RESUMEN
Many of the Earth's microbes remain uncultured and understudied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in most metagenomic studies1. Here we analysed 149,842 environmental genomes from multiple habitats2-6 and compiled a curated catalogue of 404,085 functionally and evolutionarily significant novel (FESNov) gene families exclusive to uncultivated prokaryotic taxa. All FESNov families span multiple species, exhibit strong signals of purifying selection and qualify as new orthologous groups, thus nearly tripling the number of bacterial and archaeal gene families described to date. The FESNov catalogue is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes and orders, probably representing synapomorphies that facilitated their evolutionary divergence. Using genomic context analysis and structural alignments we predicted functional associations for 32.4% of FESNov families, including 4,349 high-confidence associations with important biological processes. These predictions provide a valuable hypothesis-driven framework that we used for experimental validatation of a new gene family involved in cell motility and a novel set of antimicrobial peptides. We also demonstrate that the relative abundance profiles of novel families can discriminate between environments and clinical conditions, leading to the discovery of potentially new biomarkers associated with colorectal cancer. We expect this work to enhance future metagenomics studies and expand our knowledge of the genetic repertory of uncultivated organisms.
Asunto(s)
Archaea , Bacterias , Ecosistema , Evolución Molecular , Genes Arqueales , Genes Bacterianos , Genómica , Conocimiento , Péptidos Antimicrobianos/genética , Archaea/clasificación , Archaea/genética , Bacterias/clasificación , Bacterias/genética , Biomarcadores , Movimiento Celular/genética , Neoplasias Colorrectales/genética , Genómica/métodos , Genómica/tendencias , Metagenómica/tendencias , Familia de Multigenes , Filogenia , Reproducibilidad de los ResultadosRESUMEN
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 .
Asunto(s)
Bacterias , Programas Informáticos , Filogenia , ARN Ribosómico 16S/genética , Bacterias/genética , Genes Arqueales/genéticaRESUMEN
Many aerobic microbes can utilize alternative electron acceptors under oxygen-limited conditions. In some cases, this is mediated by extracellular electron transfer (or EET), wherein electrons are transferred to extracellular oxidants such as iron oxide and manganese oxide minerals. Here, we show that an ammonia-oxidizer previously known to be strictly aerobic, Nitrosomonas communis, may have been able to utilize a poised electrode to maintain metabolic activity in anoxic conditions. The presence and activity of multiheme cytochromes in N. communis further suggest a capacity for EET. Molecular clock analysis shows that the ancestors of ß-proteobacterial ammonia oxidizers appeared after Earth's atmospheric oxygenation when the oxygen levels were >10-4pO2 (present atmospheric level [PAL]), consistent with aerobic origins. Equally important, phylogenetic reconciliations of gene and species trees show that the multiheme c-type EET proteins in Nitrosomonas and Nitrosospira lineages were likely acquired by gene transfer from γ-proteobacteria when the oxygen levels were between 0.1 and 1 pO2 (PAL). These results suggest that ß-proteobacterial EET evolved during the Proterozoic when oxygen limitation was widespread, but oxidized minerals were abundant.
Asunto(s)
Amoníaco , Gammaproteobacteria , Oxidación-Reducción , Amoníaco/metabolismo , Electrones , Filogenia , Oxígeno , Genes Arqueales , Gammaproteobacteria/metabolismoRESUMEN
Three novel filamentous halophilic archaea, strains DFN5T, RDMS1, and QDMS1, were isolated from the coastal saline soil samples of the intertidal zones located in different regions of Jiangsu Province, China. The colonies of these strains were pinkish-white due to the presence of white spores. These three strains are extremely halophilic and grew optimally at 35-37 °C and pH 7.0-7.5. Based on 16S rRNA and rpoB' gene analysis, strains DFN5T, RDMS1, and QDMS1 gathered together in phylogenetic trees and then clustered with the current species of the genus Halocatena showing 96.9-97.4% and 82.2-82.5% similarities, respectively. Both the 16S rRNA gene-based and rpoB' gene-based phylogenies were fully supported by the phylogenomic analysis, and the overall genome-related indexes indicated that strains DFN5T, RDMS1, and QDMS1 should be a novel species of the genus Halocatena. Genome mining revealed that there are considerable differences in the genes related to ß-carotene synthesis among these three strains and the current species of Halocatena. The major polar lipids of strains DFN5T, RDMS1, and QDMS1 are PA, PG, PGP-Me, S-TGD-1, TGD-1, and TGD-2. The minor polar lipids, S-DGD-1, DGD-1, S2-DGD, and S-TeGD may be detected. According to the phenotypic characteristics, phylogenetic analysis, genomic and chemotaxonomic features, strains DFN5T (= CGMCC 1.19401 T = JCM 35422 T), RDMS1 (= CGMCC 1.19411) and QDMS1 (= CGMCC 1.19410) were classified as a novel species of the genus Halocatena with the proposed name, Halocatena marina sp. nov. This is the first report of the description of a novel filamentous haloarchaeon isolated from marine intertidal zones.
Asunto(s)
Halobacteriaceae , Halobacteriales , Glucolípidos , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , ADN de Archaea/genética , Composición de Base , Hibridación de Ácido Nucleico , Genes Arqueales , ChinaRESUMEN
The biology of Korarchaeia remains elusive due to the lack of genome representatives. Here, we reconstruct 10 closely related metagenome-assembled genomes from hot spring habitats and place them into a single species, proposed herein as Panguiarchaeum symbiosum. Functional investigation suggests that Panguiarchaeum symbiosum is strictly anaerobic and grows exclusively in thermal habitats by fermenting peptides coupled with sulfide and hydrogen production to dispose of electrons. Due to its inability to biosynthesize archaeal membranes, amino acids, and purines, this species likely exists in a symbiotic lifestyle similar to DPANN archaea. Population metagenomics and metatranscriptomic analyses demonstrated that genes associated with amino acid/peptide uptake and cell attachment exhibited positive selection and were highly expressed, supporting the proposed proteolytic catabolism and symbiotic lifestyle. Our study sheds light on the metabolism, evolution, and potential symbiotic lifestyle of Panguiarchaeum symbiosum, which may be a unique host-dependent archaeon within the TACK superphylum.
Asunto(s)
Archaea , Manantiales de Aguas Termales , Simbiosis , Simbiosis/genética , Manantiales de Aguas Termales/microbiología , Fermentación , Anaerobiosis , Aminoácidos/metabolismo , Coenzimas/metabolismo , Filogeografía , Polimorfismo de Nucleótido Simple/genética , Azufre/metabolismo , Péptidos/metabolismo , Proteolisis , Archaea/clasificación , Archaea/citología , Archaea/genética , Adhesión Celular/genética , Genes Arqueales , Regulación de la Expresión Génica Arqueal , Genoma Arqueal , Metagenómica , MetagenomaRESUMEN
Archaea inhabit a wide variety of habitats and are well-placed to provide insights into the origins of eukaryotes. In this primer, we examine the available model archaeal genetic systems. We consider the limitations and barriers involved in genetically modifying different archaeal species, the techniques and breakthroughs that have contributed to their tractability, and potential areas for future development.
Asunto(s)
Archaea , Eucariontes , Archaea/genética , Eucariontes/genética , Genes ArquealesRESUMEN
Thermophiles and hyperthermophiles are immensely useful in understanding the evolution of life, besides their utility in environmental and industrial biotechnology. Advancements in sequencing technologies have revolutionized the field of microbial genomics. The massive generation of data enhances the sequencing coverage multi-fold and allows to analyse the entire genomic features of microbes efficiently and accurately. The mandate of a pure isolate can also be bypassed where whole metagenome-assembled genomes and single cell-based sequencing have fulfilled the majority of the criteria to decode various attributes of microbial genomes. A boom has, therefore, been seen in analysing the extremophilic bacteria and archaea using sequence-based approaches. Due to extensive sequence analysis, it becomes easier to understand the gene flow and their evolution among the members of bacteria and archaea. For instance, sequencing unveiled that Thermotoga maritima shares around 24% of genes of archaeal origin. Comparative and functional genomics provide an analytical view to understanding the microbial diversity of thermophilic bacteria and archaea, their interactions with other microbes, their adaptations, gene flow, and evolution over time. In this review, the genomic features of thermophilic bacteria and archaea are dealt with comprehensively.
Asunto(s)
Archaea , Bacterias , Archaea/genética , Bacterias/genética , Genes Arqueales , Genómica , Metagenoma , FilogeniaRESUMEN
Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.
Asunto(s)
Toxinas Bacterianas/metabolismo , Sistemas CRISPR-Cas , Haloarcula/genética , Halobacterium/genética , ARN Pequeño no Traducido/metabolismo , ARN de Transferencia de Isoleucina/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Ingeniería Celular , Genes Arqueales , Genes Bacterianos , Biosíntesis de Proteínas , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , ARN de Transferencia de Arginina/metabolismoRESUMEN
tRNAscan-SE has been widely used for transfer RNA (tRNA) gene prediction for over twenty years, developed just as the first genomes were decoded. With the massive increase in quantity and phylogenetic diversity of genomes, the accurate detection and functional prediction of tRNAs has become more challenging. Utilizing a vastly larger training set, we created nearly one hundred specialized isotype- and clade-specific models, greatly improving tRNAscan-SE's ability to identify and classify both typical and atypical tRNAs. We employ a new comparative multi-model strategy where predicted tRNAs are scored against a full set of isotype-specific covariance models, allowing functional prediction based on both the anticodon and the highest-scoring isotype model. Comparative model scoring has also enhanced the program's ability to detect tRNA-derived SINEs and other likely pseudogenes. For the first time, tRNAscan-SE also includes fast and highly accurate detection of mitochondrial tRNAs using newly developed models. Overall, tRNA detection sensitivity and specificity is improved for all isotypes, particularly those utilizing specialized models for selenocysteine and the three subtypes of tRNA genes encoding a CAU anticodon. These enhancements will provide researchers with more accurate and detailed tRNA annotation for a wider variety of tRNAs, and may direct attention to tRNAs with novel traits.
Asunto(s)
ARN de Transferencia/genética , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Genes Arqueales , Genes Bacterianos , Genes FúngicosRESUMEN
Arsenic (As) metabolism genes are generally present in soils, but their diversity, relative abundance, and transcriptional activity in response to different As concentrations remain unclear, limiting our understanding of the microbial activities that control the fate of an important environmental pollutant. To address this issue, we applied metagenomics and metatranscriptomics to paddy soils showing a gradient of As concentrations to investigate As resistance genes (ars) including arsR, acr3, arsB, arsC, arsM, arsI, arsP, and arsH as well as energy-generating As respiratory oxidation (aioA) and reduction (arrA) genes. Somewhat unexpectedly, the relative DNA abundances and diversities of ars, aioA, and arrA genes were not significantly different between low and high (â¼10 versus â¼100 mg kg-1) As soils. Compared to available metagenomes from other soils, geographic distance rather than As levels drove the different compositions of microbial communities. Arsenic significantly increased ars gene abundance only when its concentration was higher than 410 mg kg-1. In contrast, metatranscriptomics revealed that relative to low-As soils, high-As soils showed a significant increase in transcription of ars and aioA genes, which are induced by arsenite, the dominant As species in paddy soils, but not arrA genes, which are induced by arsenate. These patterns appeared to be community wide as opposed to taxon specific. Collectively, our findings advance understanding of how microbes respond to high As levels and the diversity of As metabolism genes in paddy soils and indicated that future studies of As metabolism in soil or other environments should include the function (transcriptome) level. IMPORTANCE Arsenic (As) is a toxic metalloid pervasively present in the environment. Microorganisms have evolved the capacity to metabolize As, and As metabolism genes are ubiquitously present in the environment even in the absence of high concentrations of As. However, these previous studies were carried out at the DNA level; thus, the activity of the As metabolism genes detected remains essentially speculative. Here, we show that the high As levels in paddy soils increased the transcriptional activity rather than the relative DNA abundance and diversity of As metabolism genes. These findings advance our understanding of how microbes respond to and cope with high As levels and have implications for better monitoring and managing an important toxic metalloid in agricultural soils and possibly other ecosystems.
Asunto(s)
Arsénico/metabolismo , Genes Arqueales , Genes Bacterianos , Microbiología del Suelo , Contaminantes del Suelo/metabolismo , Archaea/genética , Archaea/metabolismo , Arsénico/análisis , Bacterias/genética , Bacterias/metabolismo , Biodegradación Ambiental , Metales Pesados/análisis , Oryza , ARN Ribosómico 16S , Contaminantes del Suelo/análisisRESUMEN
Essential genes are required for the reproduction and survival of an organism. Rapid identification of essential genes has practical application value in biomedicine. Information theory is a discipline that studies information transmission. Based on the similarity between heredity and information transmission, measures derived from information theory can be applied to genetic sequence analysis on different scales. In this study, we employed 114 features extracted by information theory methods to construct an essential gene prediction model. We applied a backpropagation neural network to construct a classifier and employed it to predict essential genes of 37 prokaryotes. The performance of the classifier was evaluated by applying intra-organism prediction and leave-one-species-out prediction. Among 37 prokaryotes, intra-organism prediction and leave-one-species-out prediction yielded average AUC scores of 0.791 and 0.717, respectively. Considering the potential redundancy in the feature set, we performed feature selection and constructed a key feature subset. In the above two prediction methods, the average AUC scores of 37 organisms obtained by using key features were 0.786 and 0.714, respectively. The results show the potential and universality of information-theoretic features in the study of prokaryotic essential gene prediction.
Asunto(s)
Genes Esenciales , Genómica/métodos , Modelos Teóricos , Algoritmos , Biología Computacional , Genes Arqueales , Genes Bacterianos , Redes Neurales de la ComputaciónRESUMEN
A novel mesophilic and aerobic ammonia-oxidizing archaeon of the phylum Thaumarchaeota, strain NM25T, was isolated from coastal eelgrass zone sediment sampled in Shimoda (Japan). The cells were rod-shaped with an S-layer cell wall. The temperature range for growth was 20-37 °C, with an optimum at 30 °C. The pH range for growth was pH 6.1-7.7, with an optimum at pH 7.1. The salinity range for growth was 5-40â%, with an optimum range of 15-32â%. Cells obtained energy from ammonia oxidation and used bicarbonate as a carbon source. Utilization of urea was not observed for energy generation and growth. Strain NM25T required a hydrogen peroxide scavenger, such as α-ketoglutarate, pyruvate or catalase, for sustained growth on ammonia. Growth of strain NM25T was inhibited by addition of low concentrations of some organic compounds and organic mixtures, including complete inhibition by glycerol, peptone and yeast extract. Phylogenetic analysis of four concatenated housekeeping genes (16S rRNA, rpoB, rpsI and atpD) and concatenated AmoA, AmoB, AmoC amino acid sequences indicated that the isolate is similar to members of the genus Nitrosopumilus. The closest relative is Nitrosopumilus ureiphilus PS0T with sequence similarities of 99.5â% for the 16S rRNA gene and 97.2â% for the amoA gene. Genome relatedness between strain NM25T and N. ureiphilus PS0T was assessed by average nucleotide identity and digital DNA-DNA hybridization, giving results of 85.4 and 40.2â%, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strain NM25T represents a novel species of the genus Nitrosopumilus, for which the name sp. nov, is proposed. The type strain is NM25T (=NBRC 111181T=ATCC TSD-147T).
Asunto(s)
Amoníaco , Archaea , Sedimentos Geológicos/microbiología , Filogenia , Humedales , Archaea/clasificación , Archaea/aislamiento & purificación , Genes Arqueales , Japón , Hibridación de Ácido Nucleico , Oxidación-Reducción , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Chromosome organization in archaea has long been enigmatic due, in part, to the typically small cell size of archaea and the extremophilic nature of many of the model archaeal species studies, rendering live-cell imaging technically challenging. To circumvent these problems, we recently applied chromosome conformation capture combined with biotin enrichment and deep sequencing (Hi-C) to members of hyperthermophilic archaeal genus Sulfolobus. Our optimized Hi-C protocol described here permits delineation of how Sulfolobus species organize their chromosomes. For complete details on the use and execution of this protocol, please refer to Takemata et al. (2019).
Asunto(s)
Biotina/metabolismo , Cromosomas de Archaea , Sulfolobus acidocaldarius/metabolismo , Genes Arqueales , Análisis de Secuencia de ADN/métodos , Sulfolobus acidocaldarius/genéticaRESUMEN
The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and nonsubstrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.
Asunto(s)
Isomerasas de Aminoácido/metabolismo , Proteínas Arqueales/metabolismo , Thermococcus/enzimología , Isomerasas de Aminoácido/química , Secuencia de Aminoácidos , Proteínas Arqueales/química , Electroforesis en Gel de Poliacrilamida , Genes Arqueales , Chaperonas Moleculares/metabolismo , Filogenia , Especificidad por Sustrato , Thermococcus/genéticaRESUMEN
Essential genes, which form the basis of life activities, are crucial for the survival of organisms. Essential genes tend to be located in operons, but how they are distributed in operons is still unclear for most prokaryotes. In order to clarify the general rule of position preference of essential genes in operons, an index of the average position of genes in an operon was proposed, and the distributions of essential and non-essential genes in operons in 51 bacterial genomes and two archaeal genomes were analyzed based on this new index. Consequently, essential genes were found to preferentially occupy the front positions of the operons, which tend to be expressed at higher levels.
Asunto(s)
Archaea/genética , Bacterias/genética , Genes Arqueales , Genes Bacterianos , Operón , Biología Computacional/métodos , Bases de Datos Genéticas , Genes EsencialesRESUMEN
The last eukaryote common ancestor (LECA) possessed mitochondria and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors, yet the timing of mitochondrial acquisition and the role of mitochondria in the origin of eukaryote complexity remain debated. Here, we report evidence from gene duplications in LECA indicating an early origin of mitochondria. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes, we identify 713 gene duplication events that occurred in LECA. LECA's bacterial-derived genes include numerous mitochondrial functions and were duplicated significantly more often than archaeal-derived and eukaryote-specific genes. The surplus of bacterial-derived duplications in LECA most likely reflects the serial copying of genes from the mitochondrial endosymbiont to the archaeal host's chromosomes. Clustering, phylogenies and likelihood ratio tests for 22.4 million genes from 5,655 prokaryotic and 150 eukaryotic genomes reveal no evidence for lineage-specific gene acquisitions in eukaryotes, except from the plastid in the plant lineage. That finding, and the functions of bacterial genes duplicated in LECA, suggests that the bacterial genes in eukaryotes are acquisitions from the mitochondrion, followed by vertical gene evolution and differential loss across eukaryotic lineages, flanked by concomitant lateral gene transfer among prokaryotes. Overall, the data indicate that recurrent gene transfer via the copying of genes from a resident mitochondrial endosymbiont to archaeal host chromosomes preceded the onset of eukaryotic cellular complexity, favoring mitochondria-early over mitochondria-late hypotheses for eukaryote origin.
Asunto(s)
Evolución Biológica , Eucariontes/genética , Duplicación de Gen , Mitocondrias/genética , Evolución Molecular , Transferencia de Gen Horizontal , Genes Arqueales , Genes BacterianosRESUMEN
Climate warming is known to impact ecosystem composition and functioning. However, it remains largely unclear how soil microbial communities respond to long-term, moderate warming. In this study, we used Illumina sequencing and microarrays (GeoChip 5.0) to analyze taxonomic and functional gene compositions of the soil microbial community after 14 years of warming (at 0.8-1.0 °C for 10 years and then 1.5-2.0 °C for 4 years) in a Californian grassland. Long-term warming had no detectable effect on the taxonomic composition of soil bacterial community, nor on any plant or abiotic soil variables. In contrast, functional gene compositions differed between warming and control for bacterial, archaeal, and fungal communities. Functional genes associated with labile carbon (C) degradation increased in relative abundance in the warming treatment, whereas those associated with recalcitrant C degradation decreased. A number of functional genes associated with nitrogen (N) cycling (e.g., denitrifying genes encoding nitrate-, nitrite-, and nitrous oxidereductases) decreased, whereas nifH gene encoding nitrogenase increased in the warming treatment. These results suggest that microbial functional potentials are more sensitive to long-term moderate warming than the taxonomic composition of microbial community.
Asunto(s)
Archaea/genética , Bacterias/clasificación , Hongos/genética , Análisis de Secuencia de ARN/métodos , Bacterias/genética , Bacterias/aislamiento & purificación , Carbono/metabolismo , Perfilación de la Expresión Génica , Genes Arqueales , Genes Bacterianos , Genes Fúngicos , Calentamiento Global , Pradera , Secuenciación de Nucleótidos de Alto Rendimiento , Nitrógeno/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , ARN Ribosómico 16S/genética , Microbiología del SueloRESUMEN
The genome of the hyperthermophilic and piezophilic euryarchaeaon Thermococcus barophilus Ch5 encodes three putative alcohol dehydrogenases (Tba ADHs). Herein, we characterized Tba ADH547 biochemically and probed its catalytic mechanism by mutational studies. Our data demonstrate that Tba ADH547 can oxidize ethanol and reduce acetaldehyde at high temperature with the same optimal temperature (75 °C) and exhibit similar thermostability for oxidization and reduction reactions. However, Tba ADH547 has different optimal pH for oxidation and reduction: 8.5 for oxidation and 7.0 for reduction. Tba ADH547 is dependent on a divalent ion for its oxidation activity, among which Mn2+ is optimal. However, Tba ADH547 displays about 20% reduction activity without a divalent ion, and the maximal activity with Fe2+. Furthermore, Tba ADH547 showcases a strong substrate preference for 1-butanol and 1-hexanol over ethanol and other alcohols. Similarly, Tba ADH547 prefers butylaldehyde to acetaldehyde as its reduction substrate. Mutational studies showed that the mutations of residues D195, H199, H262 and H274 to Ala result in the significant activity loss of Tba ADH547, suggesting that residues D195, H199, H262 and H274 are responsible for catalysis. Overall, Tba ADH547 is a thermoactive ADH with novel biochemical characteristics, thereby allowing this enzyme to be a potential biocatalyst.
Asunto(s)
Aldehído Oxidorreductasas/aislamiento & purificación , Proteínas Arqueales/aislamiento & purificación , Thermococcus/enzimología , Alcoholes/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , Cationes/farmacología , Dicroismo Circular , Secuencia Conservada , Genes Arqueales , Calor , Concentración de Iones de Hidrógeno , Cinética , Mutagénesis Sitio-Dirigida , Filogenia , Desnaturalización Proteica , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Thermococcales/enzimología , Thermococcales/genética , Thermococcus/genéticaRESUMEN
CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division (cdvA), transcription (rpo8), and RNA metabolism (smAP2) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius. Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis (slaB) and translation (aif5A). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III - based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Genes Arqueales , Genes Esenciales , Genes Letales , Sulfolobales/genética , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , División Celular/genética , Orden Génico , Marcación de Gen , Vectores Genéticos/genética , Mutación , Operón , Fenotipo , ARN Guía de Kinetoplastida , Sulfolobales/metabolismoRESUMEN
CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria. Organisms of Sulfolobales, an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum, usually contain both type I and type III CRISPR-Cas systems. Two species, Saccharolobus solfataricus and Sulfolobus islandicus, have been important models for CRISPR study in archaea, and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps: adaptation, expression and crRNA processing, and interference. Four subtypes of CRISPR-Cas systems are common in these organisms, including I-A, I-D, III-B, and III-D. These cas genes form functional modules, e.g., all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and iCas modules. Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea, and these methodologies are useful in demonstration of the protospacer-adjacent motif (PAM)-dependent DNA interference by I-A interference modules and multiple interference activities by III-B Cmr systems. Ribonucleoprotein effector complexes have been isolated for Sulfolobales III-B and III-D systems, and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.