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Bacterial populations that originate from a single bacterium are not strictly clonal and often contain subgroups with distinct phenotypes 1 . Bacteria can generate heterogeneity through phase variation-a preprogrammed, reversible mechanism that alters gene expression levels across a population 1 . One well-studied type of phase variation involves enzyme-mediated inversion of specific regions of genomic DNA 2 . Frequently, these DNA inversions flip the orientation of promoters, turning transcription of adjacent coding regions on or off 2 . Through this mechanism, inversion can affect fitness, survival or group dynamics 3,4 . Here, we describe the development of PhaVa, a computational tool that identifies DNA inversions using long-read datasets. We also identify 372 'intragenic invertons', a novel class of DNA inversions found entirely within genes, in genomes of bacterial and archaeal isolates. Intragenic invertons allow a gene to encode two or more versions of a protein by flipping a DNA sequence within the coding region, thereby increasing coding capacity without increasing genome size. We validate ten intragenic invertons in the gut commensal Bacteroides thetaiotaomicron, and experimentally characterize an intragenic inverton in the thiamine biosynthesis gene thiC., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

In the post-genomic era, phylogenomics is a powerful and routinely-used tool to discover evolutionary relationships between microorganisms. Inferring phylogenomic trees by concatenating core gene sequences into a supermatrix is the standard method. The previously released up-to-date bacterial core gene (UBCG) tool provides a pipeline to infer phylogenomic trees using single-copy core genes for the Bacteria domain. In this study, we established up-to-date archaeal core gene (UACG), comprising 128 genes suitable for inferring archaeal phylogenomic trees. To test the gene set, we selected the Haloarcula genus and scrutinized its phylogeny. The phylogeny inferred using the UACG tool was consistent with the orthoANIu dendrogram, whereas the 16S rRNA gene phylogeny showed high intragenomic heterogeneity resulting in phylogenetic discrepancies. The software tool using the UACG set is available at https://www.ezbiocloud.net/tools/uacg ., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Genome analysis points to N-glycosylation as being an almost universal posttranslational modification in Archaea. Although such predictions have been confirmed in only a limited number of species, such studies are making it increasingly clear that the N-linked glycans which decorate archaeal glycoproteins present diversity in terms of both glycan composition and architecture far beyond what is seen in the other two domains of life. In addition to continuing to decipher pathways of N-glycosylation, recent efforts have revealed how Archaea exploit this variability in novel roles. As well as encouraging glycoprotein synthesis, folding and assembly into properly functioning higher ordered complexes, N-glycosylation also provides Archaea with a strategy to cope with changing environments. Archaea can, moreover, exploit the apparent species-specific nature of N-glycosylation for selectivity in mating, and hence, to maintain species boundaries, and in other events where cell-selective interactions are required. At the same time, addressing components of N-glycosylation pathways across archaeal phylogeny offers support for the concept of an archaeal origin for eukaryotes. In this MicroReview, these and other recent discoveries related to N-glycosylation in Archaea are considered., (© 2020 John Wiley & Sons Ltd.)

CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N 2 ) fixation were targeted for dCas9-mediated repression. First, the nif operon ( nifHI 1 I 2 DKEN ) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD Remarkably, growth of M. acetivorans with N 2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N 2 Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 , encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N 2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens., (Copyright © 2020 American Society for Microbiology.)

In the years following its discovery and characterization, the CRISPR-Cas system has been modified and converted into a multitude of applications for eukaryotes and bacteria, such as genome editing and gene regulation. Since no such method has been available for archaea, we developed a tool for gene repression in the haloarchaeon Haloferax volcanii by repurposing its endogenous type I-B CRISPR-Cas system. Here, we present the two possible approaches for gene repression as well as our workflow to achieve and assess gene knockdown, offer recommendations on protospacer selection and give some examples of genes we have successfully silenced., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The haloarchaeon Haloferax volcanii was found to grow on D-galactose as carbon and energy source. Here we report a comprehensive analysis of D-galactose catabolism in H. volcanii. Genome analyses indicated a cluster of genes encoding putative enzymes of the DeLey-Doudoroff pathway for D-galactose degradation including galactose dehydrogenase, galactonate dehydratase, 2-keto-3-deoxygalactonate kinase and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase. The recombinant galactose dehydrogenase and galactonate dehydratase showed high specificity for D-galactose and galactonate, respectively, whereas KDPGal aldolase was promiscuous in utilizing KDPGal and also the C4 epimer 2-keto-3-deoxy-6-phosphogluconate as substrates. Growth studies with knock-out mutants indicated the functional involvement of galactose dehydrogenase, galactonate dehydratase and KDPGal aldolase in D-galactose degradation. Further, the transcriptional regulator GacR was identified, which was characterized as an activator of genes of the DeLey-Doudoroff pathway. Finally, genes were identified encoding components of an ABC transporter and a knock-out mutant of the substrate binding protein indicated the functional involvement of this transporter in D-galactose uptake. This is the first report of D-galactose degradation via the DeLey-Doudoroff pathway in the domain of archaea., (© FEMS 2020.)

Several species of Sulfolobales have been used as model organisms in the study of response mechanisms to ultraviolet (UV) irradiation in hyperthermophilic crenarchaea. To date, the transcriptional responses of genes involved in the initiation of DNA replication, transcriptional regulation, protein phosphorylation, and hypothetical function have been observed in Sulfolobales species after UV irradiation. However, due to the absence of knockout experiments, the functions of these genes under in situ UV irradiation have not yet been demonstrated. In the present study, we constructed five gene knockout strains (cdc6-2, tfb3, rio1, and two genes encoding the hypothetical proteins, Saci_0951 and Saci_1302) of Sulfolobus acidocaldarius and examined their sensitivities to UV irradiation. The knockout strains exhibited significant sensitivities to UV-B irradiation, indicating that the five UV-regulated genes play an important role in responses to UV irradiation in vivo. Furthermore, Δcdc6-2, Δrio1, ΔSaci_0951, and Δtfb3 were sensitive to a wide variety of helix-distorting DNA lesions, including UV-induced DNA damage, an intra-strand crosslink, and bulky adducts. These results reveal that cdc6-2, tfb3, rio1, and Saci_0951 are play more important roles in broad responses to helix-distorting DNA damage than in specific responses to UV irradiation.

Subglacial ecosystems harbor diverse chemoautotrophic microbial communities in areas with limited organic carbon, and lithological H 2 produced during glacial erosion has been considered an important energy source in these ecosystems. To verify the H 2 -utilizing potential there and to identify the related energy-converting metabolic mechanisms of these communities, we performed metagenomic analysis on subglacial sediment samples from East Antarctica with and without H 2 supplementation. Genes coding for several [NiFe]-hydrogenases were identified in raw sediment and were enriched after H 2 incubation. All genes in the dissimilatory nitrate reduction and denitrification pathways were detected in the subglacial community, and the genes coding for these pathways became enriched after H 2 was supplied. Similarly, genes transcribing key enzymes in the Calvin cycle were detected in raw sediment and were also enriched. Moreover, key genes involved in H 2 oxidization, nitrate reduction, oxidative phosphorylation, and the Calvin cycle were identified within one metagenome-assembled genome belonging to a Polaromonas sp. As suggested by our results, the microbial community in the subglacial environment we investigated consisted of chemoautotrophic populations supported by H 2 oxidation. These results further confirm the importance of H 2 in the cryosphere.

TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis ( trp ) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis , we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli , and the protein, here designated Tar ( T hermococcalesa romatic amino acid r egulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant ( K d ) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis ( tyr-phe ) and aro operons. The protein recognized a palindromic sequence (TGGACA-N 8 -TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp , tyr-phe , and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage., (Copyright © 2019 Yamamoto et al.)

The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.

Objective: Sulfodiicoccus acidiphilus HS-1 T is the type species of the genus Sulfodiicoccus, a thermoacidophilic archaeon belonging to the order Sulfolobales (class Thermoprotei; phylum Crenarchaeota). While S. acidiphilus HS-1 T shares many common physiological and phenotypic features with other Sulfolobales species, the similarities in their 16S rRNA gene sequences are less than 89%. In order to know the genomic features of S. acidiphilus HS-1 T in the order Sulfolobales, we determined and characterized the genome of this strain., Results: The circular genome of S. acidiphilus HS-1 T is comprised of 2353,189 bp with a G+C content of 51.15 mol%. A total of 2459 genes were predicted, including 2411 protein coding and 48 RNA genes. The notable genomic features of S. acidiphilus HS-1 T in Sulfolobales species are the absence of genes for polB3 and the autotrophic carbon fixation pathway, and the distribution pattern of essential genes and sequences related to genomic replication initiation. These insights contribute to an understanding of archaeal genomic diversity and evolution.

Methanogens are an important biogenic source of methane, especially in estuarine waters across a river-to-sea gradient. However, the diversity and trophic strategy of methanogens in this gradient are not clear. In this study, the diversity and trophic strategy of methanogens in sediments across the Yellow River (YR) to the Bohai Sea (BS) gradient were investigated by high-throughput sequencing based on the 16S rRNA gene. The results showed that the diversity of methanogens in sediments varied from multitrophic communities in YR samples to specific methylotrophic communities in BS samples. The methanogenic community in YR samples was dominated by Methanosarcina, while that of BS samples was dominated by methylotrophic Methanococcoides. The distinct methanogens suggested that the methanogenic community of BS sediments did not originate from YR sediment input. High-throughput sequencing of the mcrA gene revealed that active Methanococcoides dominated in the BS enrichment cultures with trimethylamine as the substrate, and methylotrophic Methanolobus dominated in the YR enrichment cultures, as detected to a limited amount in in situ sediment samples. Methanosarcina were also detected in this gradient sample. Furthermore, the same species of Methanosarcina mazei, which was widely distributed, was isolated from the area across a river-to-sea gradient by the culture-dependent method. In summary, our results showed that a distribution of diverse methanogens across a river-to-sea gradient may shed light on adaption strategies and survival mechanisms in methanogens.

The Dallol geothermal area in the northern part of the Danakil Depression (up to 124-155 meter below sea level) is deemed one of the most extreme environments on Earth. The area is notable for being part of the Afar Depression, an incipient seafloor-spreading center located at the triple junction, between Nubian, Somali and Arabian plates, and for hosting environments at the very edge of natural physical-chemical extremities. The northern part of the Danakil Depression is dominated by the Assale salt plain (an accumulation of marine evaporite deposits) and hosts the Dallol volcano. Here, the interaction between the evaporitic deposit and the volcanisms have created the unique Dallol hot springs, which are highly acidic (pH ~ 0) and saline (saturation) with maximum temperatures ranging between 90 and 109 °C. Here we report for the first time evidence of life existing with these hot springs using a combination of morphological and molecular analyses. Ultra-small structures are shown to be entombed within mineral deposits, which are identified as members of the Order Nanohaloarchaea. The results from this study suggest the microorganisms can survive, and potential live, within this extreme environment, which has implications for understanding the limits of habitability on Earth and on (early) Mars.

Anaerobic oxidation of methane greatly contributes to global carbon cycling, yet the anaerobic oxidation of non-methane alkanes by archaea was only recently detected in lab enrichments. The distribution and activity of these archaea in natural environments are not yet reported and understood. Here, a combination of metagenomic and metatranscriptomic approaches was utilized to understand the ecological roles and metabolic potentials of methyl-coenzyme M reductase (MCR)-based alkane oxidizing (MAO) archaea in Guaymas Basin sediments. Diverse MAO archaea, including multi-carbon alkane oxidizer Ca. Syntrophoarchaeum spp., anaerobic methane oxidizing archaea ANME-1 and ANME-2c as well as sulfate-reducing bacteria HotSeep-1 and Seep-SRB2 that potentially involved in MAO processes, coexisted and showed activity in Guaymas Basin sediments. High-quality genomic bins of Ca. Syntrophoarchaeum spp., ANME-1 and ANME-2c were retrieved. They all contain and expressed mcr genes and genes in Wood-Ljungdahl pathway for the complete oxidation from alkane to CO 2 in local environment, while Ca. Syntrophoarchaeum spp. also possess beta-oxidation genes for multi-carbon alkane degradation. A global survey of potential multi-carbon alkane metabolism archaea shows that they are usually present in organic rich environments but are not limit to hydrothermal or marine ecosystems. Our study provided new insights into ecological and metabolic potentials of MAO archaea in natural environments., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Ammonia oxidation, mainly driven by ammonia-oxidizing archaea (AOA) and bacteria (AOB), plays an important role in determining the rate of nitrification in riparian zones. However, the underlying factors driving the distribution and activity of AOA and AOB in riparian zones, especially in the rhizosphere of Phragmites communis remain unknown. This study revealed the dominance of AOA in ammonium oxidization with higher abundance and activity in both rhizosphere and bulk soil in summer and winter over AOB in riparian zones, based on molecular methods and double-inhibitors method. Phylogenetic analysis showed that 54d9 cluster and Nitrososphaera dominated the AOA community and Nitrosospira dominated the AOB, respectively. For the distribution of AOA and AOB, it was the spatial heterogeneity of physicochemical properties that had the most significant effect. Specifically, TOM & TC were the main physicochemical variables accounting for the difference in abundance and community composition of AOA, and TN had an important influence on AOB in the sediment/soil in riparian zones. For abundance and activity, seasonal heterogeneity and P. communis rhizosphere had a significant impact on the archaeal activity and abundance, respectively, but did not show significant influencing on AOB. These findings suggest that the small-scale environmental heterogeneities in riparian zones are important in shaping the community composition and abundance of AOA and AOB., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Methane is a primary greenhouse gas which is responsible for global warming. The sulfate-dependent anaerobic methane oxidation (S-AOM) process catalyzed by an aerobic me thanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB) is a vital link connecting the global carbon and sulfur cycles, and it is considered to be the overriding methane sink in marine ecosystem. However, there have been few studies regarding the role of S-AOM process and the distribution of ANME archaea in intertidal ecosystem. The intertidal zone is a buffer zone between sea and land and plays an important role in global geochemical cycle. In the present study, the abundance, potential methane oxidation rate, and community structure of ANME archaea in the intertidal zone were studied by quantitative PCR, stable isotope tracing method and high-throughput sequencing. The results showed that the potential S-AOM activity ranged from 0 to 0.77 nmol 13 CO 2 g -1 (dry sediment) day -1 The copy number of 16S rRNA gene of ANME archaea reached 10 6 ∼ 10 7 copies g -1 (dry sediment). The average contribution of S-AOM to total anaerobic methane oxidation was up to 34.5%, while denitrifying anaerobic methane oxidation accounted for the rest, which implied that S-AOM process was an essential methane sink that cannot be overlooked in intertidal ecosystem. The simulated column experiments also indicated that ANME archaea were sensitive to oxygen and preferred anaerobic environmental conditions. This study will help us gain a better understanding of the global carbon-sulfur cycle and greenhouse gas emission reduction and introduce a new perspective into the enrichment of ANME archaea. IMPORTANCE The sulfate-dependent anaerobic methane oxidation (S-AOM) process catalyzed by an aerobic me thanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB) is a vital link connecting the global carbon and sulfur cycles. We conducted a research into the spatial-temporal pattern of S-AOM process and the distribution of ANME archaea in coastal sediments collected from the intertidal zone. The results implied that S-AOM process was a methane sink that cannot be overlooked in the intertidal ecosystem. We also found that ANME archaea were sensitive to oxygen and preferred anaerobic environmental conditions. This study will help us gain a better understanding of the global carbon-sulfur cycle and greenhouse gas emission reduction and introduce a new perspective into the enrichment of ANME archaea., (Copyright © 2019 American Society for Microbiology.)

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (P hhp ) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulence-associated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (P hmtB ), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter P gdh The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ 1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles. IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles., (Copyright © 2019 American Society for Microbiology.)

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O 2 ) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O 2 . Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P) + from NAD(P)H. We present a plausible mechanism by which O 2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O 2 -mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

Diversity and distribution pattern of ammonia-oxidizing archaea (AOA) were studied across a salinity gradient in the water column of Cochin Estuary (CE), a tropical monsoonal estuary along the southeast Arabian Sea. The water column of CE was found to be nutrient rich with high bacterial (3.7-6.7 × 10 8  cells L -1 ) and archaeal abundance (1.9-4.5 × 10 8  cells L -1 ). Diversity and seasonal variation in the distribution pattern of AOA were studied using clone library analysis and Denaturing gradient gel electrophoresis (DGGE). Clone library analysis of both the amoA and 16S rRNA gene sequences showed similar diversity pattern, however the diversity was more clear when the 16S rRNA gene sequences were analyzed. More than 70% of the sequences retrieved were clustered under uncultured Thaumarchaeota group 1 lineage and the major fractions of the remaining sequences were grouped into the Nitrosopumilus lineage and Nitrosopelagicus lineage. The AOA community in the CE was less adaptable to changing environmental conditions and its distribution showed seasonal variations within the DGGE banding pattern with higher diversity during the pre-monsoon period. The distribution of AOA also showed its preference to intermediate salinity for their higher diversity. Summer monsoon associated runoff and flushing played a critical role in regulating the seasonality of AOA distribution.

We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii , exhibited over 4 times higher N -acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N -acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity. IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes., (Copyright © 2018 American Society for Microbiology.)

Phosphorus (P) is a nutrient of primary importance in all living systems, and it is especially important in streams and rivers which are sensitive to anthropogenic P inputs and eutrophication. Microbes are accepted as the primary mineralizers and solubilizers of P improving bioavailability for organisms at all trophic levels. Here, we use a genomics approach with metagenome sequencing of 24 temperate streams and rivers representing a total P (TP) gradient to identify relationships between functional genes, functional gene groupings, P, and organisms within the P biogeochemical cycle. Combining information from network analyses, functional groupings, and system P levels, we have constructed a System Relational Overview of Gene Groupings (SROGG) which is a cohesive system level representation of P cycle gene and nutrient relationships. Using SROGG analysis in concert with other statistical approaches, we found that the compositional makeup of P cycle genes is strongly correlated to environmental P whereas absolute abundance of P genes shows no significant correlation to environmental P. We also found orthophosphate (PO 4 3- ) to be the dominant factor correlating with system P cycle gene composition with little evidence of a strong organic phosphorous correlation present even in more oligotrophic streams.

This study investigated the community structure of ammonia-oxidizing bacteria /archaea (AOB and AOA), as well as the effects of four aquatic plants (namely Ceratophyllum demersum, Hydrilla verticillata, Potamogeton crispus, and Nymphaea tetragona) rhizospheres on the abundance of AOB amoA, AOA amoA, anammox 16S rRNA, nirK, and nirS in Lake Liangzi, China. Phylogenetic analysis revealed that most AOB groups were Nitrosospira and Nitrosomonas, in which Nitrosospira was dominant. The AOA amoA were affiliated with two branches of classical sequences which belonging to Thaumarchaeota: water/sediments branch and soil/sediments branch. The abundance of AOA amoA in the rhizospheres of aquatic plants were higher than in the non-rhizosphere (p < 0.05), indicating that aquatic plants may promote the growth of AOA. However, the anammox 16S rRNA showed the opposite trend relative to AOA amoA (p < 0.05). Redundancy analysis (RDA) showed that the differences in abundance of AOB, AOA, anammox bacteria, and denitrifying bacteria are very likely related to the different contents of ammonia nitrogen (NH 4 + -N), pH and dissolved oxygen (DO) and thus to the rhizosphere states of aquatic plants., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ 2603 in Pyrococcus abyssi 23S rRNA and of Ψ 55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ 55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ 2607 (Ψ 2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.

Polar permafrost is at the forefront of climate change, yet only a few studies have enriched the native methane-producing microbes that might provide positive feedbacks to climate change. Samples Ant1 and Ant2, collected in Antarctic Miers Valley from permafrost sediments, with and without biogenic methane, respectively, were evaluated for methanogenic activity and presence of methanogens. After a one-year incubation of both samples under anaerobic conditions, methane production was observed only at room temperature in microcosm Ant1 with CO2/H2 (20/80) as carbon and energy sources and was monitored during the subsequent 10 years. The concentration of methane in the headspace of microcosm Ant1 changed from 0.8% to a maximum of 45%. Archaeal 16S rRNA genes from microcosm Ant1 were related to psychrotolerant Methanosarcina lacustris. Repeated efforts at achieving a pure culture of this organism were unsuccessful. Metagenomic reads obtained for the methane-producing microcosm Ant1 were assembled and resulted in a 99.84% complete genome affiliated with the genus Methanosarcina. The metagenome assembled genome contained cold-adapted enzymes and pathways suggesting that the novel uncultured Methanosarcina sp. Ant1 is adapted to sub-freezing conditions in permafrost. This is the first methanogen genome reported from the 15 000 years old permafrost of the Antarctic Dry Valleys.

Background: Haloarchaea, a major group of archaea, are able to metabolize sugars and to live in oxygenated salty environments. Their physiology and lifestyle strongly contrast with that of their archaeal ancestors. Amino acid optimizations, which lowered the isoelectric point of haloarchaeal proteins, and abundant lateral gene transfers from bacteria have been invoked to explain this deep evolutionary transition. We use network analyses to show that the evolution of novel genes exclusive to Haloarchaea also contributed to the evolution of this group., Results: We report the creation of 320 novel composite genes, both early in the evolution of Haloarchaea during haloarchaeal genesis and later in diverged haloarchaeal groups. One hundred and twenty-six of these novel composite genes derived from genetic material from bacterial genomes. These latter genes, largely involved in metabolic functions but also in oxygenic lifestyle, constitute a different gene pool from the laterally acquired bacterial genes formerly identified. These novel composite genes were likely advantageous for their hosts, since they show significant residence times in haloarchaeal genomes-consistent with a long phylogenetic history involving vertical descent and lateral gene transfer-and encode proteins with optimized isoelectric points., Conclusions: Overall, our work encourages a systematic search for composite genes across all archaeal major groups, in order to better understand the origins of novel prokaryotic genes, and in order to test to what extent archaea might have adjusted their lifestyles by incorporating and recycling laterally acquired bacterial genetic fragments into new archaeal genes.

In the present study, the shotgun high throughput metagenomic sequencing was implemented to globally capture the features of Taxus rhizosphere microbiome. Total reads could be assigned to 6925 species belonging to 113 bacteria phyla and 301 species of nine fungi phyla. For archaea and virus, 263 and 134 species were for the first time identified, respectively. More than 720,000 Unigenes were identified by clean reads assembly. The top five assigned phyla were Actinobacteria (363,941 Unigenes), Proteobacteria (182,053), Acidobacteria (44,527), Ascomycota (fungi; 18,267), and Chloroflexi (15,539). KEGG analysis predicted numerous functional genes; 7101 Unigenes belong to "Xenobiotics biodegradation and metabolism." A total of 12,040 Unigenes involved in defense mechanisms (e.g., xenobiotic metabolism) were annotated by eggNOG. Talaromyces addition could influence not only the diversity and structure of microbial communities of Taxus rhizosphere, but also the relative abundance of functional genes, including metabolic genes, antibiotic resistant genes, and genes involved in pathogen-host interaction, bacterial virulence, and bacterial secretion system. The structure and function of rhizosphere microbiome could be sensitive to non-native microbe addition, which could impact on the pollutant degradation. This study, complementary to the amplicon sequencing, more objectively reflects the native microbiome of Taxus rhizosphere and its response to environmental pressure, and lays a foundation for potential combination of phytoremediation and bioaugmentation., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mangrove species are adapted to grow at specific zones in a tidal gradient. Here we tested the hypothesis that the archaeal and bacterial ammonia-oxidizing microbial communities differ in soils dominated by the mangrove species Avicennia germinans and Rhizophora mangle. Two of the sampling locations were tidal locations, while the other location was impounded. Differences in the community compositions of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were analyzed by denaturing gradient gel electrophoresis (DGGE) of amoA genes and by MiSeq 16S rRNA gene-sequencing. The abundances of AOA and AOB were established by quantitative PCR of amoA genes. In addition, we analyzed the total microbial community composition based on 16S rRNA genes and explored the influence of soil physicochemical properties underneath Avicennia germinans and Rhizophora mangle on microbial communities. AOA were always more abundant than AOB, but the effect of mangrove species on total numbers of ammonia oxidizers was location-specific. The microbial communities including the ammonia oxidizers in soils associated with A. germinans and R. mangle differed only at the tidal locations. In conclusion, potential site-specific effects of mangrove species on soil microbial communities including those of the AOA and AOB are apparently overruled by the absence or presence of tide.

Methanogenic archaea survive under aerated soil conditions in paddy fields, and their community is stable under these conditions. Changes in the abundance and composition of an active community of methanogenic archaea were assessed by analyzing mcrA gene (encoding α subunit of methyl-coenzyme M reductase) and transcripts during a prolonged drained period in a paddy-upland rotational field. Paddy rice (Oryza sativa L.) was planted in the flooded field and rotated with soybean (Glycine max [L.] Merr.) under upland soil conditions. Soil samples were collected from the rotational plot in the first year, with paddy rice, and in the two successive years, with soybean, at six time points, before seeding, during cultivation, and after harvest as well as from a consecutive paddy (control) plot. By the time that soybean was grown in the second year, the methanogenic archaeal community in the rotational plot maintained high mcrA transcript levels, comparable with those of the control plot community, but the levels drastically decreased by over three orders of magnitude after 2 years of upland conversion. The composition of active methanogenic archaeal communities that survived upland conversion in the rotational plot was similar to that of the active community in the control plot. These results revealed that mcrA gene transcription of methanogenic archaeal community in the rotational field was affected by a prolonged non-flooding period, longer than 1 year, indicating that unknown mechanisms maintain the stability of methanogenic archaeal community in paddy fields last up to 1 year after the onset of drainage.

Microbial nitrification is a key process in the nitrogen cycle in the continental shelf ecosystems. The genotype compositions and abundance of the ammonia monooxygenase gene, amoA, derived from ammonia-oxidizing archaea (AOA) and bacteria (AOB) in two size fractions (2-10 and 0.2-2 µm), were investigated in the East China Sea (ECS) in May 2008 using PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR). Four sites were selected across the continental shelf edge: continental shelf water (CSW), Kuroshio branch water (KBW), transition between CSW and KBW (TCSKB) and coastal KBW (CKBW). The gene copy numbers of AOA-amoA were higher than those of AOB-amoA in ECS. The relative abundance of amoA to the total 16S rRNA gene level reached approximately 15% in KBW and CKBW for the free-living fraction of AOA, whereas the level was less than 0.01% throughout ECS for the AOB. A cluster analysis of the AOA-amoA-DGGE band pattern showed distinct genotype compositions in CSW in both the size fractions and in the surface of the TCSKB and KBW. Sequences of the DGGE bands were assigned to two clades. One of the clades exclusively consisted of sequences derived from the 2-10-µm fraction. This study revealed that AOA-amoA abundance dominated over AOB-amoA throughout the ECS, whereas the genotype composition of AOA-amoA were distributed heterogeneously across the water masses. Additionally, this is the first report showing the distribution of AOA-amoA genotypes characteristic to particle-associated AOA in the offshore of the East China Sea.

Chromosomal DNA replication starts at a specific region called an origin of replication. Until recently, all organisms were thought to require origins to replicate their chromosomes. It was recently discovered that some archaeal species do not utilize origins of replication under laboratory growth conditions., (Published by Elsevier Ltd.)

Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the l ast a rchaeal e ukaryote c ommon a ncestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD + transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp, whereas argD marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of S. islandicus gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism. IMPORTANCE Current procedures for the construction of deletion mutants of S. islandicus are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in S. islandicus , providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the Sulfolobus genus that are recombinogenic., (Copyright © 2017 American Society for Microbiology.)

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu 2+ resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.

SILVA (lat. forest) is a comprehensive web resource, providing services around up to date, high-quality datasets of aligned ribosomal RNA gene (rDNA) sequences from the Bacteria, Archaea, and Eukaryota domains. SILVA dates back to the year 1991 when Dr. Wolfgang Ludwig from the Technical University Munich started the integrated software workbench ARB (lat. tree) to support high-quality phylogenetic inference and taxonomy based on the SSU and LSU rDNA marker genes. At that time, the ARB project maintained both, the sequence reference datasets and the software package for data analysis. In 2005, with the massive increase of DNA sequence data, the maintenance of the software system ARB and the corresponding rRNA databases SILVA was split between Munich and the Microbial Genomics and Bioinformatics Research Group in Bremen. ARB has been continuously developed to include new features and improve the usability of the workbench. Thousands of users worldwide appreciate the seamless integration of common analysis tools under a central graphical user interface, in combination with its versatility. The first SILVA release was deployed in February 2007 based on the EMBL-EBI/ENA release 89. Since then, full SILVA releases offering the database content in various flavours are published at least annually, complemented by intermediate web-releases where only the SILVA web dataset is updated. SILVA is the only rDNA database project worldwide where special emphasis is given to the consistent naming of clades of uncultivated (environmental) sequences, where no validly described cultivated representatives are available. Also exclusive for SILVA is the maintenance of both comprehensive aligned 16S/18S rDNA and 23S/28S rDNA sequence datasets. Furthermore, the SILVA alignments and trees were designed to include Eukaryota, another unique feature among rDNA databases. With the termination of the European Ribosomal RNA Database Project in 2007, the SILVA database has become the authoritative rDNA database project for Europe. The application spectrum of ARB and SILVA ranges from biodiversity analysis, medical diagnostics, to biotechnology and quality control for academia and industry., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Origin of new biological functions is a complex phenomenon ranging from single-nucleotide substitutions to the gain of new genes via horizontal gene transfer or duplication. Neofunctionalization and subfunctionalization of proteins is often attributed to the emergence of paralogs that are subject to relaxed purifying selection or positive selection and thus evolve at accelerated rates. Such phenomena potentially could be detected as anomalies in the phylogenies of the respective gene families. We developed a computational pipeline to search for such anomalies in 1,834 orthologous clusters of archaeal genes, focusing on lineage-specific subfamilies that significantly deviate from the expected rate of evolution. Multiple potential cases of neofunctionalization and subfunctionalization were identified, including some ancient, house-keeping gene families, such as ribosomal protein S10, general transcription factor TFIIB and chaperone Hsp20. As expected, many cases of apparent acceleration of evolution are associated with lineage-specific gene duplication. On other occasions, long branches in phylogenetic trees correspond to horizontal gene transfer across long evolutionary distances. Significant deceleration of evolution is less common than acceleration, and the underlying causes are not well understood; functional shifts accompanied by increased constraints could be involved. Many gene families appear to be "highly evolvable," that is, include both long and short branches. Even in the absence of precise functional predictions, this approach allows one to select targets for experimentation in search of new biology., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public domain in the US.)

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f , cas7f , and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element., Competing Interests: The authors declare no conflict of interest.

Methanosarcina mazei Go1 is a heterotrophic methanogenic archaean contributing a significant role in global methane cycling and biomethanation process. Phylogenomic relatedness and metabolic discrepancy of this genome were described herein by comparing its whole genome sequence, intergenomic distance, genome function, synteny homologs and origin of replication, and marker genes with very closely related genomes, Methanosarcina acetivorans and Methanosarcina barkeri. Phylogenomic analysis of this study revealed that genome functional feature and metabolic core of M. mazei and M. barkeri could be originated from M. acetivorans. The metabolic core of these genomes shares a common evolutionary origin to perform the metabolic activity at different environmental niches. Genome expansion, dynamics and gene collinearity were constrained and restrained the conservation of the metabolic core genes by duplication events occurring across methanosarcinal genomes. The Darwinian positive selection was an evolutionary constraint to purify the function of core metabolic genes. Using genome-wide metabolic survey, we found the existence of four novel putative metabolic pathways such as complete methanogenesis from acetate, indole-3-acetate biosynthesis V, 4-aminobutyrate degradation III, galactosamine biosynthesis I and siroheme biosynthesis. Overall, the present study would provide a stand point to revisit its phylogenomic status in order to understand the origin and evolution history of this organism., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Archaea constitute the third domain of life, but studies on their physiology and other features have lagged behind bacteria and eukarya, largely due to the challenging biology of archaea and concomitant difficulties in methods development. The use of genome-wide en masse insertion mutagenesis is one of the most efficient means to discover the genes behind various biological functions, and such a methodology is described in this chapter for a model archaeon Haloferax volcanii. The strategy successfully employs efficient in vitro transposition in combination with gene targeting in vivo via homologous recombination. The methodology is general and should be transferable to other archaeal species.

Archaeal and eukaryotic organisms contain sets of C/D box s(no)RNAs with guide sequences that determine ribose 2'-O-methylation sites of target RNAs. The composition of these C/D box sRNA sets is highly variable between organisms and results in varying RNA modification patterns which are important for ribosomal RNA folding and stability. Little is known about the genomic organization of C/D box sRNA genes in archaea. Here, we aimed to obtain first insights into the biogenesis of these archaeal C/D box sRNAs and analyzed the genetic context of more than 300 archaeal sRNA genes. We found that the majority of these genes do not possess independent promoters but are rather located at positions that allow for co-transcription with neighboring genes and their start or stop codons were frequently incorporated into the conserved boxC and D motifs. The biogenesis of plasmid-encoded C/D box sRNA variants was analyzed in vivo in Sulfolobus acidocaldarius. It was found that C/D box sRNA maturation occurs independent of their genetic context and relies solely on the presence of intact RNA kink-turn structures. The observed plasticity of C/D box sRNA biogenesis is suggested to enable their accelerated evolution and, consequently, allow for adjustments of the RNA modification landscape., (© 2016 John Wiley & Sons Ltd.)

The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on carbon monoxide (CO) and its H2 production rates have been improved through metabolic engineering. In this study, we applied adaptive evolution to enhance H2 productivity. After over 150 serial transfers onto CO medium, cell density, CO consumption rate and H2 production rate increased. The underlying mechanism for those physiological changes could be explained by using multi-omics approaches including genomic, transcriptomic and epigenomic analyses. A putative transcriptional regulator was newly identified to regulate the expression levels of genes related to CO oxidation. Transcriptome analysis revealed significant changes in the transcript levels of genes belonging to the categories of transcription, translation and energy metabolism. Our study presents the first genome-scale methylation pattern of hyperthermophilic archaea. Adaptive evolution led to highly enhanced H2 productivity at high CO flow rates using synthesis gas produced from coal gasification.

We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla-Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis.

The genomes of the 21 completely sequenced Thermococcales display a characteristic high level of rearrangements. As a result, the prediction of their origin and termination of replication on the sole basis of chromosomal DNA composition or skew is inoperative. Using a different approach based on biologically relevant sequences, we were able to determine oriC position in all 21 genomes. The position of dif, the site where chromosome dimers are resolved before DNA segregation could be predicted in 19 genomes. Computation of the core genome uncovered a number of essential gene clusters with a remarkably stable chromosomal position across species, in sharp contrast with the scrambled nature of their genomes. The active chromosomal reorganization of numerous genes acquired by horizontal transfer, mainly from mobile elements, could explain this phenomenon., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

A systematic comparative genomic analysis of all archaeal membrane proteins that have been projected to the last archaeal common ancestor gene set led to the identification of several novel components of predicted secretion, membrane remodeling, and protein glycosylation systems. Among other findings, most crenarchaea have been shown to encode highly diverged orthologs of the membrane insertase YidC, which is nearly universal in bacteria, eukaryotes, and euryarchaea. We also identified a vast family of archaeal proteins, including the C-terminal domain of N-glycosylation protein AglD, as membrane flippases homologous to the flippase domain of bacterial multipeptide resistance factor MprF, a bifunctional lysylphosphatidylglycerol synthase and flippase. Additionally, several proteins were predicted to function as membrane transporters. The results of this work, combined with our previous analyses, reveal an unexpected diversity of putative archaeal membrane-associated functional systems that remain to be functionally characterized. A more general conclusion from this work is that the currently available collection of archaeal (and bacterial) genomes could be sufficient to identify (almost) all widespread functional modules and develop experimentally testable predictions of their functions., (Published by Elsevier B.V.)

Leaderless translation is prevalent in haloarchaea, with many of these leaderless transcripts possessing short 5'-untranslated regions (UTRs) less than 10 nucleotides. Whereas, little is known about the function of this very short 5'-UTR. Our previous studies determined that just four nucleotides preceded the start codon of hsp70 mRNA in Natrinema sp. J7, with residues -3A and +4G, relative to the A of the ATG start codon, acting as the preferred bases around the start codon of all known haloarchaeal hsp70 genes. Here, we examined the effects of nucleotides flanking the start codon on gene expression. The results revealed that shortening and deletion of the short 5'-UTR enhanced transcript levels; however, it led to significant reductions in overall translational efficiency. AUG was efficiently used as start codons, in both the presence and absence of short 5'-UTRs. GUG also could initiate translation, even though it was so inefficient that it would not be detected without considerably elevated transcript. Nucleotide substitutions at position -4 to +6 were shown to affect gene expression by transcript and/or translational levels. Notably, -3A and A/U nucleotides at position +4~+6 were more optimal for gene expression. Nucleotide transversions of -3A to -3C and +4G to +4T with hsp70 promoter from either Haloferax volcanii DS70 or Halobacterium salinarum NRC-1 showed the same effects on gene expression as that of Natrinema sp. J7. Taken together, our results suggest that the nucleotides flanking the start codon in hsp70 mRNAs with very short 5'-UTRs play an important role in haloarchaeal gene expression.

Recent studies have suggested that conductive iron oxide minerals can facilitate syntrophic metabolism of the methanogenic degradation of organic matter, such as ethanol, propionate and butyrate, in natural and engineered microbial ecosystems. This enhanced syntrophy involves direct interspecies electron transfer (DIET) powered by microorganisms exchanging metabolic electrons through electrically conductive minerals. Here, we evaluated the possibility that conductive iron oxides (hematite and magnetite) can stimulate the methanogenic degradation of benzoate, which is a common intermediate in the anaerobic metabolism of aromatic compounds. The results showed that 89-94% of the electrons released from benzoate oxidation were recovered in CH4 production, and acetate was identified as the only carbon-bearing intermediate during benzoate degradation. Compared with the iron-free controls, the rates of methanogenic benzoate degradation were enhanced by 25% and 53% in the presence of hematite and magnetite, respectively. This stimulatory effect probably resulted from DIET-mediated methanogenesis in which electrons transfer between syntrophic partners via conductive iron minerals. Phylogenetic analyses revealed that Bacillaceae, Peptococcaceae, and Methanobacterium are potentially involved in the functioning of syntrophic DIET. Considering the ubiquitous presence of iron minerals within soils and sediments, the findings of this study will increase the current understanding of the natural biological attenuation of aromatic hydrocarbons in anaerobic environments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Understanding how major organismal lineages originated is fundamental for understanding processes by which life evolved. Major evolutionary transitions, like eukaryogenesis, merging genetic material from distantly related organisms, are rare events, hence difficult ones to explain causally. If most archaeal lineages emerged after massive acquisitions of bacterial genes, a rule however arises: metabolic bacterial genes contributed to all major evolutionary transitions., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The origin of the eukaryotic cell is one of the most important transitions in the history of life. However, the emergence and early evolution of eukaryotes remains poorly understood. Recent data have shown that the last eukaryotic common ancestor (LECA) was much more complex than previously thought. The LECA already had the genetic machinery encoding the endomembrane apparatus, spliceosome, nuclear pore, and myosin and kinesin cytoskeletal motors. It is unclear, however, when the functional regulation of these cellular components evolved. Here, we address this question by analyzing the origin and evolution of the ubiquitin (Ub) signaling system, one of the most important regulatory layers in eukaryotes. We delineated the evolution of the whole Ub, Small-Ub-related MOdifier (SUMO), and Ub-fold modifier 1 (Ufm1) signaling networks by analyzing representatives from all major eukaryotic, bacterial, and archaeal lineages. We found that the Ub toolkit had a pre-eukaryotic origin and is present in three extant archaeal groups. The pre-eukaryotic Ub toolkit greatly expanded during eukaryogenesis, through massive gene innovation and diversification of protein domain architectures. This resulted in a LECA with essentially all of the Ub-related genes, including the SUMO and Ufm1 Ub-like systems. Ub and SUMO signaling further expanded during eukaryotic evolution, especially labeling and delabeling enzymes responsible for substrate selection. Additionally, we analyzed protein domain architecture evolution and found that multicellular lineages have the most complex Ub systems in terms of domain architectures. Together, we demonstrate that the Ub system predates the origin of eukaryotes and that a burst of innovation during eukaryogenesis led to a LECA with complex posttranslational regulation., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The mechanisms that underlie the origin of major prokaryotic groups are poorly understood. In principle, the origin of both species and higher taxa among prokaryotes should entail similar mechanisms--ecological interactions with the environment paired with natural genetic variation involving lineage-specific gene innovations and lineage-specific gene acquisitions. To investigate the origin of higher taxa in archaea, we have determined gene distributions and gene phylogenies for the 267,568 protein-coding genes of 134 sequenced archaeal genomes in the context of their homologues from 1,847 reference bacterial genomes. Archaeal-specific gene families define 13 traditionally recognized archaeal higher taxa in our sample. Here we report that the origins of these 13 groups unexpectedly correspond to 2,264 group-specific gene acquisitions from bacteria. Interdomain gene transfer is highly asymmetric, transfers from bacteria to archaea are more than fivefold more frequent than vice versa. Gene transfers identified at major evolutionary transitions among prokaryotes specifically implicate gene acquisitions for metabolic functions from bacteria as key innovations in the origin of higher archaeal taxa.

It was long assumed that translation initiation in prokaryotes generally occurs via the so-called Shine Dalgarno (SD) mechanism. Recently, it became clear that translation initiation in prokaryotes is more heterogeneous. In the haloarchaeon Haloferax volcanii, the majority of transcripts is leaderless and most transcripts with a 5'-UTR lack a SD motif. Nevertheless, a bioinformatic analysis predicted that 20-30% of all genes are preceded by a SD motif in haloarchaea. To analyze the importance of the SD mechanism for translation initiation in haloarchaea experimentally the monocistronic sod gene was chosen, which contains a 5'-UTR with an extensive SD motif of seven nucleotides and a length of 19 nt, the average length of 5'UTRs in this organism. A translational fusion of part of the sod gene with the dhfr reporter gene was constructed. A mutant series was generated that matched the SD motif from zero to eight positions, respectively. Surprisingly, there was no correlation between the base pairing ability between transcripts and 16S rRNA and translational efficiency in vivo under several different growth conditions. Furthermore, complete replacement of the SD motif by three unrelated sequences did not reduce translational efficiency. The results indicate that H. volcanii does not make use of the SD mechanism for translation initiation in 5'-UTRs. A genome analysis revealed that while the number of SD motifs in 5'-UTRs is rare, their fraction within open reading frames is high. Possible biological functions for intragenic SD motifs are discussed, including re-initiation of translation at distal genes in operons.

A screening system for directed evolution of DNA polymerases employing a fluorescent Scorpion probe as a reporter has been developed. The screening system has been validated in a directed evolution experiment of a distributive polymerase from the Y-polymerase family (Dpo4 from Sulfolobus solfataricus) which was improved in elongation efficiency of consecutive mismatches. The engineering campaign yielded improved Dpo4 polymerase variants one of which was successfully benchmarked in a sequence saturation mutagenesis experiment especially with regard to the desirable consecutive transversion mutations ([2.5-fold increase in frequency relative to a reference library prepared with Dpo4 WT). The Scorpion probe screening system enables to reengineer polymerases with low processivity and fidelity, and no secondary activities (i.e. exonuclease activity or strand displacement activity) to match demands in diversity generation for directed protein evolution.