Cuniculiplasmataceae, their ecogenomic and metabolic patterns, and interactions with 'ARMAN'.

Recently, the order Thermoplasmatales was expanded through the cultivation and description of species Cuniculiplasma divulgatum and corresponding family Cuniculiplasmataceae. Initially isolated from acidic streamers, signatures of these archaea were ubiquitously found in various low-pH settings. Eight genomes with various levels of completeness are currently available, all of which exhibit very high sequence identities and genomic conservation. Co-existence of Cuniculiplasmataceae with archaeal Richmond Mine acidophilic nanoorganisms ('ARMAN')-related archaea representing an intriguing group within the "microbial dark matter" suggests their common fundamental environmental strategy and metabolic networking. The specific case of "Candidatus Mancarchaeum acidiphilum" Mia14 phylogenetically affiliated with "Ca. Micrarchaeota" from the superphylum "Ca. Diapherotrites" along with the presence of other representatives of 'DPANN' with significantly reduced genomes points at a high probability of close interactions between the latter and various Thermoplasmatales abundant in situ. This review critically assesses our knowledge on specific functional role and potential of the members of Cuniculiplasmataceae abundant in acidophilic microbiomes through the analysis of distribution, physiological and genomic patterns, and their interactions with 'ARMAN'-related archaea.

The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma.

Characterization of the replication initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus.

Eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at or very near origins in G1 phase, which licenses origin firing in S phase. The archaeal DNA replication machinery broadly resembles the eukaryal apparatus, though simpler in form. The eukaryotic replication initiator origin recognition complex (ORC), which serially recruits Cdc6 and other pre-RC proteins, comprises six components, Orc1-6. In archaea, a single gene encodes a protein similar to both the eukaryotic Cdc6 and the Orc1 subunit of the eukaryotic ORC, with most archaea possessing one to three Orc1/Cdc6 orthologs. Genome sequence analysis of the extreme acidophile Picrophilus torridus revealed a single Orc1/Cdc6 (PtOrc1/Cdc6). Biochemical analyses show MBP-tagged PtOrc1/Cdc6 to preferentially bind ORB (origin recognition box) sequences. The protein hydrolyzes ATP in a DNA-independent manner, though DNA inhibits MBP-PtOrc1/Cdc6-mediated ATP hydrolysis. PtOrc1/Cdc6 exists in stable complex with PCNA in Picrophilus extracts, and MBP-PtOrc1/Cdc6 interacts directly with PCNA through a PIP box near its C terminus. Furthermore, PCNA stimulates MBP-PtOrc1/Cdc6-mediated ATP hydrolysis in a DNA-dependent manner. This is the first study reporting a direct interaction between Orc1/Cdc6 and PCNA in archaea. The bacterial initiator DnaA is converted from an active to an inactive form by ATP hydrolysis, a process greatly facilitated by the bacterial ortholog of PCNA, the ß subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNA-interacting protein motifs in several archaeal PCNAs suggest the possibility of a similar mechanism of regulation existing in archaea. This mechanism may involve other yet to be identified archaeal proteins.

A moderately thermophilic mixed microbial culture for bioleaching of chalcopyrite concentrate at high pulp density.

Three kinds of samples (acid mine drainage, coal mine wastewater, and thermal spring) derived from different sites were collected in China. Thereafter, these samples were combined and then inoculated into a basal salts solution in which different substrates (ferrous sulfate, elemental sulfur, and chalcopyrite) served as energy sources. After that, the mixed cultures growing on different substrates were pooled equally, resulting in a final mixed culture. After being adapted to gradually increasing pulp densities of chalcopyrite concentrate by serial subculturing for more than 2 years, the final culture was able to efficiently leach the chalcopyrite at a pulp density of 20% (wt/vol). At that pulp density, the culture extracted 60.4% of copper from the chalcopyrite in 25 days. The bacterial and archaeal diversities during adaptation were analyzed by denaturing gradient gel electrophoresis and constructing clone libraries of the 16S rRNA gene. The results show that the culture consisted mainly of four species, including Leptospirillum ferriphilum, Acidithiobacillus caldus, Sulfobacillus acidophilus, and Ferroplasma thermophilum, before adapting to a pulp density of 4%. However, L. ferriphilum could not be detected when the pulp density was greater than 4%. Real-time quantitative PCR was employed to monitor the microbial dynamics during bioleaching at a pulp density of 20%. The results show that A. caldus was the predominant species in the initial stage, while S. acidophilus rather than A. caldus became the predominant species in the middle stage. F. thermophilum accounted for the greatest proportion in the final stage.

Internal Proton Transfer in the Activation of Heliorhodopsin.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, contains a single counterion of the protonated Schiff base, E108 in HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR). Upon light absorption, the M and O intermediates form in HeRs, as well as type-1 microbial rhodopsins, indicating that the proton transfer from the Schiff base leads to the activation of HeRs. The present flash photolysis study of TaHeR in the presence of a pH-sensitive dye showed that TaHeR contains a proton-accepting group (PAG) inside protein. Comprehensive mutation study of TaHeR found the E108D mutant abolishing the M formation, which is not only at pH 8, but also at pH 9 and 10. The lack of M observation does not originate from the short lifetime of the M intermediate in E108D, as FTIR spectroscopy revealed that a red-shifted K-like intermediate is long lived in E108D. It is likely that the K-like intermediate returns to the unphotolyzed state without internal proton transfer in E108D. E108 and D108 are the Schiff base counterions of the wild-type and E108D mutant TaHeR, respectively, whereas small difference in length of side chains determine internal proton transfer reaction from the Schiff base. Based on the present finding, we propose that the internal water cluster (four water molecules) constitutes PAG in the M intermediate of TaHeR. In the wild type TaHeR, a protonated water cluster is stabilized by forming a salt bridge with E108. In contrast, slightly shortened counterion (D108) cannot stabilize the protonated water cluster in E108D, and thus impairs internal proton transfer from the Schiff base.

Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea.

BACKGROUND: Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. RESULTS: We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. CONCLUSION: The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic differences, coupled with unknown differences in gene expression, distinguish these organisms enough to allow for co-existence. Overall this study reveals shared features of organisms from the Thermoplasmatales lineage and provides new insights into the functioning of AMD communities.

Quantitative tracking of isotope flows in proteomes of microbial communities.

Stable isotope probing (SIP) has been used to track nutrient flows in microbial communities, but existing protein-based SIP methods capable of quantifying the degree of label incorporation into peptides and proteins have been demonstrated only by targeting usually less than 100 proteins per sample. Our method automatically (i) identifies the sequence of and (ii) quantifies the degree of heavy atom enrichment for thousands of proteins from microbial community proteome samples. These features make our method suitable for comparing isotopic differences between closely related protein sequences, and for detecting labeling patterns in low-abundance proteins or proteins derived from rare community members. The proteomic SIP method was validated using proteome samples of known stable isotope incorporation levels at 0.4%, ∼50%, and ∼98%. The method was then used to monitor incorporation of (15)N into established and regrowing microbial biofilms. The results indicate organism-specific migration patterns from established communities into regrowing communities and provide insights into metabolism during biofilm formation. The proteomic SIP method can be extended to many systems to track fluxes of (13)C or (15)N in microbial communities.

Characterization of retinal chromophore and protonated Schiff base in Thermoplasmatales archaeon heliorhodopsin using solid-state NMR spectroscopy.

Heliorhodopsin (HeR) is a seven-helical transmembrane protein with a retinal chromophore that corresponds to a new rhodopsin family. HeR from the archaebacterium Thermoplasmatales archaeon (TaHeR) exhibits unique features, such as the inverted protein orientation in the membrane compared to other rhodopsins and a long photocycle. Here, we used solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate the 13C and 15N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20-13C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20-13C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15N RPSB/λmax plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15N chemical shift anisotropy (CSA) suggested that Ser112 and Ser234 polar residues distinguish the electronic environment tendencies of RPSB from those of other microbial rhodopsins. Our NMR results revealed that the retinal chromophore and the RPSB in TaHeR exhibit unique electronic environments.

The importance of biofilm formation for cultivation of a Micrarchaeon and its interactions with its Thermoplasmatales host.

Micrarchaeota is a distinctive lineage assigned to the DPANN archaea, which includes poorly characterised microorganisms with reduced genomes that likely depend on interactions with hosts for growth and survival. Here, we report the enrichment of a stable co-culture of a member of the Micrarchaeota (Ca. Micrarchaeum harzensis) together with its Thermoplasmatales host (Ca. Scheffleriplasma hospitalis), as well as the isolation of the latter. We show that symbiont-host interactions depend on biofilm formation as evidenced by growth experiments, comparative transcriptomic analyses and electron microscopy. In addition, genomic, metabolomic, extracellular polymeric substances and lipid content analyses indicate that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host. Our study of the cell biology and physiology of a Micrarchaeon and its host adds to our limited knowledge of archaeal symbioses.

Iron homeostasis and responses to iron limitation in extreme acidophiles from the Ferroplasma genus.

Extremely acidophilic archaea from the genus Ferroplasma inhabit iron-rich biomining environments and are important constituents of naturally occurring microbial consortia that catalyze the production of acid mine drainage. A combined bioinformatic, transcript profiling, and proteomic approach was used to elucidate iron homeostasis mechanisms in "F. acidarmanus" Fer1 and F. acidiphilum Y(T) . Bioinformatic analysis of the "F. acidarmanus" Fer1 genome sequence revealed genes encoding proteins hypothesized to be involved in iron-dependent gene regulation and siderophore biosynthesis; the Fhu and NRAMP cation acquisition systems; iron storage proteins; and the SUF machinery for the biogenesis of Fe-S clusters. A subset of homologous genes was identified on the F. acidiphilum Y(T) chromosome by direct PCR probing. In both strains, some of the genes appeared to be regulated in a ferrous/ferric iron-dependent manner, as indicated by RT-PCR. A detailed gel-based proteomics analysis of responses to iron depletion showed that a putative isochorismatase, presumably involved in siderophore biosynthesis, and the SufBCD system were upregulated under iron-limiting conditions. No evidence was obtained for iron sparing response during iron limitation. This study constitutes the first detailed investigation of iron homeostasis in extremely acidophilic archaea.

Proteomic analysis of the extremely thermoacidophilic archaeon Picrophilus torridus at pH and temperature values close to its growth limit.

The thermoacidophilic archaeon Picrophilus torridus belongs to the Thermoplasmatales order and is the most acidophilic organism known to date, growing under extremely acidic conditions around pH 0 (pH(opt) 1) and simultaneously at high temperatures up to 65°C. Some genome features that may be responsible for survival under these harsh conditions have been concluded from the analysis of its 1.55 megabase genome sequence. A proteomic map was generated for P. torridus cells grown to the mid-exponential phase. The soluble fraction of the cells was separated by isoelectric focusing in the pH ranges 4-7 and 3-10, followed by a two dimension (2D) on SDS-PAGE gels. A total of 717 Coomassie collodial-stained protein spots from both pH ranges (pH 4-7 and 3-10) were excised and subjected to LC-MS/MS, leading to the identification of 665 soluble protein spots. Most of the enzymes of the central carbon metabolism were identified on the 2D gels, corroborating biochemically the metabolic pathways predicted from the P. torridus genome sequence. The 2D master gels elaborated in this study represent useful tools for physiological studies of this thermoacidophilic organism. Based on quantitative 2D gel electrophoresis, a proteome study was performed to find pH- or temperature-dependent differences in the proteome composition under changing growth conditions. The proteome expression patterns at two different temperatures (50 and 70°C) and two different pH conditions (pH 0.5 and 1.8) were compared. Several proteins were up-regulated under most stress stimuli tested, pointing to general roles in coping with stress.

Zinc Binding to Heliorhodopsin.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, has an inverted membrane topology compared to animal and microbial rhodopsins, and no ion-transport activity. The slow photocycle of HeRs suggests a light-sensor function, although the function remains unknown. HeRs exhibit no specific binding of monovalent cations or anions. Despite this, ATR-FTIR spectroscopy in the present study demonstrates binding of Zn2+ to HeR from Thermoplasmatales archaeon (TaHeR). The biding of Zn2+ to 0.2 mM Kd is accompanied by helical structural perturbations without altering its color. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn2+-binding. Similar results were obtained for HeR 48C12. These findings suggest a possible modification of HeR function by Zn2+.

An archaeal iron-oxidizing extreme acidophile important in acid mine drainage.

A new species of Archaea grows at pH approximately 0.5 and approximately 40 degrees C in slime streamers and attached to pyrite surfaces at a sulfide ore body, Iron Mountain, California. This iron-oxidizing Archaeon is capable of growth at pH 0. This species represents a dominant prokaryote in the environment studied (slimes and sediments) and constituted up to 85% of the microbial community when solution concentrations were high (conductivity of 100 to 160 millisiemens per centimeter). The presence of this and other closely related Thermoplasmales suggests that these acidophiles are important contributors to acid mine drainage and may substantially impact iron and sulfur cycles.

Application of real-time PCR to monitor population dynamics of defined mixed cultures of moderate thermophiles involved in bioleaching of chalcopyrite.

To compare oxidative dissolution rates of chalcopyrite by different consortia of moderately thermophilic acidophiles, various defined mixed cultures of three bacteria Acidithiobacillus caldus s2, Leptospirillum ferriphilum YSK, and Sulfobacillus sp. LN and one archaeon Ferroplasma thermophilum L1 were studied in batch shake flask cultures incubated at 45 degrees C. Chalcopyrite dissolution was determined by measuring variations of soluble copper, ferric iron, and pH. Microbial population dynamics involved in bioleaching process were monitored using real-time quantitative polymerase chain reaction (PCR) technology. The complex consortia containing both chemoautotrophic (L. ferriphilum and At. caldus) and chemomixotrophic (Sulfobacillus LN and F. thermophilum) moderate thermophiles were found to be the most efficient in all of those tested. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transformations of iron and sulfur and transfer of organic compound, were considered to play a critical role in promoting chalcopyrite dissolution. The real-time PCR assay was reliable to analyze population dynamics of moderate thermophiles in bioleaching systems, and the analysis results were consistent with physiological characteristics of these strains.

Insights into archaeal chaperone machinery: a network-based approach.

Molecular chaperones are a diverse group of proteins that ensure proteome integrity by helping the proteins fold correctly and maintain their native state, thus preventing their misfolding and subsequent aggregation. The chaperone machinery of archaeal organisms has been thought to closely resemble that found in humans, at least in terms of constituent players. Very few studies have been ventured into system-level analysis of chaperones and their functioning in archaeal cells. In this study, we attempted such an analysis of chaperone-assisted protein folding in archaeal organisms through network approach using Picrophilus torridus as model system. The study revealed that DnaK protein of Hsp70 system acts as hub in protein-protein interaction network. However, DnaK protein was present only in a subset of archaeal organisms and absent from many archaea, especially members of Crenarchaeota phylum. Therefore, a similar network was created for another archaeal organism, Sulfolobus solfataricus, a member of Crenarchaeota. The chaperone network of S. solfataricus suggested that thermosomes played an integral part of hub proteins in archaeal organisms, where DnaK was absent. We further compared the chaperone network of archaea with that found in eukaryotic systems, by creating a similar network for Homo sapiens. In the human chaperone network, the UBC protein, a part of ubiquitination system, was the most important module, and interestingly, this system is known to be absent in archaeal organisms. Comprehensive comparison of these networks leads to several interesting conclusions regarding similarities and differences within archaeal chaperone machinery in comparison to humans.

Another extreme genome: how to live at pH 0.

Many Archaea live under conditions that challenge the physico-chemical limits to life: low or high temperature, extremes of pH, elevated pressure and high salt concentration. A recent paper reports the genome sequence of another record-setting archaeon, Picrophilus torridus, that thrives at 65 degrees C and pH 0. The genomic sequence provides several hints of the mechanisms used for adaptation to such hostile environment, but most secrets remain hidden and await further analysis to be disclosed.

Biology of archaea from a novel family Cuniculiplasmataceae (Thermoplasmata) ubiquitous in hyperacidic environments.

The order Thermoplasmatales (Euryarchaeota) is represented by the most acidophilic organisms known so far that are poorly amenable to cultivation. Earlier culture-independent studies in Iron Mountain (California) pointed at an abundant archaeal group, dubbed 'G-plasma'. We examined the genomes and physiology of two cultured representatives of a Family Cuniculiplasmataceae, recently isolated from acidic (pH 1-1.5) sites in Spain and UK that are 16S rRNA gene sequence-identical with 'G-plasma'. Organisms had largest genomes among Thermoplasmatales (1.87-1.94 Mbp), that shared 98.7-98.8% average nucleotide identities between themselves and 'G-plasma' and exhibited a high genome conservation even within their genomic islands, despite their remote geographical localisations. Facultatively anaerobic heterotrophs, they possess an ancestral form of A-type terminal oxygen reductase from a distinct parental clade. The lack of complete pathways for biosynthesis of histidine, valine, leucine, isoleucine, lysine and proline pre-determines the reliance on external sources of amino acids and hence the lifestyle of these organisms as scavengers of proteinaceous compounds from surrounding microbial community members. In contrast to earlier metagenomics-based assumptions, isolates were S-layer-deficient, non-motile, non-methylotrophic and devoid of iron-oxidation despite the abundance of methylotrophy substrates and ferrous iron in situ, which underlines the essentiality of experimental validation of bioinformatic predictions.

Beta-D-glucopyranosyl caldarchaetidylglycerol is the main lipid of the acidophilic, mesophilic, ferrous iron-oxidising archaeon Ferroplasma acidiphilum.

Chloroform-methanol-extractable lipids account for about 5% by weight of dry cells of the acidophilic, autotrophic, mesophilic, ferrous compound-oxidising, cell wall-less archaeon Ferroplasma acidiphilum strain Y(T), about 90% of these being contributed by phospholipids and glycophospholipids. The most abundant constituent (about 55% of total lipids) was purified by DEAE cellulose and silica gel column chromatography. By means of matrix-assisted laser desorption ionisation mass spectrometry, infrared spectroscopy, (1)H-nuclear magnetic resonance spectroscopy, and chemical degradation experiments it was established to be beta-D-glucopyranosyl caldarchaetidylglycerol, the isopranyl chains of which have a cyclopentane ring each.

Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum.

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model.

Characterization of the MCM homohexamer from the thermoacidophilic euryarchaeon Picrophilus torridus.

The typical archaeal MCM exhibits helicase activity independently in vitro. This study characterizes MCM from the euryarchaeon Picrophilus torridus. While PtMCM hydrolyzes ATP in DNA-independent manner, it displays very poor ability to unwind DNA independently, and then too only under acidic conditions. The protein exists stably in complex with PtGINS in whole cell lysates, interacting directly with PtGINS under neutral and acidic conditions. GINS strongly activates MCM helicase activity, but only at low pH. In consonance with this, PtGINS activates PtMCM-mediated ATP hydrolysis only at low pH, with the amount of ATP hydrolyzed during the helicase reaction increasing more than fifty-fold in the presence of GINS. While the stimulation of MCM-mediated helicase activity by GINS has been reported in MCMs from P.furiosus, T.kodakarensis, and very recently, T.acidophilum, to the best of our knowledge, this is the first report of an MCM helicase demonstrating DNA unwinding activity only at such acidic pH, across all archaea and eukaryotes. PtGINS may induce/stabilize a conducive conformation of PtMCM under acidic conditions, favouring PtMCM-mediated DNA unwinding coupled to ATP hydrolysis. Our findings underscore the existence of divergent modes of replication regulation among archaea and the importance of investigating replication events in more archaeal organisms.