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The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor. Yet, in 2016, a new type of thioredoxin reductase was discovered in Archaea which utilize instead a reduced deazaflavin cofactor (F 420 H 2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed that these two DFTRs are highly specific for F 420 H 2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that are dependent on NADPH (NTRs). A detailed structural analysis led to the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

It has been proposed that the superphylum of Asgard Archaea may represent a historical link between the Archaea and Eukarya. Following the discovery of the Archaea, it was soon appreciated that archaeal ribosomes were more similar to those of Eukarya rather than Bacteria. Coupled with other eukaryotic-like features, it has been suggested that the Asgard Archaea may be directly linked to eukaryotes. However, the genomes of Bacteria and non-Asgard Archaea generally organize ribosome-related genes into clusters that likely function as operons. In contrast, eukaryotes typically do not employ an operon strategy. To gain further insight into conservation of the r-protein genes, the genome order of conserved ribosomal protein (r-protein) coding genes was identified in 17 Asgard genomes (thirteen complete genomes and four genomes with less than 20 contigs) and compared with those found previously in non-Asgard archaeal and bacterial genomes. A universal core of two clusters of 14 and 4 cooccurring r-proteins, respectively, was identified in both the Asgard and non-Asgard Archaea. The equivalent genes in the E. coli version of the cluster are found in the S10 and spc operons. The large cluster of 14 r-protein genes ( uS19-uL22-uS3-uL29-uS17 from the S10 operon and uL14-uL24-uL5-uS14-uS8-uL6-uL18-uS5-uL30-uL15 from the spc operon) occurs as a complete set in the genomes of thirteen Asgard genomes (five Lokiarchaeotes, three Heimdallarchaeotes, one Odinarchaeote, and four Thorarchaeotes). Four less conserved clusters with partial bacterial equivalents were found in the Asgard. These were the L30e ( str operon in Bacteria) cluster, the L18e ( alpha operon in Bacteria) cluster, the S24e-S27ae-rpoE1 cluster, and the L31e , L12..L1 cluster. Finally, a new cluster referred to as L7ae was identified. In many cases, r-protein gene clusters/operons are less conserved in their organization in the Asgard group than in other Archaea. If this is generally true for nonribosomal gene clusters, the results may have implications for the history of genome organization. In particular, there may have been an early transition to or from the operon approach to genome organization. Other nonribosomal cellular features may support different relationships. For this reason, it may be important to consider ribosome features separately., Competing Interests: All authors declare they have no competing interests. LAK and ELS were student volunteers on this work. LAK was a fresh graduate from Clements High School, and ELS had just completed his 10th grade at Obra D. Tompkins High School, who volunteered with the group of Dr. George E. Fox at the University of Houston., (Copyright © 2023 Madhan R. Tirumalai et al.)

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N 2 ) to ammonia, with some also containing vanadium (V) and iron-only (Fe) nitrogenases that lack Mo. Among diazotrophs, the regulation and usage of the alternative V-nitrogenase and Fe-nitrogenase in methanogens are largely unknown. Methanosarcina acetivorans contains nif , vnf , and anf gene clusters encoding putative Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. This study investigated nitrogenase expression and growth by M. acetivorans in response to fixed nitrogen, Mo/V availability, and CRISPRi repression of the nif , vnf , and/or anf gene clusters. The availability of Mo and V significantly affected growth of M. acetivorans with N 2 but not with NH 4 Cl. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N 2 in medium containing Mo, and the slowest growth in medium lacking Mo and V. qPCR analysis revealed the transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo, whereas vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being key. Immunoblot analysis revealed Mo-nitrogenase is detected when fixed nitrogen is depleted regardless of Mo availability, while V-nitrogenase and Fe-nitrogenase are detected only in the absence of fixed nitrogen and Mo. CRISPRi repression studies revealed that V-nitrogenase and/or Fe-nitrogenase are required for Mo-independent diazotrophy, and unexpectedly that the expression of Mo-nitrogenase is also required. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and dependent on Mo-nitrogenase expression. IMPORTANCE Methanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. Methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, including simultaneously. The results reveal components of Mo-nitrogenase regulate or are needed to produce V-nitrogenase and Fe-nitrogenase in methanogens, a result not seen in bacteria. Overall, this study provides a foundation to understand the assembly, regulation, and activity of the alternative nitrogenases in methanogens., Competing Interests: The authors declare no conflict of interest.

Proteases and lipases are significant groups of enzymes for commercialization at the global level. Earlier, the industries depended on mesophilic proteases and lipases, which remain nonfunctional under extreme conditions. The discovery of extremophilic microorganisms, especially those belonging to haloarchaea, paved a new reserve of industrially competent extremozymes. Haloarchaea or halophilic archaea are polyextremophiles of domain Archaea that grow at high salinity, elevated temperature, pH range (pH 6-12), and low a w . Interestingly, haloarchaeal proteolytic and lipolytic enzymes also perform their catalytic function in the presence of 4-5 M NaCl in vivo and in vitro. Also, they are of great interest to study due to their capacity to function and are active at elevated temperatures, tolerance to pH extremes, and in non-aqueous media. In recent years, advances have been achieved in various aspects of genomic/molecular expression methods involving homologous and heterologous processes for the overproduction of these extremozymes and their characterization from haloarchaea. A few protease and lipase extremozymes have been successfully expressed in prokaryotic systems, especially E.coli, and enzyme modification techniques have improved the catalytic properties of the recombinant enzymes. Further, in-silico methods are currently applied to elucidate the structural and functional features of salt-stable protease and lipase in haloarchaea. In this review, the production and purification methods, catalytic and biochemical properties and biotechnological applications of haloextremozymes proteases and lipases are summarized along with recent advancements in overproduction and characterization of these enzymes, concluding with the directions for further in-depth research on proteases and lipases from haloarchaea., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Dichlorodiphenyltrichloroethane (DDT), a persistent organic pollutant, has known effects on natural microbes. However, its effects on soil ammonia-oxidizing microbes, significant contributors to soil ammoxidation, remain unexplored. To address this, we conducted a 30-day microcosm experiment to systematically study the effects of DDT contamination on soil ammonia oxidation and the communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Our findings revealed that DDT inhibited soil ammonia oxidation in the early stage (0-6 days), but it gradually recovered after 16 days. The copy numbers of amoA gene of AOA decreased in all DDT-treated groups from 2 to 10 days, while that of AOB decreased from 2 to 6 days but increased from 6 to 10 days. DDT influenced the diversity and community composition of AOA but had no significant effect on AOB. Further, the dominant AOA communities comprised uncultured&#95;ammonia-oxidizing&#95;crenarchaeote and Nitrososphaera sp. JG1: while the abundance of the latter significantly and negatively correlated with NH 4 + -N (P ≤ 0.001), DDT (0.001 < P ≤ 0.01), and DDD (0.01 < P ≤ 0.05) and positively correlated with NO 3 - -N (P ≤ 0.001), that of the former significantly and positively correlated with DDT (P ≤ 0.001), DDD (P ≤ 0.001), and NH 4 + -N (0.01 < P ≤ 0.05) and negatively correlated with NO 3 - -N (P ≤ 0.001). Among AOB, the dominant group was the unclassified Nitrosomonadales in Proteobacteria, which showed significant negative correlation with NH 4 + -N (0.01 < P ≤ 0.05) and significant positive correlation with NO 3 - -N (0.001 < P ≤ 0.01). Notably, among AOB, only Nitrosospira sp. III7 exhibited significant negative correlations with DDE (0.001 < P ≤ 0.01), DDT (0.01 < P ≤ 0.05), and DDD (0.01 < P ≤ 0.05). These results indicate that DDT and its metabolites affect soil AOA and AOB, consequently affecting soil ammonia oxidation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sulfate/sulfite-reducing microorganisms (SRM) are ubiquitous in nature, driving the global sulfur cycle. A hallmark of SRM is the dissimilatory sulfite reductase encoded by the genes dsrAB. Based on analysis of 950 mainly metagenome-derived dsrAB-carrying genomes, we redefine the global diversity of microorganisms with the potential for dissimilatory sulfate/sulfite reduction and uncover genetic repertoires that challenge earlier generalizations regarding their mode of energy metabolism. We show: (i) 19 out of 23 bacterial and 2 out of 4 archaeal phyla harbor uncharacterized SRM, (ii) four phyla including the Desulfobacterota harbor microorganisms with the genetic potential to switch between sulfate/sulfite reduction and sulfur oxidation, and (iii) the combination as well as presence/absence of different dsrAB-types, dsrL-types and dsrD provides guidance on the inferred direction of dissimilatory sulfur metabolism. We further provide an updated dsrAB database including > 60% taxonomically resolved, uncultured family-level lineages and recommendations on existing dsrAB-targeted primers for environmental surveys. Our work summarizes insights into the inferred ecophysiology of newly discovered SRM, puts SRM diversity into context of the major recent changes in bacterial and archaeal taxonomy, and provides an up-to-date framework to study SRM in a global context., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Antibiotics are widely used drugs in the world and pose serious threats to ecosystems and human health. Although it has been reported that ammonia oxidizing bacteria (AOB) can cometabolize antibiotics, little has been reported on how AOB would respond to the exposure of antibiotics on extracellular and enzymatic levels, as well as the impact of antibiotics on the bioactivity of AOB. Therefore, in this study, a typical antibiotic, sulfadiazine (SDZ), was selected, and a series short-term batch tests using enriched AOB sludge were conducted to investigate the intracellular and extracellular responses of AOB along the cometabolic degradation process of SDZ. The results showed the cometabolic degradation of AOB made the main contribution to SDZ removal. When the enriched AOB sludge was exposed to SDZ, ammonium oxidation rate, ammonia monooxygenase activity, adenosine triphosphate concentration and dehydrogenases activity were negatively affected. The amoA gene abundance increased 1.5 folds within 24 h, which may enhance the uptake and utilization of substrates and maintain stable metabolic activity. In the tests with and without ammonium, the concentration of total EPS increased from 264.9 to 231.1 mg/gVSS to 607.7 and 538.2 mg/gVSS, respectively, under the exposure to SDZ, which was mainly contributed by the increase of proteins in tightly bound extracellular polymeric substances (EPS) and polysacharides in tightly bound EPS and soluble microbial products. The proportion of tryptophan-like protein and humic acid-like organics in EPS also increased. Moreover, SDZ stress stimulated the secretion of three quorum sensing signal molecules, C4-HSL (from 140.3 to 164.9 ng/L), 3OC6-HSL (from 17.8 to 42.4 ng/L) and C8-HSL (from 35.8 to 95.9 ng/L) in the enriched AOB sludge. Among them, C8-HSL may be a key signal molecule that promoted the secretion of EPS. The findings of this study could shed more light on the cometabolic degradation of antibiotics by AOB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ammonia oxidising archaea are among the most abundant living organisms on Earth and key microbial players in the global nitrogen cycle. They carry out oxidation of ammonia to nitrite, and their activity is relevant for both food security and climate change. Since their discovery nearly 20 years ago, major insights have been gained into their nitrogen and carbon metabolism, growth preferences and their mechanisms of adaptation to the environment, as well as their diversity, abundance and activity in the environment. Despite significant strides forward through the cultivation of novel organisms and omics-based approaches, there are still many knowledge gaps on their metabolism and the mechanisms which enable them to adapt to the environment. Ammonia oxidising microorganisms are typically considered metabolically streamlined and highly specialised. Here we review the physiology of ammonia oxidising archaea, with focus on aspects of metabolic versatility and regulation, and discuss these traits in the context of nitrifier ecology., (© 2023. The Author(s).)

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

In terrestrial ecosystems, the nitrogen dynamics, including N 2 O production, are majorly regulated by a complex consortium of microbes favored by different substrates and environmental conditions. To better predict the daily, seasonal and annual variation in N 2 O fluxes, it is critical to estimate the temperature sensitivity of different microbial groups for N 2 O fluxes under oxic and suboxic conditions prevalent in soil and wetlands. Here, we studied the temperature sensitivity of two groups of ammonia oxidizers, archaea (AOA) and bacteria (AOB), in relation to N 2 O fluxes through both nitrification and nitrifier-denitrification pathways across a wide temperature gradient (10-55 °C). Using square root theory (SQRT) and macromolecular rate theory (MMRT) models, we estimated thermodynamic parameters and cardinal temperatures, including maximum temperature sensitivity (T Smax ). The distinction between N 2 O pathways was facilitated by microbial-specific inhibitors (PTIO and C 2 H 2 ) and controlled oxygen supply environments (oxic: ambient level; and suboxic: ∼4%). We found that nitrification supported by AOA (Nt A ) and AOB (Nt B ) dominated N 2 O production in an oxic climate, while only AOB-supported nitrifier-denitrification (ND B ) majorly contributed (>90%) to suboxic N 2 O budget. The models predicted significantly higher optimum temperature (T opt ) and T Smax for Nt A and ND B compared to Nt B . Intriguingly, both Nt B and ND B exhibited significantly wider temperature ranges than Nt A. Altogether, our results suggest that temperature and oxygen supply control the dominance of specific AOA- and AOB-supported N 2 O pathways in soil and sediments. This emergent understanding can potentially contribute toward novel targeted N 2 O inhibitors for GHG mitigation under global warming., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Considering the scarcity of data in the literature regarding phylogenetic and metabolic composition of different inocula, especially those from thermophilic conditions, this research aimed at characterizing the microbial community and preferable metabolic pathways of an UASB reactor sludge applied to the thermophilic treatment (55°C) of sugarcane vinasse, by means of shotgun metagenomics. After its metabolic potential was depicted, it was possible to observe several genes encoding enzymes that are of great importance to anaerobic digestion processes with different wastes as substrate, especially regarding the biodegradation of carbohydrates and ligninolytic compounds, glycerolypids, volatile fatty acids and alcohols metabolism and biogas (H 2 and CH 4 ) production. The genera identified in higher relative abundances for Bacteria domain were Sulfirimonas (37.52 ± 1.8%), possibly related to the sludge endogenic activity due to its strong relation with a peptidoglycan lyase enzymes family, followed by Fluviicola (5.01 ± 1.0%), Defluviitoga (4.36 ± 0.2%), Coprothermobacter (4.32 ± 0.5%), Fervidobacterium (2.93 ± 0.3%), Marinospirillum (2.75 ± 0.2%), Pseudomonas (2.14 ± 0.2%) and Flavobacterium (1.78 ± 0.1%), mostly related with carbohydrates fermentations and/or H 2 production. For Archaea domain, Methanosarcina (0.61 ± 0.1%), Methanothermobacter (0.38 ± 0.0%), Methanoculleus (0.30 ± 0.1%), Thermococcus (0.03 ± 0.0%), Methanolobus (0.02 ± 1.8%), Methanobacterium (0.013 ± 0.0%), Aciduliprofundum and Pyrococcus (0.01 ± 0.0%) were the most dominant ones, being Methanosarcina the most related with methanogenesis. It was concluded that the robust inoculum description performed in this study may subside future biotechnological researches by using similar inocula (UASB sludges), focusing on the obtainment of value-added by-products by means of anaerobic digestion, such as volatile fatty acids, alcohols and biogas (H 2 and CH 4 ), by using several types of waste as substrate.

Archaea are microorganisms with great ability to colonize some of the most inhospitable environments in nature, managing to survive in places with extreme characteristics for most microorganisms. Its proteins and enzymes are stable and can act under extreme conditions in which other proteins and enzymes would degrade. These attributes make them ideal candidates for use in a wide range of biotechnological applications. This review describes the most important applications, both current and potential, that archaea present in Biotechnology, classifying them according to the sector to which the application is directed. It also analyzes the advantages and disadvantages of its use., Competing Interests: Conflict of interest The authors declare that they do not have any conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that Δ SisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in Δ SisarnA . The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro . Δ SisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro . These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo . Interestingly, Δ SisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR., Competing Interests: The authors declare no conflict of interest.

An important role of nitric oxide (NO) as either a free intermediate in the NH 3 oxidation pathway or a potential oxidant for NH 3 or NH 2 OH has been proposed for ammonia-oxidizing bacteria (AOB) and archaea (AOA), respectively. However, tracing NO metabolism at low concentrations remains notoriously difficult. Here, we use electrochemical sensors and the mild NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) to trace apparent NO concentration and determine production rates at low micromolar concentrations in the model AOB strain Nitrosomonas europaea. In agreement with previous studies, we found that PTIO does not affect NH 3 oxidation instantaneously in both Nitrosospira briensis and Nitrosomonas europaea, unlike inhibitors for ammonia oxidation such as allylthiourea and acetylene, although it effectively scavenged NO from the cell suspensions. Quantitative analysis showed that NO production by N. europaea amounted to 3.15% to 6.23% of NO 2 - production, whereas N. europaea grown under O 2 limitation produced NO equivalent to up to 40% of NO 2 - production at high substrate concentrations. In addition, we found that PTIO addition to N. europaea grown under O 2 limitation abolished N 2 O production. These results indicate different turnover rates of NO during NH 3 oxidation under O 2 -replete and O 2 -limited growth conditions in AOB. The results suggest that NO may not be a free intermediate or remain tightly bound to iron centers of enzymes during hydroxylamine oxidation and that only NH 3 saturation and adaptation to O 2 limitation may lead to significant dissociation of NO from hydroxylamine dehydrogenase. IMPORTANCE Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria (AOB) is thought to contribute significantly to global nitrous oxide (N 2 O) emissions and leaching of oxidized nitrogen, particularly through their activity in nitrogen (N)-fertilized agricultural production systems. Although substantial efforts have been made to characterize the N metabolism in AOB, recent findings suggest that nitric oxide (NO) may play an important mechanistic role as a free intermediate of hydroxylamine oxidation in AOB, further implying that besides hydroxylamine dehydrogenase (HAO), additional enzymes may be required to complete the ammonia oxidation pathway. However, the NO spin trap PTIO was found to not inhibit ammonia oxidation in AOB. This study provides a combination of physiological and spectroscopic evidence that PTIO indeed scavenges only free NO in AOB and that significant amounts of free NO are produced only during incomplete hydroxylamine oxidation or nitrifier denitrification under O 2 -limited growth conditions., Competing Interests: The authors declare no conflict of interest.

Methanogenic archaea are the only organisms that produce CH 4 as part of their energy-generating metabolism. They are ubiquitous in oxidant-depleted, anoxic environments such as aquatic sediments, anaerobic digesters, inundated agricultural fields, the rumen of cattle, and the hindgut of termites, where they catalyze the terminal reactions in the degradation of organic matter. Methanogenesis is the only metabolism that is restricted to members of the domain Archaea . Here, we discuss the importance of model organisms in the history of methanogen research, including their role in the discovery of the archaea and in the biochemical and genetic characterization of methanogenesis. We also discuss outstanding questions in the field and newly emerging model systems that will expand our understanding of this uniquely archaeal metabolism., Competing Interests: The authors declare no conflict of interest.

Recently, various metabolites derived from host microbes have been reported to modulate the immune system, with potential involvement in health or diseases. Archaea, prokaryotic organisms, are present in the human body, but their connection with the host is largely unknown when compared to other microorganisms such as bacteria. This study focused on unique glycerolipids from symbiotic methanogenic archaea and evaluated their activities toward an innate immune receptor. The results revealed that archaeal lipids were recognized by the C-type lectin receptor Mincle and induced immune responses. A concurrent structure-activity relationship study identified the key structural features of archaeal lipids required for recognition by Mincle. Subsequent gene expression profiling suggested qualitative differences between the symbiotic archaeal lipid and the pathogenic bacteria-derived lipid. These findings have broad implications for understanding the function of symbiotic archaea in host health and diseases.

Application of inorganic nitrogen (N) fertilizers in agriculture can increase emissions of nitrous oxide, a potent greenhouse gas, leaching of nitrate (NO 3 - ), a groundwater contaminant hazardous to human health, and soil acidification. Soil amendment with biochar potentially mitigates these losses and undesirable outcomes. However, there have been considerable inconsistencies in reported impacts, likely owing to variable physiochemical characteristics of the biochar materials and/or the soil environment. This study methodically evaluated the impact of biochar soil incorporation on N transformation and underlying microbial processes using soils with varying biochar types, soil texture, soil moisture, and manure compost co- amendments. Laboratory incubations were conducted to monitor the fate of urea fertilizer N spiked in biochar amended and unamended soils by assaying soil ammonium (NH 4 + ), nitrite (NO 2 - ), and NO 3 - concentrations, pH, and abundances of soil nitrifiers; ammonia oxidizing bacteria and archaea (AOB and AOA) and Nitrospira with the capacity to perform complete ammonia oxidation (comammox). Soil moisture was a critical factor affecting N transformation processes, more so than biochar, but biochar did result in significantly different concentrations of N species in response to urea application. Biochar enhanced nitrification, more significantly in drier conditions and in sandy soil. Biochar offered some buffering potential in the neutral-alkaline, unsaturated soils, preventing >1 unit drop in pH compared to unamended soils. Co-application of biochar with manure composts enhanced nitrification slightly, which was evidenced by higher abundances of some soil nitrifiers at 4 weeks, although increases in nitrification rates were not statistically significant. Soil nitrifier populations tended to increase in response to a pinewood biochar, but trends differed for saturated soil, in soils of differing textures, or when different biochar materials were evaluated. Thus, when evaluating implications of biochar on the fate of mineral N fertilizer, soil moisture and other environment conditions should be considered., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Suduan Gao reports financial support was provided by United States Department of Agriculture Agricultural Research Service., (Published by Elsevier B.V.)

Selenium oxyanions are released into environments by natural and anthropogenic activities and are present in agricultural and glass manufacturing wastewater in several locations worldwide. Excessive amounts of this metalloid have adverse effects on the health of living organisms. Halophilic and halotolerant microorganisms were selected for selenium oxyanions remediation due to presence of significant amount of salt in selenium-containing wastewater. Effects of aeration, carbon sources, competitive electron acceptors, and reductase inhibitors were investigated on SeO 3 2- bio-removal. Additionally, NO 3 - -containing wastewater were exploited to investigate SeO 3 2- remediation in synthetic agricultural effluents. The results showed that the SeO 3 2- removal extent is maximum in aerobic conditions with succinate as a carbon source. SO 4 2- and PO 4 3- do not significantly interfere with SeO 3 2- reduction, while WO 4 2- and TeO 3 2- decrease the SeO 3 2- removal percentage (up to 35 and 37%, respectively). Furthermore, NO 3 - had an adverse effect on SeO 3 2- biotransformation by our consortia. All consortia reduced SeO 3 2- in synthetic agricultural wastewaters with a 45-53% removal within 120 h. This study suggests that consortia of halophilic/halotolerant bacteria and yeasts could be applied to treat SeO 3 2- -contaminated drainage water. In addition, sulphates, and phosphates do not interfere with selenite bioreduction by these consortia, which makes them suitable candidates for the bioremediation of selenium-containing wastewater., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The interaction among microorganisms in the anaerobic digestion of food waste (ADFW) reactors lead to the degradation of organics and the recycling of energy. Viruses are an important component of the microorganisms involved in ADFW, but are rarely investigated. Furthermore, little is known about how viruses affect methanogenesis., Results: Thousands of viral sequences were recovered from five full-scale ADFW reactors. Gene-sharing networks indicated that the ADFW samples contained substantial numbers of unexplored anaerobic-specific viruses. Moreover, the viral communities in five full-scale reactors exhibited both commonalities and heterogeneities. The lab-scale dynamic analysis of typical ADFW scenarios suggested that the viruses had similar kinetic characteristics to their prokaryotic hosts. By associating with putative hosts, a majority of the bacteria and archaea phyla were found to be infected by viruses. Viruses may influence prokaryotic ecological niches, and thus methanogenesis, by infecting key functional microorganisms, such as sulfate-reducing bacteria (SRB), syntrophic acetate-oxidizing bacteria (SAOB), and methanogens. Metabolic predictions for the viruses suggested that they may collaborate with hosts at key steps of sulfur and long-chain fatty acid (LCFA) metabolism and could be involved in typical methanogenesis pathways to participate in methane production., Conclusions: Our results expanded the diversity of viruses in ADFW systems and suggested two ways that viral manipulated ADFW biochemical processes. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Aims: One of the main challenges of culture-independent soil microbiology is distinguishing the microbial community's viable fraction from dead matter. Propidium monoazide (PMA) binds the DNA of dead cells, preventing its amplification. This dye could represent a robust means to overcome the drawbacks of other selective methods, such as ribonucleic acid-based analyses., Methods and Results: We quantified functional genes from viable archaea and bacteria in soil by combining the use of PMA and quantitative polymerase chain reaction. Four N-cycle-related functional genes (bacterial and archaeal ammonia monooxygenase, nitrate reductase, and nitrite reductase) were successfully quantified from the living fraction of bacteria and archaea of a paddy soil. The protocol was also tested with pure bacterial cultures and soils with different physical and chemical properties., Conclusions: The experiment results revealed a contrasting impact of mineral and organic fertilizers on the abundance of microbial genes related to the N-cycle in paddy soil., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

The haloarchaeon Haloferax volcanii degrades D-glucose via the semiphosphorylative Entner-Doudoroff pathway and D-fructose via a modified Embden-Meyerhof pathway. Here, we report the identification of GfcR, a novel type of transcriptional regulator that functions as an activator of both D-glucose and D-fructose catabolism. We find that in the presence of D-glucose, GfcR activates gluconate dehydratase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase and also acts as activator of the phosphotransferase system and of fructose-1,6-bisphosphate aldolase, which are involved in uptake and degradation of D-fructose. In addition, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase are activated by GfcR in the presence of D-fructose and also during growth on D-galactose and glycerol. Electrophoretic mobility shift assays indicate that GfcR binds directly to promoters of regulated genes. Specific intermediates of the degradation pathways of the three hexoses and of glycerol were identified as inducer molecules of GfcR. GfcR is composed of a phosphoribosyltransferase (PRT) domain with an N-terminal helix-turn-helix motif and thus shows homology to PurR of Gram-positive bacteria that is involved in the transcriptional regulation of nucleotide biosynthesis. We propose that GfcR of H. volcanii evolved from a PRT-like enzyme to attain a function as a transcriptional regulator of central sugar catabolic pathways in archaea., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Both eukaryotes and prokaryotes (archaea and bacteria) encode an arsenal of immune systems that protect the host against mobile genetic elements (MGEs) including viruses, plasmids, and transposons. Whereas Argonaute proteins (Agos) are best known for post-transcriptional gene silencing in eukaryotes, in all domains of life, members from the highly diverse Argonaute protein family act as programmable immune systems. To this end, Agos are programmed with small single-stranded RNA or DNA guides to detect and silence complementary MGEs. Across and within the different domains of life, Agos function in distinct pathways and MGE detection can trigger various mechanisms that provide immunity. In this review, we delineate the diverse immune pathways and underlying mechanisms for both eukaryotic Argonautes (eAgos) and prokaryotic Argonautes (pAgos)., Competing Interests: Declaration of Competing Interest D.C.S. has submitted several patent applications regarding the utilization of pAgos for nucleic acid detection and modification., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

S-adenosylmethionine decarboxylase (AdoMetDC/SpeD) is a key polyamine biosynthetic enzyme required for conversion of putrescine to spermidine. Autocatalytic self-processing of the AdoMetDC/SpeD proenzyme generates a pyruvoyl cofactor from an internal serine. Recently, we discovered that diverse bacteriophages encode AdoMetDC/SpeD homologs that lack AdoMetDC activity and instead decarboxylate L-ornithine or L-arginine. We reasoned that neofunctionalized AdoMetDC/SpeD homologs were unlikely to have emerged in bacteriophages and were probably acquired from ancestral bacterial hosts. To test this hypothesis, we sought to identify candidate AdoMetDC/SpeD homologs encoding L-ornithine and L-arginine decarboxylases in bacteria and archaea. We searched for the anomalous presence of AdoMetDC/SpeD homologs in the absence of its obligatory partner enzyme spermidine synthase, or the presence of two AdoMetDC/SpeD homologs encoded in the same genome. Biochemical characterization of candidate neofunctionalized genes confirmed lack of AdoMetDC activity, and functional presence of L-ornithine or L-arginine decarboxylase activity in proteins from phyla Actinomycetota, Armatimonadota, Planctomycetota, Melainabacteria, Perigrinibacteria, Atribacteria, Chloroflexota, Sumerlaeota, Omnitrophota, Lentisphaerota, and Euryarchaeota, the bacterial candidate phyla radiation and DPANN archaea, and the δ-Proteobacteria class. Phylogenetic analysis indicated that L-arginine decarboxylases emerged at least three times from AdoMetDC/SpeD, whereas L-ornithine decarboxylases arose only once, potentially from the AdoMetDC/SpeD-derived L-arginine decarboxylases, revealing unsuspected polyamine metabolic plasticity. Horizontal transfer of the neofunctionalized genes appears to be the more prevalent mode of dissemination. We identified fusion proteins of bona fide AdoMetDC/SpeD with homologous L-ornithine decarboxylases that possess two, unprecedented internal protein-derived pyruvoyl cofactors. These fusion proteins suggest a plausible model for the evolution of the eukaryotic AdoMetDC., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Selenium-binding proteins represent a ubiquitous protein family and recently SBP1 was described as a new stress response regulator in plants. SBP1 has been characterized as a methanethiol oxidase, however its exact role remains unclear. Moreover, in mammals, it is involved in the regulation of anti-carcinogenic growth and progression as well as reduction/oxidation modulation and detoxification. In this work, we delineate the functional potential of certain motifs of SBP in the context of evolutionary relationships. The phylogenetic profiling approach revealed the absence of SBP in the fungi phylum as well as in most non eukaryotic organisms. The phylogenetic tree also indicates the differentiation and evolution of characteristic SBP motifs. Main evolutionary events concern the CSSC motif for which Acidobacteria, Fungi and Archaea carry modifications. Moreover, the CC motif is harbored by some bacteria and remains conserved in Plants, while modified to CxxC in Animals. Thus, the characteristic sequence motifs of SBPs mainly appeared in Archaea and Bacteria and retained in Animals and Plants. Our results demonstrate the emergence of SBP from bacteria and most likely as a methanethiol oxidase., (© 2023. The Author(s).)

Considering the link between neurodegenerative diseases and impaired proteasome function, and the neuro-protective impact of enhanced proteasome activity in animal models, it's crucial to understand proteasome activation mechanisms. A hydrophobic-tyrosine-any residue (HbYX) motif on the C-termini of proteasome-activating complexes independently triggers gate-opening of the 20S core particle for protein degradation; however, the causal allosteric mechanism remains unclear. Our study employs a structurally irreducible dipeptide HbYX mimetic to investigate the allosteric mechanism of gate-opening in the archaeal proteasome. High-resolution cryo-EM structures pinpoint vital residues and conformational changes in the proteasome α-subunit implicated in HbYX-dependent activation. Using point mutations, we simulated the HbYX-bound state, providing support for our mechanistic model. We discerned four main mechanistic elements triggering gate-opening: 1) back-loop rearrangement adjacent to K66, 2) intra- and inter- α subunit conformational changes, 3) occupancy of the hydrophobic pocket, and 4) a highly conserved isoleucine-threonine pair in the 20S channel stabilizing the open and closed states, termed the "IT switch." Comparison of different complexes unveiled convergent and divergent mechanism of 20S gate-opening among HbYX-dependent and independent activators. This study delivers a detailed molecular model for HbYX-dependent 20S gate-opening, enabling the development of small molecule proteasome activators that hold promise to treat neurodegenerative diseases., (© 2023. The Author(s).)

Flagellar motility has independently arisen three times during evolution: in bacteria, archaea, and eukaryotes. In prokaryotes, the supercoiled flagellar filaments are composed largely of a single protein, bacterial or archaeal flagellin, although these two proteins are not homologous, while in eukaryotes, the flagellum contains hundreds of proteins. Archaeal flagellin and archaeal type IV pilin are homologous, but how archaeal flagellar filaments (AFFs) and archaeal type IV pili (AT4Ps) diverged is not understood, in part, due to the paucity of structures for AFFs and AT4Ps. Despite having similar structures, AFFs supercoil, while AT4Ps do not, and supercoiling is essential for the function of AFFs. We used cryo-electron microscopy to determine the atomic structure of two additional AT4Ps and reanalyzed previous structures. We find that all AFFs have a prominent 10-strand packing, while AT4Ps show a striking structural diversity in their subunit packing. A clear distinction between all AFF and all AT4P structures involves the extension of the N-terminal α-helix with polar residues in the AFFs. Additionally, we characterize a flagellar-like AT4P from Pyrobaculum calidifontis with filament and subunit structure similar to that of AFFs which can be viewed as an evolutionary link, showing how the structural diversity of AT4Ps likely allowed for an AT4P to evolve into a supercoiling AFF.

To date, multiple studies have shown that the accumulation of microplastics (MPs)/nanoplastics (NPs) in the environment may lead to various problems. However, the effects of MPs/NPs on microbial communities and biogeochemical processes, particularly methane metabolism in cold seep sediments, have not been well elucidated. In this study, an indoor microcosm experiment for a period of 120 days exposure of MPs/NPs was conducted. The results showed that MPs/NPs addition did not significantly influence bacterial and archaeal richness in comparison with the control (p > 0.05), whereas higher levels of NPs (1 %, w/w) had a significant adverse effect on bacterial diversity (p < 0.05). Moreover, the bacterial community was more sensitive to the addition of MPs/NPs than the archaea, and Epsilonbacteraeota replaced Proteobacteria as the dominant phylum in the MPs/NPs treatments (except 0.2 % NPs). With respect to the co-occurrence relationships, network analysis showed that the presence of NPs, in comparison with MPs, reduced microbial network complexity. Finally, the presence of MPs/NPs decreased the abundance of mcrA, while promoting the abundance of pmoA. This study will help elucidate the responses of microbial communities to MPs/NPs and evaluate their effects on methane metabolism in cold seep ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Self-assembling protein filaments are at the heart of cell function. Among them, tubulin-like proteins are essential for cell division, DNA segregation and cytoskeletal functions across the domains of life. FtsZ and tubulin share their core structures, a characteristic nucleotide-binding pocket and similar protofilament architecture. GTP hydrolysis between consecutive subunits drives their assembly dynamics. Two recent studies provide previously missing, filament atomic structures of bacterial FtsZ and a recently discovered archaeal tubulin in their nucleotide triphosphate-bound states. Both filament structures reveal strikingly conserved interfacial GTPase active sites, with Mg 2+ and K + /Na + cations and an NxDxxD/E triad of catalytic residues, probably inherited from the common ancestor of FtsZs and tubulins. Moreover, both proteins exhibit nucleotide-regulated subunit association mediated by interfacial water bridges, as well as polymerization-induced structural changes, likely enabling related dynamic assembly mechanisms., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The major archaeal membrane glycerolipids are distinguished from those of bacteria and eukaryotes by the contrasting stereochemistry of their glycerol backbones, and by the use of ether-linked isoprenoid-based alkyl chains rather than ester-linked fatty acyl chains for their hydrophobic moieties. These fascinating compounds play important roles in the extremophile lifestyles of many species, but are also present in the growing numbers of recently discovered mesophilic archaea. The past decade has witnessed significant advances in our understanding of archaea in general and their lipids in particular. Much of the new information has come from the ability to screen large microbial populations via environmental metagenomics, which has revolutionised our understanding of the extent of archaeal biodiversity that is coupled with a strict conservation of their membrane lipid compositions. Significant additional progress has come from new culturing and analytical techniques that are gradually enabling archaeal physiology and biochemistry to be studied in real time. These studies are beginning to shed light on the much-discussed and still-controversial process of eukaryogenesis, which probably involved both bacterial and archaeal progenitors. Puzzlingly, although eukaryotes retain many attributes of their putative archaeal ancestors, their lipid compositions only reflect their bacterial progenitors. Finally, elucidation of archaeal lipids and their metabolic pathways have revealed potentially interesting applications that have opened up new frontiers for biotechnological exploitation of these organisms. This review is concerned with the analysis, structure, function, evolution and biotechnology of archaeal lipids and their associated metabolic pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier Ltd.)

Iron oxides and sulfate are usually abundant in paddy soil, but their role in reducing methane emissions is little known. In this work, paddy soil was anaerobically cultivated with ferrihydrite and sulfate for 380 days. An activity assay, inhibition experiment, and microbial analysis were conducted to evaluate the microbial activity, possible pathways, and community structure, respectively. The results showed that anaerobic oxidation of methane (AOM) was active in the paddy soil. The AOM activity was much higher with ferrihydrite than sulfate, and an extra 10% of AOM activity was stimulated when ferrihydrite and sulfate coexisted. The microbial community was highly similar to the duplicates but totally different with different electron acceptors. The microbial abundance and diversity decreased due to the oligotrophic condition, but mcrA-carrying archaea increased 2-3 times after 380 days. Both the microbial community and the inhibition experiment implied that there was an intersection between iron and sulfur cycles. A "cryptic sulfur cycle" might link the two cycles, in which sulfate was quickly regenerated by iron oxides, and it might contribute 33% of AOM in the tested paddy soil. Complex links between methane, iron, and sulfur geochemical cycles occur in paddy soil, which may be significant in reducing methane emissions from rice fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This study proposes a novel method to enhance methane production from anaerobic digestion using an amino acid-derived ionic liquid, glycine hydrochloride, ([Gly][Cl]), as an exogenous additive. After 40 days of digestion with 5% [Gly][Cl], the cumulative methane production was 115.56 mL/g VS, which was 73% higher than that of the control group (without additive). Specifically, the peak activities of cellulase, xylanase, and lignin peroxidase were significantly higher than those of the control group. The addition of [Gly][Cl] increased bacterial diversity and reduced archaeal diversity. Synergistota represented by Syner-01, Fibrobacterota represented by BBMC-4, Bacteroides, and unclassified&#95;f&#95;&#95;Lachnospiraceae significantly increased in relative abundance. It suggested that [Gly][Cl] stimulated the activities of protein-hydrolyzing and acid-producing bacteria. [Gly][Cl] also increased the abundance of methanogens and archaea, converting more lignocellulose to methane. Methanobacterium, that metabolizes H 2 and CO 2 to CH 4 , was more abundant. Therefore, [Gly][Cl] can improve methane yield as an anaerobic digestion additive., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Bathyarchaeota, known as key participants of global elements cycling, is highly abundant and diverse in the sedimentary environments. Bathyarchaeota has been the research spotlight on sedimentary microbiology; however, its distribution in arable soils is far from understanding. Paddy soil is a habitat similar to freshwater sediments, while the distribution and composition of Bathyarchaeota in paddy soils have largely been overlooked. In this study, we collected 342 in situ paddy soil sequencing data worldwide to illuminate the distribution patterns of Bathyarchaeota and explore their potential ecological functions in paddy soils. The results showed that Bathyarchaeota is the dominant archaeal lineage, and Bathy-6 is the most predominant subgroup in paddy soils. Based on random forest analysis and construction of a multivariate regression tree, the mean annual precipitation and mean annual temperature are identified as the factors significantly influencing the abundance and composition of Bathyarchaeota in paddy soils. Bathy-6 was abundant in temperate environments, while other subgroups were more abundant in sites with higher rainfall. There are highly frequent associations between Bathyarchaeota and methanogens and ammonia-oxidizing archaea. The interactions between Bathyarchaeota and microorganisms involved in carbon and nitrogen metabolism imply a potential syntrophy between these microorganisms, suggesting that members of Bathyarchaeota could be important participants of geochemical cycle in paddy soils. These results shed light on the ecological lifestyle of Bathyarchaeota in paddy soils, and provide some baseline for further understanding Bathyarchaeota in arable soils. IMPORTANCE Bathyarchaeota, the dominant archaeal lineage in sedimentary environments, has been the spotlight of microbial research due to its vital role in carbon cycling. Although Bathyarchaeota has been also detected in paddy soils worldwide, its distribution in this environment has not yet been investigated. In this study, we conducted a global scale meta-analysis and found that Bathyarchaeota is also the dominant archaeal lineage in paddy soils with significant regional abundance differences. Bathy-6 is the most predominant subgroup in paddy soils, which differs from sediments. Furthermore, Bathyarchaeota are highly associated with methanogens and ammonia-oxidizing archaea, suggesting that they may be involved in the carbon and nitrogen cycle in paddy soil. These interactions provide insight into the ecological functions of Bathyarchaeota in paddy soils, which will be the foundation of future studies regarding the geochemical cycle in arable soils and global climate change., Competing Interests: The authors declare no conflict of interest.

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO 2 , polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA ( V max of 8.4 μmol mg -1  min -1 and K m of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA ( V max of 0.4 μmol mg -1  min -1 and K m of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar V max values for both directions ( V max,acetate of 0.9 μmol mg -1  min -1 versus V max,acetyl-CoA of 0.3 μmol mg -1  min -1 ). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13 C 2 -labeled acetate produced 3 μmol of [ 13 C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight] -1 min -1 ). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d., Competing Interests: The authors declare no conflict of interest.

Recent discoveries of methyl-coenzyme M reductase-encoding genes ( mcr ) in uncultured archaea beyond traditional euryarchaeotal methanogens have reshaped our view of methanogenesis. However, whether any of these nontraditional archaea perform methanogenesis remains elusive. Here, we report field and microcosm experiments based on 13 C-tracer labeling and genome-resolved metagenomics and metatranscriptomics, revealing that nontraditional archaea are predominant active methane producers in two geothermal springs. Archaeoglobales performed methanogenesis from methanol and may exhibit adaptability in using methylotrophic and hydrogenotrophic pathways based on temperature/substrate availability. A five-year field survey found Candidatus Nezhaarchaeota to be the predominant mcr -containing archaea inhabiting the springs; genomic inference and mcr expression under methanogenic conditions strongly suggested that this lineage mediated hydrogenotrophic methanogenesis in situ. Methanogenesis was temperature-sensitive , with a preference for methylotrophic over hydrogenotrophic pathways when incubation temperatures increased from 65° to 75°C. This study demonstrates an anoxic ecosystem wherein methanogenesis is primarily driven by archaea beyond known methanogens, highlighting diverse nontraditional mcr -containing archaea as previously unrecognized methane sources.

The biological carbon pump (BCP) in the Southern Ocean is driven by phytoplankton productivity and is a significant organic matter sink. However, the role of particle-attached (PA) and free-living (FL) prokaryotes (bacteria and archaea) and their diversity in influencing the efficiency of the BCP is still unclear. To investigate this, we analyzed the metagenomes linked to suspended and sinking marine particles from the Sub-Antarctic Southern Ocean Time Series (SOTS) by deploying a Marine Snow Catcher (MSC), obtaining suspended and sinking particulate material, determining organic carbon and nitrogen flux, and constructing metagenome-assembled genomes (MAGs). The suspended and sinking particle-pools were dominated by bacteria with the potential to degrade organic carbon. Bacterial communities associated with the sinking fraction had more genes related to the degradation of complex organic carbon than those in the suspended fraction. Archaea had the potential to drive nitrogen metabolism via nitrite and ammonia oxidation, altering organic nitrogen concentration. The data revealed several pathways for chemoautotrophy and the secretion of recalcitrant dissolved organic carbon (RDOC) from CO 2 , with bacteria and archaea potentially sequestering particulate organic matter (POM) via the production of RDOC. These findings provide insights into the diversity and function of prokaryotes in suspended and sinking particles and their role in organic carbon/nitrogen export in the Southern Ocean. IMPORTANCE The biological carbon pump is crucial for the export of particulate organic matter in the ocean. Recent studies on marine microbes have shown the profound influence of bacteria and archaea as regulators of particulate organic matter export. Yet, despite the importance of the Southern Ocean as a carbon sink, we lack comparable insights regarding microbial contributions. This study provides the first insights regarding prokaryotic contributions to particulate organic matter export in the Southern Ocean. We reveal evidence that prokaryotic communities in suspended and sinking particle fractions harbor widespread genomic potential for mediating particulate organic matter export. The results substantially enhance our understanding of the role played by microorganisms in regulating particulate organic matter export in suspended and sinking marine fractions in the Southern Ocean., Competing Interests: The authors declare no conflict of interest.

To adapt and proliferate, bacteria must sense and respond to the ever-changing environment. Transmembrane transcription regulators (TTRs) are a family of one-component transcription regulators that respond to extracellular information and influence gene expression from the cytoplasmic membrane. How TTRs function to modulate expression of their target genes while localized to the cytoplasmic membrane remains poorly understood. In part, this is due to a lack of knowledge regarding the prevalence of TTRs among prokaryotes. Here, we show that TTRs are highly diverse and prevalent throughout bacteria and archaea. Our work demonstrates that TTRs are more common than previously appreciated and are enriched within specific bacterial and archaeal phyla and that many TTRs have unique transmembrane region properties that can facilitate association with detergent-resistant membranes. IMPORTANCE One-component signal transduction systems are the major class of signal transduction systems among bacteria and are commonly cytoplasmic. TTRs are a group of unique one-component signal transduction systems that influence transcription from the cytoplasmic membrane. TTRs have been implicated in a wide array of biological pathways critical for both pathogens and human commensal organisms but were considered to be rare. Here, we demonstrate that TTRs are in fact highly diverse and broadly distributed in bacteria and archaea. Our findings suggest that transcription factors can access the chromosome and influence transcription from the membrane in both archaea and bacteria. This study challenges thus the commonly held notion that signal transduction systems require a cytoplasmic transcription factor and highlights the importance of the cytoplasmic membrane in directly influencing signal transduction., Competing Interests: The authors declare no conflict of interest.

The marine thermophilic archaeon Nanoarchaeum equitans possesses a monomeric primase encompassing the conserved domains of the small catalytic and the large regulatory subunits of archaeoeukaryotic heterodimeric primases in one protein chain. The recombinant protein primes on templates containing a triplet with a central thymidine, thus displaying a pronounced sequence specificity typically observed with bacterial type primases only. The N. equitans primase (NEQ395) is a highly active primase enzyme synthesizing short RNA primers. Termination occurs preferentially at about nine nucleotides, as determined by HPLC analysis and confirmed with mass spectrometry. Possibly, the compact monomeric primase NEQ395 represents the minimal archaeoeukaryotic primase and could serve as a functional and structural model of the heterodimeric archaeoeukaryotic primases, whose study is hindered by engagement in protein assemblies and rather low activity., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Complete ammonia oxidizers (Comammox) are of great significance for studying nitrification and expanding the understanding of the nitrogen cycle. Moreover, Comammox bacteria are also crucial in natural and engineered environments due to their role in wastewater treatment and maintaining the flux of greenhouse gases to the atmosphere. However, only few studies are there regarding the Comammox bacteria and their role in ammonia and nitrite oxidation in the environment. This review mainly focuses on summarizing the genomes of Nitrospira in the NCBI database. Ecological distribution of Nitrospira was also reviewed and the influence of environmental parameters on genus Nitrospira in different environments has been summarized. Furthermore, the role of Nitrospira in carbon cycle, nitrogen cycle, and sulfur cycle were discussed, especially the comammox Nitrospira. In addition, the overviews of current research and development regarding comammox Nitrospira, were summarized along with the scope of future research. KEY POINTS: • Most of Comammox Nitrospira are widely distributed in both aquatic and terrestrial ecosystems, but it has been studied less frequently in the extreme environments. • Comammox Nitrospira can be involved in different nitrogen transformation process, but rarely involved in nitrogen fixation. • The stable isotope and transcriptome techniques are important methods to study the metabolic function of comammox Nitrospira., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Coastal zones are among the world's most productive ecosystems. They store vast amounts of organic carbon, as 'blue carbon' reservoirs, and impact global climate change. Archaeal communities are integral components of coastal microbiomes but their ecological roles are often overlooked. However, archaeal diversity, metabolism, evolution, and interactions, revealed by recent studies using rapidly developing cutting-edge technologies, place archaea as important players in coastal carbon biogeochemical cycling. We here summarize the latest advances in the understanding of archaeal carbon cycling processes in coastal ecosystems, specifically, archaeal involvement in CO 2 fixation, organic biopolymer transformation, and methane metabolism. We also showcase the potential to use of archaeal communities to increase carbon sequestration and reduce methane production, with implications for mitigating climate change., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The adaptation of microbial community to the long-term contamination of hexabromocyclododecanes (HBCDs) has not been well studied. Our previous study found that the HBCDs contamination in the microcosms constructed of sediments from two different mangrove forests in 8 months resulted in serious acidification (pH2-3). This study reanalyzed previous sequencing data and compared them with data after 20 months to investigate the adaptive properties of microbial communities in the stress of HBCDs and acidification. It hypothesized that the reassembly was based on the fitness of taxa. The results indicated that eukaryotes and fungi might have better adaptive capacity to these deteriorated habitats. Eukaryotic taxa Eufallia and Syncystis, and fungal taxa Wickerhamomyces were only detected after 20 months of contamination. Moreover, eukaryotic taxa Caloneis and Nitzschia, and fungal taxa Talaromyces were dominant in most of microbial communities (14.467-95.941%). The functional compositions were sediment-dependent and more divergent than community reassemblies. Network and co-occurrence analysis suggested that acidophiles such as Acidisoma and Acidiphilium were gaining more positive relations in the long-term stress. The acidophilic taxa and genes involved in resistance to the acidification and toxicity of HBCDs were enriched, for example, bacteria Acidisoma and Acidiphilium, archaea Thermogymnomonas, and eukaryotes Nitzschia, and genes kdpC, odc1, polA, gst, and sod-2. These genes involved in oxidative stress response, energy metabolism, DNA damage repair, potassium transportation, and decarboxylation. It suggested that the microbial communities might cope with the stress from HBCDs and acidification via multiple pathways. The present research shed light on the evolution of microbial communities under the long-term stress of HBCDs contamination and acidification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Northern lakes disproportionately influence the global carbon cycle, and may do so more in the future depending on how their microbial communities respond to climate warming. Microbial communities can change because of the direct effects of climate warming on their metabolism and the indirect effects of climate warming on groundwater connectivity from thawing of surrounding permafrost, especially at lower landscape positions. Here we used shotgun metagenomics to compare the taxonomic and functional gene composition of sediment microbes in 19 peatland lakes across a 1600-km permafrost transect in boreal western Canada. We found microbes responded differently to the loss of regional permafrost cover than to increases in local groundwater connectivity. These results suggest that both the direct and indirect effects of climate warming, which were respectively associated with loss of permafrost and subsequent changes in groundwater connectivity interact to change microbial composition and function. Archaeal methanogens and genes involved in all major methanogenesis pathways were more abundant in warmer regions with less permafrost, but higher groundwater connectivity partly offset these effects. Bacterial community composition and methanotrophy genes did not vary with regional permafrost cover, and the latter changed similarly to methanogenesis with groundwater connectivity. Finally, we found an increase in sugar utilization genes in regions with less permafrost, which may further fuel methanogenesis. These results provide the microbial mechanism for observed increases in methane emissions associated with loss of permafrost cover in this region and suggest that future emissions will primarily be controlled by archaeal methanogens over methanotrophic bacteria as northern lakes warm. Our study more generally suggests that future predictions of aquatic carbon cycling will be improved by considering how climate warming exerts both direct effects associated with regional-scale permafrost thaw and indirect effects associated with local hydrology., (© 2023 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

The Cdv system is the protein machinery that performs cell division and other membrane-deforming processes in a subset of archaea. Evolutionarily, the system is closely related to the eukaryotic ESCRT machinery, with which it shares many structural and functional similarities. Since its first description 15 years ago, the understanding of the Cdv system progressed rather slowly, but recent discoveries sparked renewed interest and insights. The emerging physical picture appears to be that CdvA acts as a membrane anchor, CdvB as a scaffold that localizes division to the mid-cell position, CdvB1 and CvdB2 as the actual constriction machinery, and CdvC as the ATPase that detaches Cdv proteins from the membrane. This paper provides a comprehensive overview of the research done on Cdv and explains how this relatively understudied machinery acts to perform its cell-division function. Understanding of the Cdv system helps to better grasp the biophysics and evolution of archaea, and furthermore provides new opportunities for the bottom-up building of a divisome for synthetic cells., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria , is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria , previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade " Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that " Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of " Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate " Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that " Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other " Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny " Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis., Competing Interests: The authors declare no conflict of interest.

Oxygen-deficient marine waters referred to as oxygen minimum zones (OMZs) or anoxic marine zones (AMZs) are common oceanographic features. They host both cosmopolitan and endemic microorganisms adapted to low oxygen conditions. Microbial metabolic interactions within OMZs and AMZs drive coupled biogeochemical cycles resulting in nitrogen loss and climate active trace gas production and consumption. Global warming is causing oxygen-deficient waters to expand and intensify. Therefore, studies focused on microbial communities inhabiting oxygen-deficient regions are necessary to both monitor and model the impacts of climate change on marine ecosystem functions and services. Here we present a compendium of 5,129 single-cell amplified genomes (SAGs) from marine environments encompassing representative OMZ and AMZ geochemical profiles. Of these, 3,570 SAGs have been sequenced to different levels of completion, providing a strain-resolved perspective on the genomic content and potential metabolic interactions within OMZ and AMZ microbiomes. Hierarchical clustering confirmed that samples from similar oxygen concentrations and geographic regions also had analogous taxonomic compositions, providing a coherent framework for comparative community analysis., (© 2023. The Author(s).)

A rising outbreak of waterborne diseases caused by global warming requires higher microbial stability in the drinking water distribution system (DWDS). Chloramine disinfection is gaining popularity in this context due to its good persistent stability and fewer disinfection byproducts. However, the microbiological risks may be significantly magnified by ammonia-oxidizing bacteria (AOB) in distribution systems during global warming, which is rarely noticed. Hence, this work mainly focuses on AOB to explore its impact on water quality biosafety in the context of global warming. Research indicates that global warming-induced high temperatures can directly or indirectly promote the growth of AOB, thus leading to nitrification. Further, its metabolites or cellular residues can be used as substrates for the growth of heterotrophic bacteria (e.g., waterborne pathogens). Thus, biofilm may be more persistent in the pipelines due to the presence of AOB. Breakpoint chlorination is usually applied to control such situations. However, switching between this strategy and chloramine disinfection would result in even more severe nitrification and other adverse effects. Based on the elevated microbiological risks in DWDS, the following aspects should be paid attention to in future research: (1) to understand the response of nitrifying bacteria to high temperatures and the possible association between AOB and pathogenic growth, (2) to reveal the mechanisms of AOB-mediated biofilm formation under high-temperature stress, and (3) to develop new technologies to prevent and control the occurrence of nitrification in drinking water distribution system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Utilizing biogas as a fuel for heating and power generation usually requires desulfurization pretreatment. In this study, the biogas utilization without desulfurization pretreatment in a bioelectrochemical system (BES) was explored. The results showed that the biogas-fueled BES was successfully started up within 36 d and the presence of hydrogen sulfide promoted both methane consumption and electricity generation. The optimal performance (i.e., a methane consumption of 0.523 ± 0.004 mmol/d, a peak voltage of 577 ± 1 mV, a coulomb production of 37.86 ± 0.43C/d, a coulombic efficiency of 9.37 ± 0.06 % and the maximum power density of 2.070 W/m 3 ) was obtained under bicarbonate buffer solution and 40 °C conditions. The addition of 1 mg/L sulfide and 5 mg/L L-cysteine facilitated methane consumption and electricity generation. In the anode biofilm, the dominant bacteria were Sulfurivermis, unclassified&#95;o&#95;&#95;Ignavibacteriales and Lentimicrobium, while Methanobacterium, Methanosarcina and Methanothrix were the predominant archaea. Besides, the metagenomics profiles reveal that anaerobic methane oxidation and electricity generation were closely related to sulfur metabolism. These findings provide a novel approach for utilizing biogas without desulfurization pretreatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Soil arsenic contamination is of great concern because of its toxicity to human, crops, and soil microorganisms. However, the impacts of arsenic on soil ammonia oxidizers communities remain unclear. Seven types of soil spiked with 0 or 100 mg arsenic per kg soil were incubated for 180 days and sampled at days 1, 15, 30, 90 and 180. The changes in the community composition and abundance of ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) were analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis, clone library sequencing, and quantitative PCR (qPCR) targeting amoA gene. Results revealed considerable variations in the potential ammonia oxidation (PAO) rates in different soils, but soil PAO was not consistently significantly inhibited by arsenic, probably due to the low bioavailable arsenic contents or the existence of functional redundancy between AOB and AOA. The variations in AOB and AOA communities were closely associated with the changes in arsenic fractionations. The amoA gene abundances of AOA increased after arsenic addition, whereas AOB decreased, which corroborated the notion that AOA and AOB might occupy different niches in arsenic-contaminated soils. Phylogenetic analysis of amoA gene-encoded proteins revealed that all AOB clone sequences belonged to the genus Nitrosospira, among which those belonging to Nitrosospira cluster 3a were dominant. The main AOA sequence detected belonged to Thaumarchaeal Group 1.1b, which was considered to have a high ability to adapt to environmental changes. Our results provide new insights into the impacts of arsenic on the soil nitrogen cycling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

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 13 C and 15 N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20- 13 C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20- 13 C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15 N RPSB/λ max plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15 N 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nitrate-driven anaerobic oxidation of methane (AOM), catalyzing by Candidatus Methanoperedens-like archaea, is a new addition in the global CH 4 cycle. This AOM process acts as a novel pathway for CH 4 emission reduction in freshwater aquatic ecosystems; however, its quantitative importance and regulatory factors in riverine ecosystems are nearly unknown. Here, we examined the spatio-temporal changes of the communities of Methanoperedens-like archaea and nitrate-driven AOM activity in sediment of Wuxijiang River, a mountainous river in China. These archaeal community composition varied significantly among reaches (upper, middle, and lower reaches) and between seasons (winter and summer), but their mcrA gene diversity showed no significant spatial or temporal variations. The copy numbers of Methanoperedens-like archaeal mcrA genes were 1.32 × 10 5 -2.47 × 10 7 copies g -1 (dry weight), and the activity of nitrate-driven AOM was 0.25-1.73 nmol CH 4 g -1 (dry weight) d -1 , which could potentially reduce 10.3% of CH 4 emissions from rivers. Significant spatio-temporal variations of mcrA gene abundance and nitrate-driven AOM activity were found. Both the gene abundance and activity increased significantly from upper to lower reaches in both seasons, and were significantly higher in sediment collected in summer than in winter. In addition, the variations of Methanoperedens-like archaeal communities and nitrate-driven AOM activity were largely impacted by the sediment temperature, NH 4 + and organic carbon contents. Taken together, both time and space scales need to be considered for better evaluating the quantitative importance of nitrate-driven AOM in reducing CH 4 emissions from riverine ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)