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.

Archaea are vital components of the human microbiome, yet their study within the gastrointestinal tract (GIT) is limited by the scarcity of cultured representatives. Our study presents a method for the targeted enrichment and isolation of methanogenic archaea from human fecal samples. The procedure combines methane breath testing, in silico metabolic modeling, media optimization, FACS, dilution series, and genomic sequencing through Nanopore technology. Additional analyzes include the co-cultured bacteriome, comparative genomics of archaeal genomes, functional comparisons, and structure-based protein function prediction of unknown differential traits. Successful establishment of stable archaeal cultures from 14 out of 16 fecal samples yielded nine previously uncultivated strains, eight of which are absent from a recent archaeome genome catalog. Comparative genomic and functional assessments of Methanobrevibacter smithii and Candidatus Methanobrevibacter intestini strains from individual donors revealed features potentially associated with gastrointestinal diseases. Our work broadens available archaeal representatives for GIT studies, and offers insights into Candidatus Methanobrevibacter intestini genomes\' adaptability in critical microbiome contexts.

Haloarchaeal cultures were isolated from solar salterns of Goa and Tamil Nadu and designated as BS2, BBK2 and E3. These isolates grew with a characteristic bright orange to pink pigmentation and were capable of growing in media containing upto 25% (w/vol) NaCl. Whole genome sequencing (WGS) of the three haloarchaeal strains BS2, BBK2 and E3 indicated an assembled genomic size of 4.1 Mb, 3.8 Mb and 4 Mb with G + C content of 61.8, 65.6 and 59.8% respectively. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the archaeal isolates belong to Haloarcula, Haloferax and Halogeometricum genera. Haloarcula rubripromontorii BS2  was predicted to have 4292 genes with 4242 CDS regions, 46 tRNAs, 6 rRNAs and 3 misc_RNAs. In case of Haloferax lucentense  BBK2,, 3840 genes with 3780 CDS regions were detected along with 52 tRNAs, 5 rRNAs and 3 misc_RNAs. Halogeometricum borinquense  E3 contained 4101 genes, 4043 CDS regions, 52 tRNAs, 4 rRNAs, and 2 misc_RNAs. The functional annotation and curation of the haloarchaeal genome, revealed C50 carotenoid biosynthetic genes like phytoene desaturase/carotenoid 3\' -4\' desaturase (crtI), lycopene elongase (ubiA/lyeJ) and carotenoid biosynthesis membrane protein (cruF) in the three isolates. Whereas crtD (C-3\',4\' desaturase), crtY (lycopene cyclase) and brp/blh (β-carotene dioxygenase) genes were identified only in BS2.

A mesophilic, hyperacidophilic archaeon, strain M1T, was isolated from a rock sample from Vulcano Island, Italy. Cells of this organism were cocci with an average diameter of 1 µm. Some cells possessed filaments. The strain grew in the range of temperatures between 15 and 52 °C and pH 0.5-4.0 with growth optima at 40 °C and pH 1.0. Strain M1T was aerobic and chemoorganotrophic, growing on complex substrates, such as casamino acids, trypticase, tryptone, yeast and beef extracts. No growth at expenses of oxidation of elemental sulphur or reduced sulphur compounds, pyrite, or ferrous sulphate was observed. The core lipids were glycerol dibiphytanyl glycerol tetraether lipids (membrane spanning) with 0 to 4 cyclopentane moieties and archaeol, with trace amounts of hydroxy archaeol. The dominant quinone was MK-7 : 7. The genome size of M1T was 1.67 Mbp with a G+C content of 39.76 mol%, and both characteristics were well within the common range for Thermoplasmatales. The phylogenetic analysis based on 16S rRNA gene sequence placed the strain M1T within the order Thermoplasmatales with sequence identities of 90.9, 90.3 and 90.5% to the closest SSU rRNA gene sequences from organisms with validly published names, Thermoplasma acidophilum, Thermoplasma volcanium and Thermogymnomonas acidicola, respectively. Based on the results of our genomic, phylogenetic, physiological and chemotaxonomic studies, we propose that strain M1T (=DSM 116605T=JCM 36570T) represents a new genus and species, Oxyplasma meridianum gen. nov., sp. nov., within the order Thermoplasmatales.

The archaeal isolate J.3.6.1-F.2.7.3T was obtained from an anaerobic enrichment culture, where it may play an important role in methane production during pyrite formation. The new isolate formed a species-level clade with Methanospirillum hungatei strains GP1 and SK, which is separate from the type strain JF-1T. Cultivation-independent surveys indicate the occurrence of this phylogenetic group in sediments and anaerobic digesters. The abundance of this clade appears to be negatively affected by high nitrogen loads, indicating a sensitivity to certain nitrogen compounds that is not known in M. hungatei JF-1T. The relatively large core genome of this Methanospirillum clade is indicative of niche specialization and efficient control of horizontal gene transfer. Genes for nitrogenase and F420-dependent secondary alcohol dehydrogenase contribute to the metabolic versatility of this lineage. Characteristics of the new isolate such as the ability to utilize 2-propanol as an electron donor or the requirement for acetate as a carbon source are found also in the strains GP1 and SK, but not in the type strain M. hungatei JF-1T. Based on the genomic differences to related species, a new species within the genus Methanospirillum is proposed with the name M. purgamenti sp. nov. The determined phenotypic characteristics support this proposal and indicate a metabolic adaptation to a separate ecological niche.

Microbes in the sediments across a series of seamounts along the island arc of the Yap and Mariana trenches were investigated by metagenome. In this study, we reconstructed 107 metagenome-assembled genomes (MAGs), including 100 bacteria and 7 archaea. All the MAGs exhibited >75% completeness and <10% contamination, with 26 MAGs being classified as \'nearly complete\' (completeness >90%), while 50 falling within 80-90% range and 31 between 75-80% complete. Phylogenomic analysis revealed that 86% (n = 92) of these MAGs represented new taxa at different taxonomical levels. The species composition of these MAGs was most consistent with the previous reports, with the most abundant phyla being Proteobacteria (n = 39), Methylomirabilota (n = 27), and Nitrospirota (n = 7). These draft genomes provided novel data on species diversity and function in the seamount microbial community, which will provide reference data for extensive comparative genomic studies across crucial phylogenetic groups worldwide.

Recent metagenomic studies have identified numerous lineages of hydrogen-dependent, obligately methyl-reducing methanogens. Yet, only a few representatives have been isolated in pure culture. Here, we describe six new species with this capability in the family Methanosarcinaceae (order Methanosarcinales), which makes up a substantial fraction of the methanogenic community in arthropod guts. Phylogenomic analysis placed the isolates from cockroach hindguts into the genus Methanimicrococcus (M. hacksteinii, M. hongohii, and M. stummii) and the isolates from millipede hindguts into a new genus, Methanolapillus (M. africanus, M. millepedarum, and M. ohkumae). Members of this intestinal clade, which includes also uncultured representatives from termites and vertebrates, have substantially smaller genomes (1.6-2.2 Mbp) than other Methanosarcinales. Genome reduction was accompanied by the loss of the upper part of the Wood-Ljungdahl pathway, several energy-converting membrane complexes (Fpo, Ech, and Rnf), and various biosynthetic pathways. However, genes involved in the protection against reactive oxygen species (catalase and superoxide reductase) were conserved in all genomes, including cytochrome bd (CydAB), a high-affinity terminal oxidase that may confer the capacity for microaerobic respiration. Since host-associated Methanosarcinales are nested within omnivorous lineages, we conclude that the specialization on methyl groups is an adaptation to the intestinal environment.

DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology.

The roles of Asgard archaea in eukaryogenesis and marine biogeochemical cycles are well studied, yet their contributions in soil ecosystems remain unknown. Of particular interest are Asgard archaeal contributions to methane cycling in wetland soils. To investigate this, we reconstructed two complete genomes for soil-associated Atabeyarchaeia, a new Asgard lineage, and a complete genome of Freyarchaeia, and predicted their metabolism in situ. Metatranscriptomics reveals expression of genes for [NiFe]-hydrogenases, pyruvate oxidation and carbon fixation via the Wood-Ljungdahl pathway. Also expressed are genes encoding enzymes for amino acid metabolism, anaerobic aldehyde oxidation, hydrogen peroxide detoxification and carbohydrate breakdown to acetate and formate. Overall, soil-associated Asgard archaea are predicted to include non-methanogenic acetogens, highlighting their potential role in carbon cycling in terrestrial environments.

The knowledge of the different population-level processes operating within a species, and the genetic variability of the individual prokaryotic genomes, is key to understanding the adaptability of microbial populations. Here, we characterized the flexible genome of ammonia-oxidizing archaeal (AOA) populations using a metagenomic recruitment approach and long-read (PacBio HiFi) metagenomic sequencing. In the lower photic zone of the western Mediterranean Sea (75 m deep), the genomes Nitrosopelagicus brevis CN25 and Nitrosopumilus catalinensis SPOT1 had the highest recruitment values among available complete AOA genomes. They were used to analyse the diversity of flexible genes (variable from strain to strain) by examining the long-reads located within the flexible genomic islands (fGIs) identified by their under-recruitment. Both AOA genomes had a large fGI involved in the glycosylation of exposed structures, highly variable, and rich in glycosyltransferases. N. brevis had two fGIs related to the transport of phosphorus and ammonium respectively. N. catalinensis had fGIs involved in phosphorus transportation and metal uptake. A fGI5 previously reported as \'unassigned function\' in N. brevis could be associated with defense. These findings demonstrate that the microdiversity of marine microbe populations, including AOA, can be effectively characterized using an approach that incorporates third-generation sequencing metagenomics.