The use of thermostable fluorescent proteins for live imaging in Sulfolobus acidocaldarius.

Introduction: Among hyperthermophilic organisms, in vivo protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon S. acidocaldarius has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins for live imaging in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in S. acidocaldarius. Methods: We expressed various previously published and optimized thermostable fluorescent proteins along with fusion proteins of interest and analyzed the cells using flow cytometry and (thermo-) fluorescent microscopy. Results: Of the tested proteins, thermal green protein (TGP) exhibited the brightest fluorescence when expressed in Sulfolobus cells. By optimizing the linker between TGP and a protein of interest, we could additionally successfully fuse proteins with minimal loss of fluorescence. TGP-CdvB and TGP-PCNA1 fusions displayed localization patterns consistent with previous immunolocalization experiments. Discussion: These initial results in live protein localization in S. acidocaldarius at high temperatures, combined with recent advancements in thermomicroscopy, open new avenues in the field of archaeal cell biology. This progress finally enables localization experiments in thermophilic archaea, which have so far been limited to mesophilic organisms.

ICTV Virus Taxonomy Profile: Turriviridae 2024.

The family Turriviridae includes viruses with a dsDNA genome of 16-17 kbp. Virions are spherical with a diameter of approximately 75 nm and comprise a host-derived internal lipid membrane surrounded by a proteinaceous capsid shell. Members of the family Turriviridae infect extremophilic archaea of the genera Sulfolobus and Saccharolobus. Viral infection results in cell lysis for Sulfolobus turreted icosahedral virus 1 infection but other members of the family can be temperate. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Turriviridae, which is available at ictv.global/report/turriviridae.

Functional diversity in archaeal Hsp60: a molecular mosaic of Group I and Group II chaperonin.

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, ß, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αßγ, Hsp60αß, and Hsp60ß, 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 Hsp60ß 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, Hsp60ß, and Hsp60αß 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.

Sulfide oxidation by members of the Sulfolobales.

The oxidation of sulfur compounds drives the acidification of geothermal waters. At high temperatures (>80°C) and in acidic conditions (pH <6.0), oxidation of sulfide has historically been considered an abiotic process that generates elemental sulfur (S0) that, in turn, is oxidized by thermoacidophiles of the model archaeal order Sulfolobales to generate sulfuric acid (i.e. sulfate and protons). Here, we describe five new aerobic and autotrophic strains of Sulfolobales comprising two species that were isolated from acidic hot springs in Yellowstone National Park (YNP) and that can use sulfide as an electron donor. These strains significantly accelerated the rate and extent of sulfide oxidation to sulfate relative to abiotic controls, concomitant with production of cells. Yields of sulfide-grown cultures were ∼2-fold greater than those of S0-grown cultures, consistent with thermodynamic calculations indicating more available energy in the former condition than the latter. Homologs of sulfide:quinone oxidoreductase (Sqr) were identified in nearly all Sulfolobales genomes from YNP metagenomes as well as those from other reference Sulfolobales, suggesting a widespread ability to accelerate sulfide oxidation. These observations expand the role of Sulfolobales in the oxidative sulfur cycle, the geobiological feedbacks that drive the formation of acidic hot springs, and landscape evolution.

Exploring surface structures.

The surface layer of Sulfolobus acidocaldarius consists of a flexible but stable outer protein layer that interacts with an inner, membrane-bound protein.

Capturing chromosome conformation in Crenarchaea.

While there is a considerable body of knowledge regarding the molecular and structural biology and biochemistry of archaeal information processing machineries, far less is known about the nature of the substrate for these machineries-the archaeal nucleoid. In this article, we will describe recent advances in our understanding of the three-dimensional organization of the chromosomes of model organisms in the crenarchaeal phylum.

Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius.

Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single-particle cryo electron microscopy, cryo electron tomography, and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesise that jackknife-like conformational changes in SlaA play important roles in S-layer assembly.

Archaeal GPN-loop GTPases involve a lock-switch-rock mechanism for GTP hydrolysis.

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 SaGPN for motility appears to be due to its large footprint on the archaeal proteome.

Viruses of the Turriviridae: an emerging model system for studying archaeal virus-host interactions.

Viruses have played a central role in the evolution and ecology of cellular life since it first arose. Investigations into viral molecular biology and ecological dynamics have propelled abundant progress in our understanding of living systems, including genetic inheritance, cellular signaling and trafficking, and organismal development. As well, the discovery of viral lineages that infect members of all three domains suggest that these lineages originated at the earliest stages of biological evolution. Research into these viruses is helping to elucidate the conditions under which life arose, and the dynamics that directed its early development. Archaeal viruses have only recently become a subject of intense study, but investigations have already produced intriguing and exciting results. STIV was originally discovered in Yellowstone National Park and has been the focus of concentrated research. Through this research, a viral genetic system was created, a novel lysis mechanism was discovered, and the interaction of the virus with cellular ESCRT machinery was revealed. This review will summarize the discoveries within this group of viruses and will also discuss future work.

Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius.

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.

kLa based scale-up cultivation of the extremophilic archaeon Sulfolobus acidocaldarius: from benchtop to pilot scale.

The two major scale-up criteria in continuously stirred bioreactors are 1) constant aerated power input per volume (Pg/Vl), and 2) the volumetric O2 mass transfer coefficient (kla). However, Pg/Vl is only influenced by the stirrer geometry, stirrer speed, aeration and working volume, while the kla is additionally affected by physiochemical properties of the medium (temperature, pH, salt content, etc.), sparging of gas and also by the bioreactor design. The extremophilic archaeon Sulfolobus acidocaldarius, thriving at 75°C and pH 3.0, has the potential for many biotechnological applications. However, previous studies imply that the family Sulfolobaceae might be affected by higher oxygen concentration in the headspace (>26%). Hence, adequate oxygen supply without being toxic has to be ensured throughout the different scales. In this study, the scale-up criteria Pg/Vl and kla were analyzed and compared in a 2 L chemostat cultivation of S. acidocaldarius on a defined growth medium at 75°C and a pH value of 3.0, using two different types of spargers at the same aerated power input. The scale-up criterion kLa, ensuring a high specific growth rate as well as viability, was then used for scaleup to 20 L and 200 L. By maintaining a constant kla comparable dry cell weight, specific growth rate, specific substrate uptake rates and viability were observed between all investigated scales. This procedure harbors the potential for further scale-up to industrial size bioreactors.

Heat shock response in Sulfolobus acidocaldarius and first implications for cross-stress adaptation.

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 thß encoding the ß 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, TFEß, 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.

Crystal structure of thermostable acetaldehyde dehydrogenase from the hyperthermophilic archaeon Sulfolobus tokodaii.

Aldehyde dehydrogenase (ALDH) is widely distributed in nature and its characteristics have been examined. ALDH plays an important role in aldehyde detoxification. Sources of aldehydes include incomplete combustion and emissions from paints, linoleum and varnishes in the living environment. Acetaldehyde is also considered to be carcinogenic and toxic. Thermostable ALDH from the hyperthermophilic archaeon Sulfolobus tokodaii exhibits high activity towards acetaldehyde and has potential applications as a biosensor for acetaldehyde. Thermostable ALDH displays a unique and wide adaptability. Therefore, its crystal structure can provide new insights into the catalytic mechanism and potential applications of ALDHs. However, a crystal structure of a thermostable ALDH exhibiting high activity towards acetaldehyde has not been reported to date. In this study, crystals of recombinant thermostable ALDH from S. tokodaii were prepared and the crystal structure of its holo form was determined. A crystal of the enzyme was prepared and its structure in complex with NADP was determined at a resolution of 2.2 Å. This structural analysis may facilitate further studies on catalytic mechanisms and applications.

Membrane Adaptations and Cellular Responses of Sulfolobus acidocaldarius to the Allylamine Terbinafine.

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.

Stay or Go: Sulfolobales Biofilm Dispersal Is Dependent on a Bifunctional VapB Antitoxin.

A type II VapB14 antitoxin regulates biofilm dispersal in the archaeal thermoacidophile Sulfolobus acidocaldarius through traditional toxin neutralization but also through noncanonical transcriptional regulation. Type II VapC toxins are ribonucleases that are neutralized by their proteinaceous cognate type II VapB antitoxin. VapB antitoxins have a flexible tail at their C terminus that covers the toxin's active site, neutralizing its activity. VapB antitoxins also have a DNA-binding domain at their N terminus that allows them to autorepress not only their own promoters but also distal targets. VapB14 antitoxin gene deletion in S. acidocaldarius stunted biofilm and planktonic growth and increased motility structures (archaella). Conversely, planktonic cells were devoid of archaella in the ΔvapC14 cognate toxin mutant. VapB14 is highly conserved at both the nucleotide and amino acid levels across the Sulfolobales, extremely unusual for type II antitoxins, which are typically acquired through horizontal gene transfer. Furthermore, homologs of VapB14 are found across the Crenarchaeota, in some Euryarchaeota, and even bacteria. S. acidocaldarius vapB14 and its homolog in the thermoacidophile Metallosphaera sedula (Msed_0871) were both upregulated in biofilm cells, supporting the role of the antitoxin in biofilm regulation. In several Sulfolobales species, including M. sedula, homologs of vapB14 and vapC14 are not colocalized. Strikingly, Sulfuracidifex tepidarius has an unpaired VapB14 homolog and lacks a cognate VapC14, illustrating the toxin-independent conservation of the VapB14 antitoxin. The findings here suggest that a stand-alone VapB-type antitoxin was the product of selective evolutionary pressure to influence biofilm formation in these archaea, a vital microbial community behavior. IMPORTANCE Biofilms allow microbes to resist a multitude of stresses and stay proximate to vital nutrients. The mechanisms of entering and leaving a biofilm are highly regulated to ensure microbial survival, but are not yet well described in archaea. Here, a VapBC type II toxin-antitoxin system in the thermoacidophilic archaeon Sulfolobus acidocaldarius was shown to control biofilm dispersal through a multifaceted regulation of the archaeal motility structure, the archaellum. The VapC14 toxin degrades an RNA that causes an increase in archaella and swimming. The VapB14 antitoxin decreases archaella and biofilm dispersal by binding the VapC14 toxin and neutralizing its activity, while also repressing the archaellum genes. VapB14-like antitoxins are highly conserved across the Sulfolobales and respond similarly to biofilm growth. In fact, VapB14-like antitoxins are also found in other archaea, and even in bacteria, indicating an evolutionary pressure to maintain this protein and its role in biofilm formation.

The Impact of Single-Stranded DNA-Binding Protein SSB and Putative SSB-Interacting Proteins on Genome Integrity in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius.

The study of DNA repair in hyperthermophiles has the potential to elucidate the mechanisms of genome integrity maintenance systems under extreme conditions. Previous biochemical studies have suggested that the single-stranded DNA-binding protein (SSB) from the hyperthermophilic crenarchaeon Sulfolobus is involved in the maintenance of genome integrity, namely, in mutation avoidance, homologous recombination (HR), and the repair of helix-distorting DNA lesions. However, no genetic study has been reported that elucidates whether SSB actually maintains genome integrity in Sulfolobus in vivo. Here, we characterized mutant phenotypes of the ssb-deleted strain Δssb in the thermophilic crenarchaeon S. acidocaldarius. Notably, an increase (29-fold) in mutation rate and a defect in HR frequency was observed in Δssb, indicating that SSB was involved in mutation avoidance and HR in vivo. We characterized the sensitivities of Δssb, in parallel with putative SSB-interacting protein-encoding gene-deleted strains, to DNA-damaging agents. The results showed that not only Δssb but also Δalhr1 and ΔSaci_0790 were markedly sensitive to a wide variety of helix-distorting DNA-damaging agents, indicating that SSB, a novel helicase SacaLhr1, and a hypothetical protein Saci_0790, were involved in the repair of helix-distorting DNA lesions. This study expands our knowledge of the impact of SSB on genome integrity and identifies novel and key proteins for genome integrity in hyperthermophilic archaea in vivo.

Integrated conjugative plasmid drives high frequency chromosomal gene transfer in Sulfolobus islandicus.

Gene transfer in crenarchaea has been observed within natural and experimental populations of Sulfolobus. However, the molecular factors that govern how gene transfer and recombination manifest themselves in these populations is still unknown. In this study, we examine a plasmid-mediated mechanism of gene transfer in S. islandicus that results in localized high frequency recombination within the chromosome. Through chromosomal marker exchange assays with defined donors and recipients, we find that while bidirectional exchange occurs among all cells, those possessing the integrated conjugative plasmid, pM164, mobilize a nearby locus at a significantly higher frequency when compared to a more distal marker. We establish that traG is essential for this phenotype and that high frequency recombination can be replicated in transconjugants after plasmid transfer. Mapping recombinants through genomic analysis, we establish the distribution of recombinant tracts with decreasing frequency at increasing distance from pM164. We suggest the bias in transfer is a result of an Hfr (high frequency recombination)-like conjugation mechanism in this strain. In addition, we find recombinants containing distal non-selected recombination events, potentially mediated by a different host-encoded marker exchange (ME) mechanism.

Archaella Isolation.

Swimming archaea are propelled by a filamentous structure called the archaellum. The first step for the structural characterization of this filament is its isolation. Here we provide various methods that allow for the isolation of archaella filaments from well-studied archaeal model organisms. Archaella filaments have been successfully extracted from organisms belonging to different archaeal phyla, e.g., euryarchaeal methanogens such as Methanococcus voltae, and crenarchaeal hyperthermoacidophiles like Sulfolobus acidocaldarius. The filament isolation protocols that we provide in this chapter follow one of two strategies: either the filaments are sheared or extracted from whole cells by detergent extraction, prior to further final purification by centrifugation methods.

Biological role of the major AP (abasic site) endonuclease of an archaeon from geothermal environments.

Archaea and bacteria in geothermal environments are predicted to suffer DNA depurination in vivo at high rates, which raises questions regarding the biological roles of their abasic-site-repair enzymes. Gene deletion and enzymatic assay demonstrated that the saci_0015 gene of Sulfolobus acidocaldarius encodes an AP endonuclease (Apn) accounting for as much as 95% of the assayable activity in cell extracts and is not essential for viability. To identify genetic functions of this enzyme, deletion (ΔApn) strains were examined with respect to growth, spontaneous mutation, transformation by ssDNA containing an abasic site, and conjugation. Relative to its isogenic control, the ΔApn strain did not exhibit any change in growth rate or final cell density, rate or spectrum of spontaneous mutation, transformation by DNA containing an abasic site, or efficiency of DNA transfer and recombination. The apparent lack of genetic impact of removing the major AP endonuclease was unexpected and indicated that abasic sites are rarely bypassed directly by DNA polymerases in S. acidocaldarius. AP endonuclease deficiency had no obvious effect on survival of S. acidocaldarius under several test conditions, but it accelerated the death of cells at 4º C under illumination. Our results suggest that the normal level of AP endonuclease in S. acidocaldarius is well above the minimum required for growth and cell division but not for recovery from prolonged exposure to certain low-temperature conditions. This situation illustrates a biological challenge that has not been emphasized in experimental studies of extremophiles, i.e., the problem of long-term survival under "non-extreme" conditions.

Sulfolobus islandicus Employs Orc1-2-Mediated DNA Damage Response in Defense against Infection by SSV2.

All living organisms have evolved DNA damage response (DDR) strategies in coping with threats to the integrity of their genome. In response to DNA damage, Sulfolobus islandicus activates its DDR network in which Orc1-2, an ortholog of the archaeal Orc1/Cdc6 superfamily proteins, plays a central regulatory role. Here, we show that pretreatment with UV irradiation reduced virus genome replication in S. islandicus infected with the fusellovirus SSV2. Like treatment with UV or the DNA-damaging agent 4-nitroquinoline-1-oxide (NQO), infection with SSV2 facilitated the expression of orc1-2 and significantly raised the cellular level of Orc1-2. The inhibitory effect of UV irradiation on the virus DNA level was no longer apparent in the infected culture of an S. islandicus orc1-2 deletion mutant strain. On the other hand, the overexpression of orc1-2 decreased virus genomic DNA by ~102-fold compared to that in the parent strain. Furthermore, as part of the Orc1-2-mediated DDR response genes for homologous recombination repair (HRR), cell aggregation and intercellular DNA transfer were upregulated, whereas genes for cell division were downregulated. However, the HRR pathway remained functional in host inhibition of SSV2 genome replication in the absence of UpsA, a subunit of pili essential for intercellular DNA transfer. In agreement with this finding, lack of the general transcriptional activator TFB3, which controls the expression of the ups genes, only moderately affected SSV2 genome replication. Our results demonstrate that infection of S. islandicus by SSV2 triggers the host DDR pathway that, in return, suppresses virus genome replication. IMPORTANCE Extremophiles thrive in harsh habitats and thus often face a daunting challenge to the integrity of their genome. How these organisms respond to virus infection when their genome is damaged remains unclear. We found that the thermophilic archaeon Sulfolobus islandicus became more inhibitory to genome replication of the virus SSV2 after preinfection UV irradiation than without the pretreatment. On the other hand, like treatment with UV or other DNA-damaging agents, infection of S. islandicus by SSV2 triggers the activation of Orc1-2-mediated DNA damage response, including the activation of homologous recombination repair, cell aggregation and DNA import, and the repression of cell division. The inhibitory effect of pretreatment with UV irradiation on SSV2 genome replication was no longer observed in an S. islandicus mutant lacking Orc1-2. Our results suggest that DNA damage response is employed by S. islandicus as a strategy to defend against virus infection.