Hi-C/3C-seq Data Analysis for Prokaryotic Genomes with HiC-Pro.

Hi-C and 3C-seq are powerful tools to study the 3D genomes of bacteria and archaea, whose small cell sizes and growth conditions are often intractable to detailed microscopic analysis. However, the circularity of prokaryotic genomes requires a number of tricks for Hi-C/3C-seq data analysis. Here, I provide a practical guide to use the HiC-Pro pipeline for Hi-C/3C-seq data obtained from prokaryotes.

Metabolic activities of marine ammonia-oxidizing archaea orchestrated by quorum sensing.

Ammonia-oxidizing archaea (AOA) play crucial roles in marine carbon and nitrogen cycles by fixing inorganic carbon and performing the initial step of nitrification. Evaluation of carbon and nitrogen metabolism popularly relies on functional genes such as amoA and accA. Increasing studies suggest that quorum sensing (QS) mainly studied in biofilms for bacteria may serve as a universal communication and regulatory mechanism among prokaryotes; however, this has yet to be demonstrated in marine planktonic archaea. To bridge this knowledge gap, we employed a combination of metabolic activity markers (amoA, accA, and grs) to elucidate the regulation of AOA-mediated nitrogen, carbon processes, and their interactions with the surrounding heterotrophic population. Through co-transcription investigations linking metabolic markers to potential key QS genes, we discovered that QS molecules could regulate AOA's carbon, nitrogen, and lipid metabolisms under different conditions. Interestingly, specific AOA ecotypes showed a preference for employing distinct QS systems and a distinct QS circuit involving a typical population. Overall, our data demonstrate that QS orchestrates nitrogen and carbon metabolism, including the exchange of organic metabolites between AOA and surrounding heterotrophic bacteria, which has been previously overlooked in marine AOA research.

Adaptive traits of Nitrosocosmicus clade ammonia-oxidizing archaea.

Nitrification is a core process in the global nitrogen (N) cycle mediated by ammonia-oxidizing microorganisms, including ammonia-oxidizing archaea (AOA) as a key player. Although much is known about AOA abundance and diversity across environments, the genetic drivers of the ecophysiological adaptations of the AOA are often less clearly defined. This is especially true for AOA within the genus Nitrosocosmicus, which have several unique physiological traits (e.g., high substrate tolerance, low substrate affinity, and large cell size). To better understand what separates the physiology of Nitrosocosmicus AOA, we performed comparative genomics with genomes from 39 cultured AOA, including five Nitrosocosmicus AOA. The absence of a canonical high-affinity type ammonium transporter and typical S-layer structural genes was found to be conserved across all Nitrosocosmicus AOA. In agreement, cryo-electron tomography confirmed the absence of a visible outermost S-layer structure, which has been observed in other AOA. In contrast to other AOA, the cryo-electron tomography highlighted the possibility that Nitrosocosmicus AOA may possess a glycoprotein or glycolipid-based glycocalyx cell covering outer layer. Together, the genomic, physiological, and metabolic properties revealed in this study provide insight into niche adaptation mechanisms and the overall ecophysiology of members of the Nitrosocosmicus clade in various terrestrial ecosystems. IMPORTANCE: Nitrification is a vital process within the global biogeochemical nitrogen cycle but plays a significant role in the eutrophication of aquatic ecosystems and the production of the greenhouse gas nitrous oxide (N2O) from industrial agriculture ecosystems. While various types of ammonia-oxidizing microorganisms play a critical role in the N cycle, ammonia-oxidizing archaea (AOA) are often the most abundant nitrifiers in natural environments. Members of the genus Nitrosocosmicus are one of the prevalent AOA groups detected in undisturbed terrestrial ecosystems and have previously been reported to possess a range of physiological characteristics that set their physiology apart from other AOA species. This study provides significant progress in understanding these unique physiological traits and their genetic drivers. Our results highlight how physiological studies based on comparative genomics-driven hypotheses can contribute to understanding the unique niche of Nitrosocosmicus AOA.

Navigating the archaeal frontier: insights and projections from bioinformatic pipelines.

Archaea continues to be one of the least investigated domains of life, and in recent years, the advent of metagenomics has led to the discovery of many new lineages at the phylum level. For the majority, only automatic genomic annotations can provide information regarding their metabolic potential and role in the environment. Here, genomic data from 2,978 archaeal genomes was used to perform automatic annotations using bioinformatics tools, alongside synteny analysis. These automatic classifications were done to assess how good these different tools perform in relation to archaeal data. Our study revealed that even with lowered cutoffs, several functional models do not capture the recently discovered archaeal diversity. Moreover, our investigation revealed that a significant portion of archaeal genomes, approximately 42%, remain uncharacterized. In comparison, within 3,235 bacterial genomes, a diverse range of unclassified proteins is obtained, with well-studied organisms like Escherichia coli having a substantially lower proportion of uncharacterized regions, ranging from <5 to 25%, and less studied lineages being comparable to archaea with the range of 35-40% of unclassified regions. Leveraging this analysis, we were able to identify metabolic protein markers, thereby providing insights into the metabolism of the archaea in our dataset. Our findings underscore a substantial gap between automatic classification tools and the comprehensive mapping of archaeal metabolism. Despite advances in computational approaches, a significant portion of archaeal genomes remains unexplored, highlighting the need for extensive experimental validation in this domain, as well as more refined annotation methods. This study contributes to a better understanding of archaeal metabolism and underscores the importance of further research in elucidating the functional potential of archaeal genomes.

Thaumarchaeota from deep-sea methane seeps provide novel insights into their evolutionary history and ecological implications.

BACKGROUND: Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota mediate the rate-limiting step of nitrification and remove the ammonia that inhibits the aerobic metabolism of methanotrophs. However, the AOA that inhabit deep-sea methane-seep surface sediments (DMS) are rarely studied. Here, we used global DMS metagenomics and metagenome-assembled genomes (MAGs) to investigate the metabolic activity, evolutionary history, and ecological contributions of AOA. Expression of AOA-specific ammonia-oxidizing gene (amoA) was examined in the sediments collected from the South China Sea (SCS) to identify their active ammonia metabolism in the DMS. RESULTS: Our analysis indicated that AOA contribute > 75% to the composition of ammonia-utilization genes within the surface layers (above 30 cm) of global DMS. The AOA-specific ammonia-oxidizing gene was actively expressed in the DMS collected from the SCS. Phylogenomic analysis of medium-/high-quality MAGs from 18 DMS-AOA indicated that they evolved from ancestors in the barren deep-sea sediment and then expanded from the DMS to shallow water forming an amoA-NP-gamma clade-affiliated lineage. Molecular dating suggests that the DMS-AOA origination coincided with the Neoproterozoic oxidation event (NOE), which occurred ~ 800 million years ago (mya), and their expansion to shallow water coincided with the Sturtian glaciation (~ 713 mya). Comparative genomic analysis suggests that DMS-AOA exhibit higher requirement of carbon source for protein synthesis with enhanced genomic capability for osmotic regulation, motility, chemotaxis, and utilization of exogenous organic compounds, suggesting it could be more heterotrophic compared with other lineages. CONCLUSION: Our findings provide new insights into the evolutionary history of AOA within the Thaumarchaeota, highlighting their critical roles in nitrogen cycling in the global DMS ecosystems. Video Abstract.

The metagenomic landscape of a high-altitude geothermal spring in Tajikistan reveals a novel Desulfurococcaceae member, Zestomicrobium tamdykulense gen. nov., sp. nov.

Metagenomic analysis was conducted to assess the microbial community in the high-altitude Tamdykul geothermal spring in Tajikistan. This analysis yielded six high-quality bins from the members of Thermaceae, Aquificaceae, and Halothiobacillaceae, with a 41.2%, 19.7%, and 18.1% share in the total metagenome, respectively. Minor components included Schleiferia thermophila (1.6%) and members of the archaeal taxa Pyrobaculum (1.2%) and Desulfurococcaceae (0.7%). Further analysis of the metagenome-assembled genome (MAG) from the Desulfurococcaceae family (MAG002) revealed novel taxonomy with only 80.95% closest placement average nucleotide identity value to its most closely related member of the Desulfurococcaceae family, which is part of the Thermoproteota phylum comprising hyperthermophilic members widespread in geothermal environments. MAG002 consisted of 1.3 Mbp, distributed into 48 contigs with 1504 predicted coding sequences, had an average GC content of 41.3%, a completeness and contamination rate of 98.7% and 2.6%, respectively, and branched phylogenetically between the Ignisphaera and Zestosphaera lineages. Digital DNA-DNA hybridization values compared with Ignisphaera aggregans and Zestosphaera tikiterensis were 33.7% and 19.4%, respectively, suggesting that this MAG represented a novel species and genus. Its 16S rRNA gene contained a large 421 bp intron. It encodes a complete gluconeogenesis pathway involving a bifunctional fructose-1,6-bisphosphate phosphatase/aldolase; however, the glycolysis pathway is incomplete. The ribulose monophosphate pathway enzymes could be used for pentose synthesis. MAG002 encodes several hydrogen-evolving hydrogenases, with possible roles as hydrogen sinks during fermentation. We propose the name Zestomicrobium tamdykulense gen. nov. sp. nov. for this organism; it is the first thermophilic genome reported from Tajikistan.

An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon Methanococcus maripaludis.

BACKGROUND: The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory. RESULTS: This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes. CONCLUSIONS: Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

Microbial dynamics, chemical profile, and bioactive potential of diverse Egyptian marine environments from archaeological wood to soda lake.

Halophilic archaea are a unique group of microorganisms that thrive in high-salt environments, exhibiting remarkable adaptations to survive extreme conditions. Archaeological wood and El-Hamra Lake serve as a substrate for a diverse range of microorganisms, including archaea, although the exact role of archaea in archaeological wood biodeterioration remains unclear. The morphological and chemical characterizations of archaeological wood were evaluated using FTIR, SEM, and EDX. The degradation of polysaccharides was identified in Fourier transform infrared analysis (FTIR). The degradation of wood was observed through scanning electron microscopy (SEM). The energy dispersive X-ray spectroscopy (EDX) revealed the inclusion of minerals, such as calcium, silicon, iron, and sulfur, into archaeological wood structure during burial and subsequent interaction with the surrounding environment. Archaea may also be associated with detected silica in archaeological wood since several organosilicon compounds have been found in the crude extracts of archaeal cells. Archaeal species were isolated from water and sediment samples from various sites in El-Hamra Lake and identified as Natronococcus sp. strain WNHS2, Natrialba hulunbeirensisstrain WNHS14, Natrialba chahannaoensis strain WNHS9, and Natronococcus occultus strain WNHS5. Additionally, three archaeal isolates were obtained from archaeological wood samples and identified as Natrialba chahannaoensisstrain W15, Natrialba chahannaoensisstrain W22, and Natrialba chahannaoensisstrain W24. These archaeal isolates exhibited haloalkaliphilic characteristics since they could thrive in environments with high salinity and alkalinity. Crude extracts of archaeal cells were analyzed for the organic compounds using gas chromatography-mass spectrometry (GC-MS). A total of 59 compounds were identified, including free saturated and unsaturated fatty acids, saturated fatty acid esters, ethyl and methyl esters of unsaturated fatty acids, glycerides, phthalic acid esters, organosiloxane, terpene, alkane, alcohol, ketone, aldehyde, ester, ether, and aromatic compounds. Several organic compounds exhibited promising biological activities. FTIR spectroscopy revealed the presence of various functional groups, such as hydroxyl, carboxylate, siloxane, trimethylsilyl, and long acyl chains in the archaeal extracts. Furthermore, the archaeal extracts exhibited antioxidant effects. This study demonstrates the potential of archaeal extracts as a valuable source of bioactive compounds with pharmaceutical and biomedical applications.

Niche breadth specialisation impacts ecological and evolutionary adaptation following environmental change.

None declared.Conflicts of interestEcological theory predicts that organismal distribution and abundance depend on the ability to adapt to environmental change. It also predicts that eukaryotic specialists and generalists will dominate in extreme environments or following environmental change, respectively. This theory has attracted little attention in prokaryotes, especially in archaea, which drive major global biogeochemical cycles. We tested this concept in Thaumarchaeota using pH niche breadth as a specialisation factor. Responses of archaeal growth and activity to pH disturbance were determined empirically in manipulated, long-term, pH-maintained soil plots. The distribution of specialists and generalists was uneven over the pH range, with specialists being more limited to the extreme range. Nonetheless, adaptation of generalists to environmental change was greater than that of specialists, except for environmental changes leading to more extreme conditions. The balance of generalism and specialism over longer timescales was further investigated across evolutionary history. Specialists and generalists diversified at similar rates, reflecting balanced benefits of each strategy, but a higher transition rate from generalists to specialists than the reverse was demonstrated, suggesting that metabolic specialism is more easily gained than metabolic versatility. This study provides evidence for a crucial ecological concept in prokaryotes, significantly extending our understanding of archaeal adaptation to environmental change.

A Haloarchaeal Transcriptional Regulator That Represses the Expression of CRISPR-Associated Genes.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide acquired heritable protection to bacteria and archaea against selfish DNA elements, such as viruses. These systems must be tightly regulated because they can capture DNA fragments from foreign selfish elements, and also occasionally from self-chromosomes, resulting in autoimmunity. Most known species from the halophilic archaeal genus Haloferax contain type I-B CRISPR-Cas systems, and the strongest hotspot for self-spacer acquisition by H. mediterranei was a locus that contained a putative transposable element, as well as the gene HFX_2341, which was a very frequent target for self-targeting spacers. To test whether this gene is CRISPR-associated, we investigated it using bioinformatics, deletion, over-expression, and comparative transcriptomics. We show that HFX_2341 is a global transcriptional regulator that can repress diverse genes, since its deletion results in significantly higher expression of multiple genes, especially those involved in nutrient transport. When over-expressed, HFX_2341 strongly repressed the transcript production of all cas genes tested, both those involved in spacer acquisition (cas1, 2 and 4) and those required for destroying selfish genetic elements (cas3 and 5-8). Considering that HFX_2341 is highly conserved in haloarchaea, with homologs that are present in species that do not encode the CRISPR-Cas system, we conclude that it is a global regulator that is also involved in cas gene regulation, either directly or indirectly.

The Saint-Leonard Urban Glaciotectonic Cave Harbors Rich and Diverse Planktonic and Sedimentary Microbial Communities.

The terrestrial subsurface harbors unique microbial communities that play important biogeochemical roles and allow for studying a yet unknown fraction of the Earth's biodiversity. The Saint-Leonard cave in Montreal City (Canada) is of glaciotectonic origin. Its speleogenesis traces back to the withdrawal of the Laurentide Ice Sheet 13,000 years ago, during which the moving glacier dislocated the sedimentary rock layers. Our study is the first to investigate the microbial communities of the Saint-Leonard cave. By using amplicon sequencing, we analyzed the taxonomic diversity and composition of bacterial, archaeal and eukaryote communities living in the groundwater (0.1 µm- and 0.2 µm-filtered water), in the sediments and in surface soils. We identified a microbial biodiversity typical of cave ecosystems. Communities were mainly shaped by habitat type and harbored taxa associated with a wide variety of lifestyles and metabolic capacities. Although we found evidence of a geochemical connection between the above soils and the cave's galleries, our results suggest that the community assembly dynamics are driven by habitat selection rather than dispersal. Furthermore, we found that the cave's groundwater, in addition to being generally richer in microbial taxa than sediments, contained a considerable diversity of ultra-small bacteria and archaea.

Metabolome fingerprinting reveals the presence of multiple nitrification inhibitors in biomass and root exudates of Thinopyrum intermedium.

Biological Nitrification Inhibition (BNI) encompasses primarily NH4 +-induced release of secondary metabolites to impede the rhizospheric nitrifying microbes from performing nitrification. The intermediate wheatgrass Thinopyrum intermedium (Kernza®) is known for exuding several nitrification inhibition traits, but its BNI potential has not yet been identified. We hypothesized Kernza® to evince BNI potential through the presence and release of multiple BNI metabolites. The presence of BNI metabolites in the biomass of Kernza® and annual winter wheat (Triticum aestivum) and in the root exudates of hydroponically grown Kernza®, were fingerprinted using HPLC-DAD and GC-MS/MS analyses. Growth bioassays involving ammonia-oxidizing bacteria (AOB) and archaea (AOA) strains were conducted to assess the influence of the crude root metabolome of Kernza® and selected metabolites on nitrification. In most instances, significant concentrations of various metabolites with BNI potential were observed in the leaf and root biomass of Kernza® compared to annual winter wheat. Furthermore, NH4 + nutrition triggered the exudation of various phenolic BNI metabolites. Crude root exudates of Kernza® inhibited multiple AOB strains and completely inhibited N. viennensis. Vanillic acid, caffeic acid, vanillin, and phenylalanine suppressed the growth of all AOB and AOA strains tested, and reduced soil nitrification, while syringic acid and 2,6-dihydroxybenzoic acid were ineffective. We demonstrated the considerable role of the Kernza® metabolome in suppressing nitrification through active exudation of multiple nitrification inhibitors.

Halofilins as emerging bactofilin families of archaeal cell shape plasticity orchestrators.

Bactofilins are rigid, nonpolar bacterial cytoskeletal filaments that link cellular processes to specific curvatures of the cytoplasmic membrane. Although homologs of bactofilins have been identified in archaea and eukaryotes, functional studies have remained confined to bacterial systems. Here, we characterize representatives of two families of archaeal bactofilins from the pleomorphic archaeon Haloferax volcanii, halofilin A (HalA) and halofilin B (HalB). HalA and HalB polymerize in vitro, assembling into straight bundles. HalA polymers are highly dynamic and accumulate at positive membrane curvatures in vivo, whereas HalB forms more static foci that localize in areas of local negative curvatures on the outer cell surface. Gene deletions and live-cell imaging show that halofilins are critical in maintaining morphological integrity during shape transition from disk (sessile) to rod (motile). Morphological defects in ΔhalA result in accumulation of highly positive curvatures in rods but not in disks. Conversely, disk-shaped cells are exclusively affected by halB deletion, resulting in flatter cells. Furthermore, while ΔhalA and ΔhalB cells imprecisely determine the future division plane, defects arise predominantly during the disk-to-rod shape remodeling. The deletion of halA in the haloarchaeon Halobacterium salinarum, whose cells are consistently rod-shaped, impacted morphogenesis but not cell division. Increased levels of halofilins enforced drastic deformations in cells devoid of the S-layer, suggesting that HalB polymers are more stable at defective S-layer lattice regions. Our results suggest that halofilins might play a significant mechanical scaffolding role in addition to possibly directing envelope synthesis.

Methanobrevibacter smithii cell variants in human physiology and pathology: A review.

Methanobrevibacter smithii (M. smithii), initially isolated from human feces, has been recognised as a distinct taxon within the Archaea domain following comprehensive phenotypic, genetic, and genomic analyses confirming its uniqueness among methanogens. Its diversity, encompassing 15 genotypes, mirrors that of biotic and host-associated ecosystems in which M. smithii plays a crucial role in detoxifying hydrogen from bacterial fermentations, converting it into mechanically expelled gaseous methane. In microbiota in contact with host epithelial mucosae, M. smithii centres metabolism-driven microbial networks with Bacteroides, Prevotella, Ruminococcus, Veillonella, Enterococcus, Escherichia, Enterobacter, Klebsiella, whereas symbiotic association with the nanoarchaea Candidatus Nanopusillus phoceensis determines small and large cell variants of M. smithii. The former translocate with bacteria to induce detectable inflammatory and serological responses and are co-cultured from blood, urine, and tissular abscesses with bacteria, prototyping M. smithii as a model organism for pathogenicity by association. The sources, mechanisms and dynamics of in utero and lifespan M. smithii acquisition, its diversity, and its susceptibility to molecules of environmental, veterinary, and medical interest still have to be deeply investigated, as only four strains of M. smithii are available in microbial collections, despite the pivotal role this neglected microorganism plays in microbiota physiology and pathologies.

Developing the script "degenerate primer 111" to enhance the coverage of universal primers for the small subunit rRNA gene on target microorganisms.

Amplifying small subunit (SSU) rRNA genes with universal primers in assessing microbial populations diversity, but target microorganisms are sometimes omitted due to inadequate primer coverage. Adding degenerate bases to primers can help, but existing methods are complex and time-consuming. This study introduces a user-friendly tool called "Degenerate primer 111" for adding degenerate bases to existing universal primers. By aligning one universal primer with one uncovered target microorganism's SSU rRNA gene, this tool iteratively generates a new primer, maximizing coverage for the target microorganisms. The tool was used to modify eight pairs of universal primers (515F Parada-806R Apprill, S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21, OP_F114-KP_R013, 27F-1492R, 341F-806R, OP_F066-KP_R013, 515F Parada-926R Quince, 616*F-1132R), and generated 29 new universal primers with increased coverage of specific target microorganisms without increasing coverage of non-target microorganisms. To verify the effectiveness of the improved primers, one set of original and improved primers (BA-515F-806R and BA-515F-806R-M1) was used to amplify DNA from the same sample, and high-throughput sequencing of the amplicons confirmed that the improved primers detected more microbial species compared to the original primers. Future researchers can use this tool to develop more personalized primers to meet their diverse microorganism detection needs.

Nitrous oxide production and consumption by marine ammonia-oxidizing archaea under oxygen depletion.

Ammonia-oxidizing archaea (AOA) are key players in the nitrogen cycle and among the most abundant microorganisms in the ocean, thriving even in oxygen-depleted ecosystems. AOA produce the greenhouse gas nitrous oxide (N2O) as a byproduct of ammonia oxidation. Additionally, the recent discovery of a nitric oxide dismutation pathway in the AOA isolate Nitrosopumilus maritimus points toward other N2O production and consumption pathways in AOA. AOA that perform NO dismutation when exposed to oxygen depletion, produce oxygen and dinitrogen as final products. Based on the transient accumulation of N2O coupled with oxygen accumulation, N2O has been proposed as an intermediate in this novel archaeal pathway. In this study, we spiked N2O to oxygen-depleted incubations with pure cultures of two marine AOA isolates that were performing NO dismutation. By using combinations of N compounds with different isotopic signatures (15NO2 - pool +44N2O spike and 14NO2 - pool +46N2O spike), we evaluated the N2O spike effects on the production of oxygen and the isotopic signature of N2 and N2O. The experiments confirmed that N2O is an intermediate in NO dismutation by AOA, distinguishing it from similar pathways in other microbial clades. Furthermore, we showed that AOA rapidly reduce high concentrations of spiked N2O to N2. These findings advance our understanding of microbial N2O production and consumption in oxygen-depleted settings and highlight AOA as potentially important key players in N2O turnover.

N-glycosylation in Archaea - Expanding the process, components and roles of a universal post-translational modification.

While performed by all three domains of life, N-glycosylation in Archaea is less well described than are the parallel eukaryal and bacterial processes. Still, what is known of the archaeal version of this universal post-translational modification reveals numerous seemingly domain-specific traits. Specifically, the biosynthesis of archaeal N-linked glycans relies on distinct pathway steps and components, rare sugars and sugar modifications, as well as unique lipid carriers upon which N-linked glycans are assembled. At the same time, Archaea possess the apparently unique ability to simultaneously modify their glycoproteins with very different N-linked glycans. In addition to these biochemical aspects of archaeal N-glycosylation, such post-translational modification has been found to serve a wide range of roles possibly unique to Archaea, including allowing these microorganisms to not only cope with the harsh physical conditions of the niches they can inhabit but also providing the ability to adapt to transient changes in such environments.

An animal charcoal contaminated cottage industry soil highlighted by halophilic archaea dominance and decimation of bacteria.

An animal charcoal contaminated cottage industry soil in Lagos, Nigeria (ACGT) was compared in an ex post facto study with a nearby unimpacted soil (ACGC). Hydrocarbon content was higher than regulatory limits in ACGT (180.2 mg/kg) but lower in ACGC (19.28 mg/kg). Heavy metals like nickel, cadmium, chromium and lead were below detection limit in ACGC. However, all these metals, except cadmium, were detected in ACGT, but at concentrations below regulatory limits. Furthermore, copper (253.205 mg/kg) and zinc (422.630 mg/kg) were above regulatory limits in ACGT. Next generation sequencing revealed that the procaryotic community was dominated by bacteria in ACGC (62%) while in ACGT archaea dominated (76%). Dominant phyla in ACGC were Euryarchaeota (37%), Pseudomonadota (16%) and Actinomycetota (12%). In ACGT it was Euryarchaeota (76%), Bacillota (9%), Pseudomonadota (7%) and Candidatus Nanohaloarchaeota (5%). Dominant Halobacteria genera in ACGT were Halobacterium (16%), Halorientalis (16%), unranked halophilic archaeon (13%) Salarchaeum (6%) and Candidatus Nanohalobium (5%), whereas ACGC showed greater diversity dominated by bacterial genera Salimicrobium (7%) and Halomonas (3%). Heavy metals homeostasis genes, especially for copper, were fairly represented in both soils but with bacterial taxonomic affiliations. Sites like ACGT, hitherto poorly studied and understood, could be sources of novel bioresources.

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.

Sulfated lactosyl archaeol (SLA) archaeosomes as a vaccine adjuvant.

Archaeosomes are liposomes traditionally comprised of total polar lipids or semi-synthetic glycerolipids of ether-linked isoprenoid phytanyl cores with varied glycol- and amino-head groups. We have developed a semi-synthetic archaeosome formulation based on sulfated lactosylarchaeol (SLA) that can be readily synthesized and easily formulated to induce robust humoral and cell-mediated immunity following systemic immunization, enhancing protection in models of infectious disease and cancer. Liposomes composed of SLA have been shown to be a safe and effective vaccine adjuvant to a multitude of antigens in preclinical studies including hepatitis C virus E1/E2 glycoproteins, hepatitis B surface antigen, influenza hemagglutinin, Rabbit Hemorrhagic Disease Virus antigens, and SARS-CoV-2 Spike antigens based on the ancestral strain as well as multiple variants of concern. With the COVID-19 pandemic highlighting the need for new vaccine technologies including adjuvants, this review outlines the studies conducted to date to support the development of SLA archaeosomes as a vaccine adjuvant.