.

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are archaeal monolayer membrane lipids that can provide a competitive advantage in extreme environments. Here, we identify a radical SAM protein, tetraether synthase (Tes), that participates in the synthesis of GDGTs. Attempts to generate a tes-deleted mutant in Sulfolobus acidocaldarius were unsuccessful, suggesting that the gene is essential in this organism. Heterologous expression of tes homologues leads to production of GDGT and structurally related lipids in the methanogen Methanococcus maripaludis (which otherwise does not synthesize GDGTs and lacks a tes homolog, but produces a putative GDGT precursor, archaeol). Tes homologues are encoded in the genomes of many archaea, as well as in some bacteria, in which they might be involved in the synthesis of bacterial branched glycerol dialkyl glycerol tetraethers., (© 2022. The Author(s).)

The spontaneous depurination of genomic DNA occurs frequently and generates apurinic/pyrimidinic (AP) site damage that is mutagenic or lethal to cells. Error-prone DNA polymerases are specifically responsible for the translesion synthesis (TLS) of specific DNA damage, such as AP site damage, generally with relatively low fidelity. The Y-family DNA polymerases are the main error-prone DNA polymerases, and they employ three mechanisms to perform TLS, including template-skipping, dNTP-stabilized misalignment, and misincorporation-misalignment. The bypass mechanism of the dinB homolog (Dbh), an archaeal Y-family DNA polymerase from Sulfolobus acidocaldarius , is unclear and needs to be confirmed. In this study, we show that the Dbh primarily uses template skipping accompanied by dNTP-stabilized misalignment to bypass AP site analogs, and the incorporation of the first nucleotide across the AP site is the most difficult. Furthermore, based on the reported crystal structures, we confirmed that three conserved residues (Y249, R333, and I295) in the little finger (LF) domain and residue K78 in the palm subdomain of the catalytic core domain are very important for TLS. These results deepen our understanding of how archaeal Y-family DNA polymerases deal with intracellular AP site damage and provide a biochemical basis for elucidating the intracellular function of these polymerases.

Heat shock proteins maintain protein homeostasis and facilitate the survival of an organism under stress. Archaeal heat shock machinery usually consists of only sHsps, Hsp70, and Hsp60. Moreover, Hsp70 is absent in thermophilic and hyperthermophilic archaea. In the absence of Hsp70, how aggregating protein substrates are transferred to Hsp60 for refolding remains elusive. Here, we investigated the crosstalk in the heat shock response pathway of thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In the present study, we biophysically and biochemically characterized one of the small heat shock proteins, Hsp14, of S. acidocaldarius. Moreover, we investigated its ability to interact with Hsp20 and Hsp60 to facilitate the substrate proteins' folding under stress conditions. Like Hsp20, we demonstrated that the dimer is the active form of Hsp14, and it forms an oligomeric storage form at a higher temperature. More importantly, the dynamics of the Hsp14 oligomer are maintained by rapid subunit exchange between the dimeric states, and the rate of subunit exchange increases with increasing temperature. We also tested the ability of Hsp14 to form hetero-oligomers via subunit exchange with Hsp20. We observed hetero-oligomer formation only at higher temperatures (50 °C-70 °C). Furthermore, experiments were performed to investigate the interaction between small heat shock proteins and Hsp60. We demonstrated an enthalpy-driven direct physical interaction between Hsp14 and Hsp60. Our results revealed that Hsp14 could transfer sHsp-captured substrate proteins to Hsp60, which then refolds them back to their active form., (© 2021 Federation of European Biochemical Societies.)

Synthetic biology requires well-characterized biological parts that can be combined into functional modules. One type of biological parts are transcriptional regulators and their cognate operator elements, which enable to either generate an input-specific response or are used as actuator modules. A range of regulators has already been characterized and used for orthogonal gene expression engineering, however, previous efforts have mostly focused on bacterial regulators. This work aims to design and explore the use of an archaeal TetR family regulator, FadR Sa from Sulfolobus acidocaldarius, in a bacterial system, namely Escherichia coli. This is a challenging objective given the fundamental difference between the bacterial and archaeal transcription machinery and the lack of a native TetR-like FadR regulatory system in E. coli. The synthetic σ 70 -dependent bacterial promoter proD was used as a starting point to design hybrid bacterial/archaeal promoter/operator regions, in combination with the mKate2 fluorescent reporter enabling a readout. Four variations of proD containing FadR Sa binding sites were constructed and characterized. While expressional activity of the modified promoter proD was found to be severely diminished for two of the constructs, constructs in which the binding site was introduced adjacent to the -35 promoter element still displayed sufficient basal transcriptional activity and showed up to 7-fold repression upon expression of FadR Sa . Addition of acyl-CoA has been shown to disrupt FadR Sa binding to the DNA in vitro. However, extracellular concentrations of up to 2 mM dodecanoate, subsequently converted to acyl-CoA by the cell, did not have a significant effect on repression in the bacterial system. This work demonstrates that archaeal transcription regulators can be used to generate actuator elements for use in E. coli, although the lack of ligand response underscores the challenge of maintaining biological function when transferring parts to a phylogenetically divergent host., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc -, hje -, hjm -, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm - and/or pina -deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δ pina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δ pina Δ hjc and Δ pina Δ hje exhibited severe cold sensitivity. Δ hjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

DNA methylations are one of the most well-known epigenetic modifications along with histone modifications and noncoding RNAs. They are found at specific sites along the DNA in all domains of life, with 5-mC and 6-mA/4-mC being well-characterized in eukaryotes and bacteria respectively, and they have not only been described as contributing to the structure of the double helix itself but also as regulators of DNA-based processes such as replication, transcription, and recombination. Different methods have been developed to accurately identify and/or map methylated motifs to decipher the involvement of DNA methylations in regulatory networks that affect the cellular state.Although DNA methylations have been detected along archaeal genomes, their involvement as regulators of DNA-based processes remains the least known. To highlight the importance of DNA methylations in the control of key cellular mechanisms and their dynamics in archaea cells, we have used single-molecule real-time (SMRT) sequencing. This sequencing technology allows the identification and direct mapping of the methylated motifs along the genome of an organism. In this chapter, we present a step-by-step protocol for detecting DNA methylations in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius using SMRT sequencing. This protocol can easily be adapted to other prokaryotes., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A well-functioning genetic system, which is important for studying gene functions in vivo, requires a transformation method, a vector system and a selection system. Sulfolobus acidocaldarius is a crenarchaeal model organism that grows optimally at 75 °C and a pH of 3. These extreme growth conditions cause some difficulties in developing a genetic system. With continuous efforts, versatile genetic tools have been developed for different species from the order of Sulfolobales. In this chapter, we describe the methods for the available genetic tools in S. acidocaldarius including a (1) transformation method, (2) pop in/pop out strategy to generate markerless deletion mutants and (3) a plasmid-based expression system., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Chromosome organization in archaea has long been enigmatic due, in part, to the typically small cell size of archaea and the extremophilic nature of many of the model archaeal species studies, rendering live-cell imaging technically challenging. To circumvent these problems, we recently applied chromosome conformation capture combined with biotin enrichment and deep sequencing (Hi-C) to members of hyperthermophilic archaeal genus Sulfolobus . Our optimized Hi-C protocol described here permits delineation of how Sulfolobus species organize their chromosomes. For complete details on the use and execution of this protocol, please refer to Takemata et al. (2019)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Sulfolobus acidocaldarius is a thermoacidophilic crenarchaeon with optimal growth at 80°C and pH 2 to 3. Due to its unique physiological properties, allowing life at environmental extremes, and the recent availability of genetic tools, this extremophile has received increasing interest for biotechnological applications. In order to elucidate the potential of tolerating process-related stress conditions, we investigated the response of S. acidocaldarius toward the industrially relevant organic solvent 1-butanol. In response to butanol exposure, biofilm formation of S. acidocaldarius was enhanced and occurred at up to 1.5% (vol/vol) 1-butanol, while planktonic growth was observed at up to 1% (vol/vol) 1-butanol. Confocal laser-scanning microscopy revealed that biofilm architecture changed with the formation of denser and higher tower-like structures. Concomitantly, changes in the extracellular polymeric substances with enhanced carbohydrate and protein content were determined in 1-butanol-exposed biofilms. Using scanning electron microscopy, three different cell morphotypes were observed in response to 1-butanol. Transcriptome and proteome analyses were performed comparing the response of planktonic and biofilm cells in the absence and presence of 1-butanol. In response to 1% (vol/vol) 1-butanol, transcript levels of genes encoding motility and cell envelope structures, as well as membrane proteins, were reduced. Cell division and/or vesicle formation were upregulated. Furthermore, changes in immune and defense systems, as well as metabolism and general stress responses, were observed. Our findings show that the extreme lifestyle of S. acidocaldarius coincided with a high tolerance to organic solvents. This study provides what may be the first insights into biofilm formation and membrane/cell stress caused by organic solvents in S. acidocaldarius IMPORTANCE Archaea are unique in terms of metabolic and cellular processes, as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic, and polyomics studies has provided deep insights into the physiology of some archaeal model organisms. In this study, we used S. acidocaldarius , which is adapted to the two extremes of low pH and high temperature, to study its tolerance and robustness as well as its global cellular response toward organic solvents, as exemplified by 1-butanol. We were able to identify biofilm formation as a primary cellular response to 1-butanol. Furthermore, the triggered cell/membrane stress led to significant changes in culture heterogeneity accompanied by changes in central cellular processes, such as cell division and cellular defense systems, thus suggesting a global response for the protection at the population level., (Copyright © 2021 Benninghoff et al.)

Homocitrate synthase (HCS) catalyzes the aldol condensation of α-ketoglutarate and acetyl coenzyme A to form homocitrate, which is the first committed step of lysine biosynthesis through the α-aminoadipate pathway in yeast, fungi, and some prokaryotes. We determined the crystal structure of a truncated form of HCS from a hyperthermophilic acidophilic archaeon, Sulfolobus acidocaldarius, which lacks the RAM (Regulation of amino acid metabolism) domain at the C terminus serving as the regulatory domain for the feedback inhibition by lysine, in complex with α-ketoglutarate, Mg 2+ , and CoA. This structure coupled with mutational analysis revealed that a subdomain, subdomain II, connecting the N-terminal catalytic domain and C-terminal RAM domain is involved in the recognition of acetyl-CoA. This is the first structural evidence of the function of subdomain II in the related enzyme family, which will lead to a better understanding of the catalytic mechanism of HCS. DATABASES: Structural data are available in the RCSB PDB database under the accession number 6KTQ., (© 2020 Federation of European Biochemical Societies.)

Chromosome conformation capture (3C) technologies have identified topologically associating domains (TADs) and larger A/B compartments as two salient structural features of eukaryotic chromosomes. These structures are sculpted by the combined actions of transcription and structural maintenance of chromosomes (SMC) superfamily proteins. Bacterial chromosomes fold into TAD-like chromosomal interaction domains (CIDs) but do not display A/B compartment-type organization. We reveal that chromosomes of Sulfolobus archaea are organized into CID-like topological domains in addition to previously described larger A/B compartment-type structures. We uncover local rules governing the identity of the topological domains and their boundaries. We also identify long-range loop structures and provide evidence of a hub-like structure that colocalizes genes involved in ribosome biogenesis. In addition to providing high-resolution descriptions of archaeal chromosome architectures, our data provide evidence of multiple modes of organization in prokaryotic chromosomes and yield insights into the evolution of eukaryotic chromosome conformation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius Notable observations are the frequent occurrence of ac 4 C nucleotides in thermophilic archaeal tRNAs, the presence of m 7 G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii ., (© 2020 Wolff et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Dbh is a Y-family translesion DNA polymerase from Sulfolobus acidocaldarius, an archaeal species that grows in harsh environmental conditions. Biochemically, Dbh displays a distinctive mutational profile, creating single-base deletion mutations at extraordinarily high frequencies (up to 50 %) in specific repeat sequences. In cells, however, Dbh does not appear to contribute significantly to spontaneous frameshifts in these same sequence contexts. This suggests that either the error-prone DNA synthesis activity of Dbh is reduced in vivo and/or Dbh is restricted from replicating these sequences. Here, we test the hypothesis that the propensity for Dbh to make single base deletion mutations is reduced through interaction with the S. acidocaldarius heterotrimeric sliding clamp processivity factor, PCNA-123. We first confirm that Dbh physically interacts with PCNA-123, with the interaction requiring both the PCNA-1 subunit and the C-terminal 10 amino acids of Dbh, which contain a predicted PCNA-interaction peptide (PIP) motif. This interaction stimulates the polymerase activity of Dbh, even on short, linear primer-template DNA, by increasing the rate of nucleotide incorporation. This stimulation requires an intact PCNA-123 heterotrimer and a DNA duplex length of at least 18 basepairs, the minimal length predicted from structural data to bind to both the polymerase and the clamp. Finally, we find that PCNA-123 increases the fidelity of Dbh on a single-base deletion hotspot sequence 3-fold by promoting an increase in the rate of correct, but not incorrect, nucleotide addition and propose that PCNA-123 induces Dbh to adopt a more active conformation that is less prone to creating deletions during DNA synthesis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Maltooligosyltrehalose synthase (MTSase) is a key enzyme for the production of trehalose from starch. Thermophilic MTSases offer advantages for trehalose production but suffer from low yield. In this study, directed evolution was used to increase the production of Sulfolobus acidocaldarius MTSase (SaMTSase) in Escherichia coli . Mutant libraries constructed using error-prone polymerase chain reaction were assessed using high-throughput activity assays. Three mutants with enhanced activities were obtained, the best of which (mutant D-4) exhibited 2.4 times greater activity than wild-type SaMTSase. The specific activity and catalytic efficiency of D-4 were also greater than those of wild-type SaMTSase. The D-4 activity (624.7 U·mL -1 ) produced in a 3 L fermenter was 2.0 times greater than that of wild-type SaMTSase. Because the same trehalose yield was obtained using an equal amount of either D-4 or wild-type SaMTSase activity, using D-4 will significantly lower the cost of trehalose production. The activities of the individual mutations present in the three SaMTSase mutants obtained using directed evolution were analyzed. Mutants F284V and T439A exhibited the greatest increases in enzyme activity. Homology models suggested that the decreased side-chain size, weakened hydrophobicity, and decreased interaction might enhance the flexibility of the loop containing catalytic residue Asp443, which was conducive to catalysis.

The UV-inducible pili system of Sulfolobales (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA to repair DNA double-strand breaks via homologous recombination. Substitution of the Sulfolobus acidocaldarius pilin subunits UpsA and UpsB with their homologs from Sulfolobus tokodaii showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of N -glycosylation in the recognition process. In addition, the N -glycan decorating the S-layer of S. tokodaii is different from the one of S. acidocaldarius Therefore, each Sulfolobus species seems to have developed a unique UpsA binding pocket and unique N- glycan composition to ensure aggregation and, consequently, also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination. IMPORTANCE Type IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The UV-inducible pili system of Sulfolobales (Ups) pili from the crenarchaeal Sulfolobales species are essential in establishing species-specific mating partners, thereby assisting in genome stability. With this work, we show that different Sulfolobus species have specific regions in their Ups pili subunits, which allow them to interact only with cells from the same species. Additionally, different Sulfolobus species have unique surface-layer N- glycosylation patterns. We propose that the unique features of each species allow the recognition of specific mating partners. This knowledge for the first time gives insights into the molecular basis of archaeal self-recognition., (Copyright © 2020 van Wolferen et al.)

Several species of Sulfolobales have been used as model organisms in the study of response mechanisms to ultraviolet (UV) irradiation in hyperthermophilic crenarchaea. To date, the transcriptional responses of genes involved in the initiation of DNA replication, transcriptional regulation, protein phosphorylation, and hypothetical function have been observed in Sulfolobales species after UV irradiation. However, due to the absence of knockout experiments, the functions of these genes under in situ UV irradiation have not yet been demonstrated. In the present study, we constructed five gene knockout strains (cdc6-2, tfb3, rio1, and two genes encoding the hypothetical proteins, Saci_0951 and Saci_1302) of Sulfolobus acidocaldarius and examined their sensitivities to UV irradiation. The knockout strains exhibited significant sensitivities to UV-B irradiation, indicating that the five UV-regulated genes play an important role in responses to UV irradiation in vivo. Furthermore, Δcdc6-2, Δrio1, ΔSaci_0951, and Δtfb3 were sensitive to a wide variety of helix-distorting DNA lesions, including UV-induced DNA damage, an intra-strand crosslink, and bulky adducts. These results reveal that cdc6-2, tfb3, rio1, and Saci_0951 are play more important roles in broad responses to helix-distorting DNA damage than in specific responses to UV irradiation.

The DNA repair mechanisms of hyperthermophiles can provide important insights for understanding how genetic information is maintained under extreme environments. Recent biochemical studies have identified a novel endonuclease in hyperthermophilic archaea, NucS/EndoMS, that acts on branched DNA substrates and mismatched bases. NucS/EndoMS is thought to participate in the DNA repair of helix-distorting DNA lesions, including UV-induced DNA damage and DNA adducts, and mismatched bases; however, the specific in vivo role of NucS/EndoMS in hyperthermophilic archaeal DNA repair has not been reported. To explore the role of this protein, we knocked out the nucS/endoMS gene of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. While the nucS/endoMS-deleted strain exhibited sensitivity to DNA adducts, it did not have high mutation rates or any sensitivity to UV irradiation. It has been proposed that the XPF endonuclease is involved in homologous recombination-mediated stalled-fork DNA repair. The xpf-deficient strain exhibited sensitivity to helix-distorting DNA lesions, but the sensitivity of the nucS/endoMS and xpf double knockout strain did not increase compared to that of the single knockout strains. We conclude that the endonuclease NucS/EndoMS works with XPF in homologous recombination-mediated stalled-fork DNA repair for the removal of helix-distorting DNA lesions in S. acidocaldarius.

Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life. Forkhead-associated (FHA) and von Willebrand type A (vWA) domains are involved in several phosphorylation-dependent processes of multiprotein complex assemblies. Although well-studied in eukaryotes and bacteria, the structural and functional contexts of these domains are not yet understood in Archaea. Here, we report the structural base for such an interacting pair of FHA and vWA domain-containing proteins, ArnA and ArnB, in the thermoacidophilic archaeon Sulfolobus acidocaldarius , where they act synergistically and negatively modulate motility. The structure of the FHA domain of ArnA at 1.75 Å resolution revealed that it belongs to the subclass of FHA domains, which recognizes double-pSer/pThr motifs. We also solved the 1.5 Å resolution crystal structure of the ArnB paralog vWA2, disclosing a complex topology comprising the vWA domain, a β-sandwich fold, and a C-terminal helix bundle. We further show that ArnA binds to the C terminus of ArnB, which harbors all the phosphorylation sites identified to date and is important for the function of ArnB in archaellum regulation. We also observed that expression levels of the archaellum components in response to changes in nutrient conditions are independent of changes in ArnA and ArnB levels and that a strong interaction between ArnA and ArnB observed during growth on rich medium sequentially diminishes after nutrient limitation. In summary, our findings unravel the structural features in ArnA and ArnB important for their interaction and functional archaellum expression and reveal how nutrient conditions affect this interaction., (© 2019 Hoffmann et al.)

Canonical single-stranded DNA-binding proteins (SSBs) are universally conserved helix-destabilizing proteins that play critical roles in DNA replication, recombination and repair. Many biochemical and genetic studies have demonstrated the importance of functional SSBs for all life forms. Herein, we report successful deletion of the gene encoding the only canonical SSB of the thermophilic crenarchaeon Sulfolobus acidocaldarius. Genomic sequencing of the ssb-deficient strain using illumina sequencing revealed that the canonical ssb gene is completely deleted from the genome of S. acidocaldarius. Phenotypic characterization demonstrated robust growth of the thermophilic archaeal cells lacking a canonical SSB, thereby demonstrating tolerance to the loss of a universal protein that is generally considered to be essential. Therefore, our work provides evidence that canonical SSBs are not essential for all life forms. Furthermore, on the basis of universal distribution and essentiality pattern of canonical SSBs, our findings can provide a conceptual understanding of the characteristics of early life forms before the last universal common ancestor., (© FEMS 2019.)

Fatty acid metabolism and its regulation are known to play important roles in bacteria and eukaryotes. By contrast, although certain archaea appear to metabolize fatty acids, the regulation of the underlying pathways in these organisms remains unclear. Here, we show that a TetR-family transcriptional regulator (FadR Sa ) is involved in regulation of fatty acid metabolism in the crenarchaeon Sulfolobus acidocaldarius. Functional and structural analyses show that FadR Sa binds to DNA at semi-palindromic recognition sites in two distinct stoichiometric binding modes depending on the operator sequence. Genome-wide transcriptomic and chromatin immunoprecipitation analyses demonstrate that the protein binds to only four genomic sites, acting as a repressor of a 30-kb gene cluster comprising 23 open reading frames encoding lipases and β-oxidation enzymes. Fatty acyl-CoA molecules cause dissociation of FadR Sa binding by inducing conformational changes in the protein. Our results indicate that, despite its similarity in overall structure to bacterial TetR-family FadR regulators, FadR Sa displays a different acyl-CoA binding mode and a distinct regulatory mechanism.

Maltooligosyl trehalose trehalohydrolase (MTHase, EC 3.2.1.141) catalyzes the release of trehalose, a novel food ingredient, by splitting the α-1,4-glucosidic linkage adjacent to the α-1,1-glucosidic linkage of maltooligosyl trehalose. However, the high-yield preparation of recombinant MTHase has not yet been reported. In this study, a codon-optimized synthetic gene encoding Sulfolobus acidocaldarius MTHase was expressed in Escherichia coli. In initial expression experiments conducted using pET-24a (+) and E. coli BL21 (DE3), the MTHase activity was 10.4 U/mL and a large amount of the expression product formed inclusion bodies. The familiar strategies, including addition of additives, co-expression with molecular chaperones, and expression with a fusion partner, failed to enhance soluble MTHase expression. Considering the intermolecular disulfide bond of MTHase, expression was investigated using a system comprising plasmid pET-32a (+) and host E. coli Origami (DE3), which is conducive to cytoplasmic disulfide bond formation. The MTHase activity increased to 55.0 U/mL, a 5.3-fold increase. Optimization of the induction conditions in a 3-L fermentor showed that when the lactose was fed at 0.2 g/L/h beginning at an OD 600 of 40 and the induction temperature was maintained at 30 °C, the MTHase activity reached a maximum of 204.6 U/mL. This is the first report describing a systematic effort to obtain high-efficiency MTHase production. The high yield obtained using this process provides the basis for the industrial-scale production of trehalose. This report is also expected to be valuable in the production of other enzymes containing disulfide bonds.

Archaea have many unique physiological features of which the lipid composition of their cellular membranes is the most striking. Archaeal ether-linked isoprenoidal membranes can occur as bilayers or monolayers, possess diverse polar head groups, and a multiplicity of ring structures in the isoprenoidal cores. These lipid structures are proposed to provide protection from the extreme temperature, pH, salinity, and nutrient-starved conditions that many archaea inhabit. However, many questions remain regarding the synthesis and physiological role of some of the more complex archaeal lipids. In this study, we identify a radical S -adenosylmethionine (SAM) protein in Sulfolobus acidocaldarius required for the synthesis of a unique cyclopentyl head group, known as calditol. Calditol-linked glycerol dibiphytanyl glycerol tetraethers (GDGTs) are membrane spanning lipids in which calditol is ether bonded to the glycerol backbone and whose production is restricted to a subset of thermoacidophilic archaea of the Sulfolobales order within the Crenarchaeota phylum. Several studies have focused on the enzymatic mechanism for the synthesis of the calditol moiety, but to date no protein that catalyzes this reaction has been discovered. Phylogenetic analyses of this putative calditol synthase (Cds) reveal the genetic potential for calditol-GDGT synthesis in phyla other than the Crenarchaeota, including the Korarchaeota and Marsarchaeota. In addition, we identify Cds homologs in metagenomes predominantly from acidic ecosystems. Finally, we demonstrate that deletion of calditol synthesis renders S. acidocaldarius sensitive to extremely low pH, indicating that calditol plays a critical role in protecting archaeal cells from acidic stress., Competing Interests: The authors declare no conflict of interest.

Sulfolobus species can grow on a variety of organic compounds as carbon and energy sources. These species degrade glucose to pyruvate by the modified branched Entner-Doudoroff pathway. We attempted to determine the differentially expressed genes (DEGs) under sugar-limited and sugar-rich conditions. RNA sequencing (RNA-seq) was used to quantify the expression of the genes and identify those DEGs between the S. acidocaldarius cells grown under sugar-rich (YT with glucose) and sugar-limited (YT only) conditions. The functions and pathways of the DEGs were examined using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Quantitative real-time PCR (qRT-PCR) was performed to validate the DEGs. Transcriptome analysis of the DSM 639 strain grown on sugar-limited and sugar-rich media revealed that 853 genes were differentially expressed, among which 481 were upregulated and 372 were downregulated under the glucose-supplemented condition. In particular, 70 genes showed significant changes in expression levels of ≥ twofold. GO and KEGG enrichment analyses revealed that the genes encoding components of central carbon metabolism, the respiratory chain, and protein and amino acid biosynthetic machinery were upregulated under the glucose condition. RNA-seq and qRT-PCR analyses indicated that the sulfur assimilation genes (Saci_2197-2204) including phosphoadenosine phosphosulfate reductase and sulfite reductase were significantly upregulated in the presence of glucose. The present study revealed metabolic networks in S. acidocaldarius that are induced in a glucose-dependent manner, improving our understanding of biomass production under sugar-rich conditions.

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

Exposure to UV light can result in severe DNA damage. The alternative general transcription factor (GTF) TFB3 has been proposed to play a key role in the UV stress response in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Reporter gene assays confirmed that tfb3 is upregulated 90-180 min after UV treatment. In vivo tagging and immunodetection of TFB3 confirmed the induced expression at 90 min. Analysis of a tfb3 insertion mutant showed that genes encoding proteins of the Ups pili and the Ced DNA importer are no longer induced in a tfb3 insertion mutant after UV treatment, which was confirmed by aggregation assays. Thus, TFB3 plays a crucial role in the activation of these genes. Genome wide transcriptome analysis allowed a differentiation between a TFB3-dependent and a TFB3-independent early UV response. The TFB3-dependent UV response is characterized by the early induction of TFB3, followed by TFB3-dependent expression of genes involved in e.g. Ups pili formation and the Ced DNA importer. Many genes were downregulated in the tfb3 insertion mutant confirming the hypothesis that TFB3 acts as an activator of transcription. The TFB3-independent UV response includes the repression of nucleotide metabolism, replication and cell cycle progression in order to allow DNA repair.

Non-coding RNAs (ncRNA) are involved in essential biological processes in all three domains of life. The regulatory potential of ncRNAs in Archaea is, however, not fully explored. In this study, RNA-seq analyses identified a set of 29 ncRNA transcripts in the hyperthermophilic archaeon Sulfolobus acidocaldarius that were differentially expressed in response to biofilm formation. The most abundant ncRNA of this set was found to be resistant to RNase R treatment (RNase R resistant RNA, RrrR(+)) due to duplex formation with a reverse complementary RNA (RrrR(-)). The deletion of the RrrR(+) gene resulted in significantly impaired biofilm formation, while its overproduction increased biofilm yield. RrrR(+) was found to act as an antisense RNA against the mRNA of a hypothetical membrane protein. The RrrR(+) transcript was shown to be stabilized by the presence of the RrrR(-) strand in S. acidocaldarius cell extracts. The accumulation of these RrrR duplexes correlates with an apparent absence of dsRNA degrading RNase III domains in archaeal proteins.

Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K M values of 0.4 μM for NADPH and 6.0 μM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

Two archaeal trehalase-like genes, Saci1250 and Saci1816, belonging to glycoside hydrolase family 15 (GH15) from the acidophilic Crenarchaeon Sulfolobus acidocaldarius were expressed in Escherichia coli. The gene products showed trehalose-hydrolyzing activities, and the names SaTreH1 and SaTreH2 were assigned to Saci1816 and Saci1250 gene products, respectively. These newly identified enzymes functioned within a narrow range of acidic pH values at elevated temperatures, which is similar to the behavior of Euryarchaeota Thermoplasma trehalases. SaTreH1 displayed high K M and k cat values, whereas SaTreH2 had lower K M and k cat values despite a high degree of identity in their primary structures. A mutation analysis indicated that two glutamic acid residues in SaTreH1, E374 and E574, may be involved in trehalase catalysis because SaTreH1 E374Q and E574Q showed greatly reduced trehalose-hydrolyzing activities. Additional mutations substituting G573 and H575 residues with serine and glutamic acid residues, respectively, to mimic the TVN1315 sequence resulted in a decrease in trehalase activity and thermal stability. Taken together, the results indicated that Crenarchaea trehalases adopt active site structures that are similar to Euryarchaeota enzymes but have distinct molecular features. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 trehalases as well as other family enzymes and will provide insights into archaeal trehalose metabolism.

Background: Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process., Results: Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L - 1 and 47.1 U·L - 1 ·h - 1 for SacPox and to 8700 U·L - 1 and 180.6 U·L - 1 ·h - 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications., Conclusions: In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.

Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources, such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus , only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius MW001 grown on peptides alone and peptides in the presence of d-xylose allowed for the identification of the ABC transporter for d-xylose and l-arabinose transport and the gaining of deeper insights into pentose catabolism under the respective growth conditions. The d-xylose/l-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the carbohydrate uptake transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea Single-gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for d-xylose and l-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and d-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD [also known as KDAD]; Saci_1939) was no longer able to catabolize d-xylose or l-arabinose, suggesting the absence of the aldolase-dependent branch in S. acidocaldarius IMPORTANCE Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications, as their optimal growth conditions resemble conditions used in certain biotechnologies such as industrial plant waste degradation. Because of its high genome stability, Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For use in (ligno)cellulose degradation, it was important to understand pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius MW001 on d-xylose and l-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose-inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation., (Copyright © 2018 American Society for Microbiology.)

The cell membrane of (hyper)thermophilic archaea, including the thermoacidophile Sulfolobus acidocaldarius, incorporates dibiphytanylglycerol tetraether lipids. The hydrophobic cores of such tetraether lipids can include up to eight cyclopentane rings. Presently, nothing is known of the biosynthesis of these rings. In this study, a series of S. acidocaldarius mutants deleted of genes currently annotated as encoding proteins involved in sugar/polysaccharide processing were generated and their glycolipids were considered. Whereas the glycerol-dialkyl-glycerol tetraether core of a S. acidocaldarius tetraether glycolipid considered here mostly includes four cyclopentane rings, in cells where the Saci_0421 or Saci_1201 genes had been deleted, species containing zero, two or four cyclopentane rings were observed. At the same time, in cells lacking Saci_0201, Saci_0275, Saci_1101, Saci_1249 or Saci_1706, lipids containing mostly four cyclopentane rings were detected. Although Saci_0421 and Saci_1201 are not found in proximity to other genes putatively involved in lipid biosynthesis, homologs of these sequences exist in other Archaea containing cyclopentane-containing tetraether lipids. Thus, Saci_0421 and Saci_1201 represent the first proteins described that somehow contribute to the appearance of cyclopentane rings in the core moiety of the S. acidocaldarius glycolipid considered here., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chromatin immunoprecipitation (ChIP) is a powerful method used for identifying genome-wide DNA-protein interactions in vivo. A large number of essential intracellular processes such as DNA replication, transcription regulation, chromatin stability, and others are all dependent on protein interactions with DNA. The DNA fragments enriched from the ChIP assay are analyzed by downstream applications, for example, microarray hybridization (ChIP-chip), quantitative PCR (ChIP-qPCR), or deep sequencing (ChIP-seq). This chapter presents a stepwise protocol for ChIP performed in hyperthermophilic archaea that we have successfully used in the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius , but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 10 4  ± 6.9 × 10 3 colonies/ μ g DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- ( phr- ) and arginine decarboxylase- ( argD- ) deficient strain of S. acidocaldarius . In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius .

In response to a variety of environmental cues, prokaryotes can switch between a motile and a sessile, biofilm-forming mode of growth. The regulatory mechanisms and signaling pathways underlying this switch are largely unknown in archaea but involve small winged helix-turn-helix DNA-binding proteins of the archaea-specific Lrs14 family. Here, we study the Lrs14 member AbfR1 of Sulfolobus acidocaldarius. Small-angle X-ray scattering data are presented, which are consistent with a model of dimeric AbfR1 in which dimerization occurs via an antiparallel coiled coil as suggested by homology modeling. Furthermore, solution structure data of AbfR1-DNA complexes suggest that upon binding DNA, AbfR1 induces deformations in the DNA. The wing residues tyrosine 84 and serine 87, which are phosphorylated in vivo, are crucial to establish stable protein-DNA contacts and their substitution with a negatively charged glutamate or aspartate residue inhibits formation of a nucleoprotein complex. Furthermore, mutation abrogates the cellular abundance and transcription regulatory function of AbfR1 and thus affects the resulting biofilm and motility phenotype of S. acidocaldarius. This work establishes a novel wHTH DNA-binding mode for Lrs14-like proteins and hints at an important role for protein phosphorylation as a signal transduction mechanism for the control of biofilm formation and motility in archaea., (© 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

In order to analyze in molecular terms how Sulfolobus genomes diverge, damage-induced mutations and natural polymorphisms (PMs) were identified in laboratory constructs and wild-type isolates, respectively, of Sulfolobus acidocaldarius Among wild-type isolates drawn from one local population, pairwise nucleotide divergence averaged 4 × 10 -6 , which is about 0.15% of the corresponding divergence reported for Sulfolobus islandicus The most variable features of wild-type S. acidocaldarius genomes were homopolymer (mononucleotide) tracts and longer tandem repeats, consistent with the spontaneous mutations that occur under laboratory conditions. Natural isolates, however, also revealed large insertions/deletions and inversions, which did not occur in any of the laboratory-manipulated strains. Several of the large insertions/deletions could be attributed to the integration or excision of mobile genetic elements (MGEs), and each MGE represented a distinct system of site-specific recombination. The mode of recombination associated with one MGE, a provirus related to Sulfolobus turreted icosahedral virus , was also seen in certain chromosomal inversions. Artificially induced mutations, non-MGE insertions/deletions, and small PMs exhibited different distributions over the genome, suggesting that large-scale patterning of Sulfolobus genomes begins early in the divergence process. Unlike induced mutations, natural base pair substitutions occurred in clusters, and one cluster exhibited properties expected of nonreciprocal recombination (gene conversion) between dispersed imperfect repeats. Taken together, the results identify simple replication errors, slipped-strand events promoted by tandem repeats, homologous recombination, and rearrangements promoted by MGEs as the primary sources of genetic variation for this extremely acidophilic archaeon in its geothermal environment. IMPORTANCE The optimal growth temperatures of hyperthermophilic archaea accelerate DNA decomposition, which is expected to make DNA repair especially important for their genetic stability, yet these archaea lack certain broadly conserved types of DNA repair proteins. In this study, the genome of the extreme thermoacidophile Sulfolobus acidocaldarius was found to be remarkably stable, accumulating few mutations in many (though not all) laboratory manipulations and in natural populations. Furthermore, all the genetic processes that were inferred to diversify these genomes also operate in mesophilic bacteria and eukaryotes. This suggests that a common set of mechanisms produces most of the genetic variation in all microorganisms, despite the fundamental differences in physiology, DNA repair systems, and genome structure represented in the three domains of life., (Copyright © 2017 American Society for Microbiology.)

L7Ae is a universal archaeal protein that recognizes and stabilizes kink-turn (k-turn) motifs in RNA substrates. These structural motifs are widespread in nature and are found in many functional RNA species, including ribosomal RNAs. Synthetic biology approaches utilize L7Ae/k-turn interactions to control gene expression in eukaryotes. Here, we present results of comprehensive RNA immunoprecipitation sequencing (RIP-Seq) analysis of genomically tagged L7Ae from the hyperthermophilic archaeon Sulfolobus acidocaldarius A large set of interacting noncoding RNAs was identified. In addition, several mRNAs, including the l7ae transcript, were found to contain k-turn motifs that facilitate L7Ae binding. In vivo studies showed that L7Ae autoregulates the translation of its mRNA by binding to a k-turn motif present in the 5' untranslated region (UTR). A green fluorescent protein (GFP) reporter system was established in Escherichia coli and verified conservation of L7Ae-mediated feedback regulation in Archaea Mobility shift assays confirmed binding to a k-turn in the transcript of nop5-fibrillarin , suggesting that the expression of all C/D box sRNP core proteins is regulated by L7Ae. These studies revealed that L7Ae-mediated gene regulation evolved in archaeal organisms, generating new tools for the modulation of synthetic gene circuits in bacteria. IMPORTANCE L7Ae is an essential archaeal protein that is known to structure ribosomal RNAs and small RNAs (sRNAs) by binding to their kink-turn motifs. Here, we utilized RIP-Seq methodology to achieve a first global analysis of RNA substrates for L7Ae. Several novel interactions with noncoding RNA molecules (e.g., with the universal signal recognition particle RNA) were discovered. In addition, L7Ae was found to bind to mRNAs, including its own transcript's 5' untranslated region. This feedback-loop control is conserved in most archaea and was incorporated into a reporter system that was utilized to control gene expression in bacteria. These results demonstrate that L7Ae-mediated gene regulation evolved originally in archaeal organisms. The feedback-controlled reporter gene system can easily be adapted for synthetic biology approaches that require strict gene expression control., (Copyright © 2017 Daume et al.)

The impact of a structured environment on genome evolution can be determined through comparative population genomics of species that live in the same habitat. Recent work comparing three genome sequences of Sulfolobus acidocaldarius suggested that highly structured, extreme, hot spring environments do not limit dispersal of this thermoacidophile, in contrast to other co-occurring Sulfolobus species. Instead, a high level of conservation among these three S. acidocaldarius genomes was hypothesized to result from rapid, global-scale dispersal promoted by low susceptibility to viruses that sets S. acidocaldarius apart from its sister Sulfolobus species. To test this hypothesis, we conducted a comparative analysis of 47 genomes of S. acidocaldarius from spatial and temporal sampling of two hot springs in Yellowstone National Park. While we confirm the low diversity in the core genome, we observe differentiation among S. acidocaldarius populations, likely resulting from low migration among hot spring "islands" in Yellowstone National Park. Patterns of genomic variation indicate that differing geological contexts result in the elimination or preservation of diversity among differentiated populations. We observe multiple deletions associated with a large genomic island rich in glycosyltransferases, differential integrations of the Sulfolobus turreted icosahedral virus, as well as two different plasmid elements. These data demonstrate that neither rapid dispersal nor lack of mobile genetic elements result in low diversity in the S. acidocaldarius genomes. We suggest instead that significant differences in the recent evolutionary history, or the intrinsic evolutionary rates, of sister Sulfolobus species result in the relatively low diversity of the S. acidocaldarius genome., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)