RESUMEN
Nosocomial pathogens of the Acinetobacter calcoaceticus-baumannii (ACB) complex are a cautionary example for the world-wide spread of multi- and pan-drug resistant bacteria. Aiding the urgent demand for novel therapeutic targets, comparative genomics studies between pathogens and their apathogenic relatives shed light on the genetic basis of human-pathogen interaction. Yet, existing studies are limited in taxonomic scope, sensing of the phylogenetic signal, and resolution by largely analyzing genes independent of their organization in functional gene clusters. Here, we explored more than 3,000 Acinetobacter genomes in a phylogenomic framework integrating orthology-based phylogenetic profiling and microsynteny conservation analyses. We delineate gene clusters in the type strain A. baumannii ATCC 19606 whose evolutionary conservation indicates a functional integration of the subsumed genes. These evolutionarily stable gene clusters (ESGCs) reveal metabolic pathways, transcriptional regulators residing next to their targets but also tie together sub-clusters with distinct functions to form higher-order functional modules. We shortlisted 150 ESGCs that either co-emerged with the pathogenic ACB clade or are preferentially found therein. They provide a high-resolution picture of genetic and functional changes that coincide with the manifestation of the pathogenic phenotype in the ACB clade. Key innovations are the remodeling of the regulatory-effector cascade connecting LuxR/LuxI quorum sensing via an intermediate messenger to biofilm formation, the extension of micronutrient scavenging systems, and the increase of metabolic flexibility by exploiting carbon sources that are provided by the human host. We could show experimentally that only members of the ACB clade use kynurenine as a sole carbon and energy source, a substance produced by humans to fine-tune the antimicrobial innate immune response. In summary, this study provides a rich and unbiased set of novel testable hypotheses on how pathogenic Acinetobacter interact with and ultimately infect their human host. It is a comprehensive resource for future research into novel therapeutic strategies.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter calcoaceticus , Infecciones por Acinetobacter/genética , Infecciones por Acinetobacter/microbiología , Acinetobacter calcoaceticus/genética , Carbono , Humanos , Familia de Multigenes/genética , Filogenia , VirulenciaRESUMEN
Even though high-throughput transcriptome sequencing is routinely performed in many laboratories, computational analysis of such data remains a cumbersome process often executed manually, hence error-prone and lacking reproducibility. For corresponding data processing, we introduce Curare, an easy-to-use yet versatile workflow builder for analyzing high-throughput RNA-Seq data focusing on differential gene expression experiments. Data analysis with Curare is customizable and subdivided into preprocessing, quality control, mapping, and downstream analysis stages, providing multiple options for each step while ensuring the reproducibility of the workflow. For a fast and straightforward exploration and visualization of differential gene expression results, we provide the gene expression visualizer software GenExVis. GenExVis can create various charts and tables from simple gene expression tables and DESeq2 results without the requirement to upload data or install software packages. In combination, Curare and GenExVis provide a comprehensive software environment that supports the entire data analysis process, from the initial handling of raw RNA-Seq data to the final DGE analyses and result visualizations, thereby significantly easing data processing and subsequent interpretation.
Asunto(s)
Curare , RNA-Seq , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodos , Transcriptoma , Programas Informáticos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Perfilación de la Expresión Génica/métodosRESUMEN
Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.
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Enfermedades Pulmonares/patología , Pulmón/crecimiento & desarrollo , Organoides/crecimiento & desarrollo , Células Madre/fisiología , Animales , Ataxina-1/genética , Ataxina-1/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Células Endoteliales/citología , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Células Epiteliales/citología , Fibroblastos , Humanos , Pulmón/citología , Células Madre Mesenquimatosas , Ratones , Morfogénesis/genética , Morfogénesis/fisiología , Organogénesis/fisiología , Organoides/citología , Alveolos Pulmonares/citología , Alveolos Pulmonares/crecimiento & desarrollo , ARN Mensajero/metabolismo , Regeneración/genética , Regeneración/fisiologíaRESUMEN
By investigating wet and dry age-related ripening of beef, Pseudomonas strains V3/3/4/13T and V3/K/3/5T were isolated. Strain V3/3/4/13T exhibited more than 99â% 16S rRNA gene-based similarity to Pseudomonas fragi and other members of this group, while isolate V3/K/3/5T was very close to Pseudomonas veronii and a number of relatives within the Pseudomonas fluorescens group. Additional comparisons of complete rpoB sequences and draft genomes allowed us to place isolate V3/3/4/13T close to Pseudomonas deceptionensis DSM 26521T. In the case of V3/K/3/5T the closest relative was P. veronii DSM 11331T. Average nucleotide identity (ANIb) and digital DNA-DNA hybridization (dDDH) values calculated from the draft genomes of V3/3/4/13T and P. deceptionensis DSM 26521T were 88.5 and 39.8â%, respectively. For V3/K/3/5T and its closest relative P. veronii DSM 11331T, the ANIb value was 95.1â% and the dDDH value was 60.7â%. The DNA G+C contents of V3/3/4/13T and V3/K/3/5T were 57.4 and 60.8 mol%, respectively. Predominant fatty acids were C16â:â0, C18â:â1 ω7c, C17â:â0 cyclo and summed feature C16â:â1 ω7ct/C15â:â0 iso 2OH. The main respiratory quinones were Q9, with minor proportions of Q8 and, in the case of V3/K/3/5T, additional Q10. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and, in the case of V3/K/3/5T, additional phosphatidylcholine. Based on the combined data, isolates V3/3/4/13T and V3/K/3/5T should be considered as representatives of two novel Pseudomonas species. The type strain of the newly proposed Pseudomonas kulmbachensis sp. nov. is V3/3/4/13T (=DSM 113654T=LMG 32520T), a second strain belonging to the same species is FLM 004-28 (=DSM 113604=LMG 32521); the type strain for the newly proposed Pseudomonas paraveronii sp. nov. is V3/K/3/5T (=DSM 113573T=LMG 32518T) with a second isolate FLM 11 (=DSM 113572=LMG 32519).
Asunto(s)
Pollos , Ácidos Grasos , Animales , Bovinos , Composición de Base , Ácidos Grasos/química , ARN Ribosómico 16S/genética , Filogenia , Análisis de Secuencia de ADN , ADN Bacteriano/genética , Técnicas de Tipificación Bacteriana , Pseudomonas/genética , NucleótidosRESUMEN
Genome erosion is a frequently observed result of relaxed selection in insect nutritional symbionts, but it has rarely been studied in defensive mutualisms. Solitary beewolf wasps harbor an actinobacterial symbiont of the genus Streptomyces that provides protection to the developing offspring against pathogenic microorganisms. Here, we characterized the genomic architecture and functional gene content of this culturable symbiont using genomics, transcriptomics, and proteomics in combination with in vitro assays. Despite retaining a large linear chromosome (7.3 Mb), the wasp symbiont accumulated frameshift mutations in more than a third of its protein-coding genes, indicative of incipient genome erosion. Although many of the frameshifted genes were still expressed, the encoded proteins were not detected, indicating post-transcriptional regulation. Most pseudogenization events affected accessory genes, regulators, and transporters, but "Streptomyces philanthi" also experienced mutations in central metabolic pathways, resulting in auxotrophies for biotin, proline, and arginine that were confirmed experimentally in axenic culture. In contrast to the strong A+T bias in the genomes of most obligate symbionts, we observed a significant G+C enrichment in regions likely experiencing reduced selection. Differential expression analyses revealed that-compared to in vitro symbiont cultures-"S. philanthi" in beewolf antennae showed overexpression of genes for antibiotic biosynthesis, the uptake of host-provided nutrients and the metabolism of building blocks required for antibiotic production. Our results show unusual traits in the early stage of genome erosion in a defensive symbiont and suggest tight integration of host-symbiont metabolic pathways that effectively grants the host control over the antimicrobial activity of its bacterial partner.
Asunto(s)
Antibacterianos/biosíntesis , Genoma Bacteriano , Seudogenes , Streptomyces/genética , Avispas/microbiología , Animales , Antenas de Artrópodos/metabolismo , Femenino , Chaperonas Moleculares/metabolismo , Streptomyces/metabolismo , SimbiosisRESUMEN
MOTIVATION: Antimicrobial resistance (AMR) is one of the biggest global problems threatening human and animal health. Rapid and accurate AMR diagnostic methods are thus very urgently needed. However, traditional antimicrobial susceptibility testing (AST) is time-consuming, low throughput and viable only for cultivable bacteria. Machine learning methods may pave the way for automated AMR prediction based on genomic data of the bacteria. However, comparing different machine learning methods for the prediction of AMR based on different encodings and whole-genome sequencing data without previously known knowledge remains to be done. RESULTS: In this study, we evaluated logistic regression (LR), support vector machine (SVM), random forest (RF) and convolutional neural network (CNN) for the prediction of AMR for the antibiotics ciprofloxacin, cefotaxime, ceftazidime and gentamicin. We could demonstrate that these models can effectively predict AMR with label encoding, one-hot encoding and frequency matrix chaos game representation (FCGR encoding) on whole-genome sequencing data. We trained these models on a large AMR dataset and evaluated them on an independent public dataset. Generally, RFs and CNNs perform better than LR and SVM with AUCs up to 0.96. Furthermore, we were able to identify mutations that are associated with AMR for each antibiotic. AVAILABILITY AND IMPLEMENTATION: Source code in data preparation and model training are provided at GitHub website (https://github.com/YunxiaoRen/ML-iAMR). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Animales , Humanos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Ciprofloxacina , Aprendizaje Automático , Genómica , Bacterias/genéticaRESUMEN
Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.
Asunto(s)
Paracoccus denitrificans , Acetilcoenzima A/metabolismo , Paracoccus denitrificans/genética , Paracoccus denitrificans/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Glioxilatos/metabolismoRESUMEN
The EDGAR platform, a web server providing databases of precomputed orthology data for thousands of microbial genomes, is one of the most established tools in the field of comparative genomics and phylogenomics. Based on precomputed gene alignments, EDGAR allows quick identification of the differential gene content, i.e. the pan genome, the core genome, or singleton genes. Furthermore, EDGAR features a wide range of analyses and visualizations like Venn diagrams, synteny plots, phylogenetic trees, as well as Amino Acid Identity (AAI) and Average Nucleotide Identity (ANI) matrices. During the last few years, the average number of genomes analyzed in an EDGAR project increased by two orders of magnitude. To handle this massive increase, a completely new technical backend infrastructure for the EDGAR platform was designed and launched as EDGAR3.0. For the calculation of new EDGAR3.0 projects, we are now using a scalable Kubernetes cluster running in a cloud environment. A new storage infrastructure was developed using a file-based high-performance storage backend which ensures timely data handling and efficient access. The new data backend guarantees a memory efficient calculation of orthologs, and parallelization has led to drastically reduced processing times. Based on the advanced technical infrastructure new analysis features could be implemented including POCP and FastANI genomes similarity indices, UpSet intersecting set visualization, and circular genome plots. Also the public database section of EDGAR was largely updated and now offers access to 24,317 genomes in 749 free-to-use projects. In summary, EDGAR 3.0 provides a new, scalable infrastructure for comprehensive microbial comparative gene content analysis. The web server is accessible at http://edgar3.computational.bio.
Asunto(s)
Genoma Microbiano , Genómica/métodos , Filogenia , Programas Informáticos , Bases de Datos GenéticasRESUMEN
Extracytoplasmic function σ factors (ECFs) represent one of the major bacterial signal transduction mechanisms in terms of abundance, diversity and importance, particularly in mediating stress responses. Here, we performed a comprehensive phylogenetic analysis of this protein family by scrutinizing all proteins in the NCBI database. As a result, we identified an average of â¼10 ECFs per bacterial genome and 157 phylogenetic ECF groups that feature a conserved genetic neighborhood and a similar regulation mechanism. Our analysis expands previous classification efforts â¼50-fold, enriches many original ECF groups with previously unclassified proteins and identifies 22 entirely new ECF groups. The ECF groups are hierarchically related to each other and are further composed of subgroups with closely related sequences. This two-tiered classification allows for the accurate prediction of common promoter motifs and the inference of putative regulatory mechanisms across subgroups composing an ECF group. This comprehensive, high-resolution description of the phylogenetic distribution of the ECF family, together with the massive expansion of classified ECF sequences and an openly accessible data repository called 'ECF Hub' (https://www.computational.bio.uni-giessen.de/ecfhub), will serve as a powerful hypothesis-generator to guide future research in the field.
Asunto(s)
Proteínas Bacterianas/química , Familia de Multigenes , Factor sigma/clasificación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Consenso , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Filogenia , Alineación de Secuencia , Factor sigma/genética , Transducción de Señal , Especificidad por Sustrato , Terminología como AsuntoRESUMEN
BACKGROUND: Mutualistic interactions with microbes can help insects adapt to extreme environments and unusual diets. An intriguing example is the burying beetle Nicrophorus vespilloides, which feeds and reproduces on small vertebrate carcasses. Its fungal microbiome is dominated by yeasts that potentially facilitate carcass utilization by producing digestive enzymes, eliminating cadaver-associated toxic volatiles (that would otherwise attract competitors), and releasing antimicrobials to sanitize the microenvironment. Some of these yeasts are closely related to the biotechnologically important species Yarrowia lipolytica. RESULTS: To investigate the roles of these Yarrowia-like yeast (YLY) strains in more detail, we selected five strains from two different phylogenetic clades for third-generation sequencing and genome analysis. The first clade, represented by strain B02, has a 20-Mb genome containing ~ 6400 predicted protein-coding genes. The second clade, represented by strain C11, has a 25-Mb genome containing ~ 6300 predicted protein-coding genes, and extensive intraspecific variability within the ITS-D1/D2 rDNA region commonly used for species assignments. Phenotypic microarray analysis revealed that both YLY strains were able to utilize a diverse range of carbon and nitrogen sources (including microbial metabolites associated with putrefaction), and can grow in environments with extreme pH and salt concentrations. CONCLUSIONS: The genomic characterization of five yeast strains isolated from N. vespilloides resulted in the identification of strains potentially representing new YLY species. Given their abundance in the beetle hindgut, and dominant growth on beetle-prepared carcasses, the analysis of these strains has revealed the genetic basis of a potential symbiotic relationship between yeasts and burying beetles that facilitates carcass digestion and preservation.
Asunto(s)
Escarabajos , Yarrowia , Animales , Escarabajos/genética , Genómica , Filogenia , Simbiosis , Yarrowia/genéticaRESUMEN
BACKGROUND: The technology of single cell RNA sequencing (scRNA-seq) has gained massively in popularity as it allows unprecedented insights into cellular heterogeneity as well as identification and characterization of (sub-)cellular populations. Furthermore, scRNA-seq is almost ubiquitously applicable in medical and biological research. However, these new opportunities are accompanied by additional challenges for researchers regarding data analysis, as advanced technical expertise is required in using bioinformatic software. RESULTS: Here we present WASP, a software for the processing of Drop-Seq-based scRNA-Seq data. Our software facilitates the initial processing of raw reads generated with the ddSEQ or 10x protocol and generates demultiplexed gene expression matrices including quality metrics. The processing pipeline is realized as a Snakemake workflow, while an R Shiny application is provided for interactive result visualization. WASP supports comprehensive analysis of gene expression matrices, including detection of differentially expressed genes, clustering of cellular populations and interactive graphical visualization of the results. The R Shiny application can be used with gene expression matrices generated by the WASP pipeline, as well as with externally provided data from other sources. CONCLUSIONS: With WASP we provide an intuitive and easy-to-use tool to process and explore scRNA-seq data. To the best of our knowledge, it is currently the only freely available software package that combines pre- and post-processing of ddSEQ- and 10x-based data. Due to its modular design, it is possible to use any gene expression matrix with WASP's post-processing R Shiny application. To simplify usage, WASP is provided as a Docker container. Alternatively, pre-processing can be accomplished via Conda, and a standalone version for Windows is available for post-processing, requiring only a web browser.
Asunto(s)
Análisis de la Célula Individual , Programas Informáticos , Biología Computacional , Perfilación de la Expresión Génica , RNA-Seq , Análisis de Secuencia de ARNRESUMEN
Whole genome sequencing of bacteria has become daily routine in many fields. Advances in DNA sequencing technologies and continuously dropping costs have resulted in a tremendous increase in the amounts of available sequence data. However, comprehensive in-depth analysis of the resulting data remains an arduous and time-consuming task. In order to keep pace with these promising but challenging developments and to transform raw data into valuable information, standardized analyses and scalable software tools are needed. Here, we introduce ASA3P, a fully automatic, locally executable and scalable assembly, annotation and analysis pipeline for bacterial genomes. The pipeline automatically executes necessary data processing steps, i.e. quality clipping and assembly of raw sequencing reads, scaffolding of contigs and annotation of the resulting genome sequences. Furthermore, ASA3P conducts comprehensive genome characterizations and analyses, e.g. taxonomic classification, detection of antibiotic resistance genes and identification of virulence factors. All results are presented via an HTML5 user interface providing aggregated information, interactive visualizations and access to intermediate results in standard bioinformatics file formats. We distribute ASA3P in two versions: a locally executable Docker container for small-to-medium-scale projects and an OpenStack based cloud computing version able to automatically create and manage self-scaling compute clusters. Thus, automatic and standardized analysis of hundreds of bacterial genomes becomes feasible within hours. The software and further information is available at: asap.computational.bio.
Asunto(s)
Biología Computacional/métodos , Análisis de Secuencia de ADN/métodos , Algoritmos , Bacterias/genética , Secuencia de Bases/genética , Mapeo Cromosómico/métodos , Nube Computacional , Genoma Bacteriano/genética , Análisis de Secuencia de ADN/estadística & datos numéricos , Programas Informáticos , Secuenciación Completa del Genoma/métodosRESUMEN
A polyphasic approach was applied to investigate the diversity of microbiota that evolved during cold storage beef ripening. Isolate V4/DAB/S4/2aT with a unique BOX-rep-PCR fingerprint profile revealed more than 99â% nucleotide identities upon pairwise comparisons of 16S rDNA sequences from the type strains Pseudomonas versuta DSM 101070T, Pseudomonas saxonica DSM 108989T, Pseudomonas deceptionensis DSM 26521T and Pseudomonas weihenstephanensis DSM 29166T, placing it within the Pseudomonas fragi / lundensis branch of the genus Pseudomonas. Additional rpoB based comparison revealed P. versuta DSM 101070T as the nearest relative, with 98.5â% nucleotide identity. Calculation of ANIb values of the V4/DAB/S4/2aT draft genome identified P. versuta DSM 101070T with 90.1â%, P. deceptionensis DSM 26521T with 85.1â%, P. fragi DSM 3456T with 84.4â%, Pseudomonas psychrophila DSM 17535T and Pseudomonas bubulae DSM 107389T with 84.2â% similarities each. Pairwise genome-to-genome distance calculations [digital DNA-DNA hybridization (dDDH)] resulted in values of 47.1, 35.1, 34.8, 34.2 and 34.1â%, respectively. A second isolate was detected years later in ground beef and showed ANIb values of 99.3â% and dDDH of 96.1â% relatedness to V4/DAB/S4/2aT. The DNA G+C content was 58.6 mol% for both isolates. The predominant cellular fatty acids of V4/DAB/S4/2aT were C16â:â0, C18â:â1ω7c, C17â:â0 cyclo and a summed feature containing C16â:â1ω7c and/or C15â:â0 iso 2-OH. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, the major respiratory quinone was Q9, with a small portion of Q8. The combined data on genotypic and phenotypic features support the proposal of a novel species, for which the name Pseudomonas paraversuta sp. nov. is proposed. The type strain is V4/DAB/S4/2aT (=DSM 111361T=LMG 31844T) and a second isolate is UBT376 (=DSM 111360=LMG 31845).
Asunto(s)
Microbiología de Alimentos , Filogenia , Pseudomonas/clasificación , Carne Roja/microbiología , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , Bovinos , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Alemania , Hibridación de Ácido Nucleico , Fosfolípidos/química , Pseudomonas/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ubiquinona/químicaRESUMEN
During a project focusing on the diversity of meat microbiota associated with beef ripening, a Pseudomonas strain was isolated exhibiting high 16S rRNA gene sequence similarities (>99â%) to Pseudomonas carnis DSM 107652T, P. lactis DSM 29167T, P. paralactis DSM 29164T and P. azotoformans DSM 18862T. Phylogenetic analysis of the complete rpoB gene sequences of the isolate V5/DAB/2/5T indicated a separate branch with about 99.0â% nucleotide identities to the closest relatives P. carnis DSM 107652T, P. lactis DSM 29167T and P. paralactis DSM 29164T, while average nucleotide identities (ANIb) calculated from the draft genomes were 94.8, 94.2 and 90.2â%, respectively. Pairwise genome-to-genome distance calculations (GGDC) resulted in values of 67.7, 63.5 and 45.7â%, respectively, lying below the actual species demarcation line as well. A second isolate, UBT403, was detected some years later by using matrix-assisted laser desorption ionization-time of flight MS of the microbiota of minced beef. The fatty acid profile of V5/DAB/2/5T consisted of C16â:â0, summed feature C 16â:â1 ω7c/iso-C15â:â0 2-OH, C18â:â1 ω7c, C17â:â0 cyclo, C12â:â0, C12â:â0 3-OH, C10â:â0 3-OH and C12â:â0 2-OH. The major cellular lipids were aminopholipids, phospholipids, phosphatidylethanolamine and phosphatidylglycerol; the major quinone was Q9 with a minor proportion of Q8. Based on phenotypic and chemotaxonomic characterizations, the isolates can be considered as representing a novel species, for which the name Pseudomonas paracarnis sp. nov. is proposed. The type strain is V5/DAB/2/5T (=DSM 111363T=LMG 31846T); a second strain is UBT403 (=DSM 111362=LMG 31847).
Asunto(s)
Microbiología de Alimentos , Filogenia , Pseudomonas/clasificación , Carne Roja/microbiología , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , Bovinos , ADN Bacteriano/genética , Ácidos Grasos/química , Hibridación de Ácido Nucleico , Fosfolípidos/química , Pseudomonas/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMEN
RNA editing by adenosine deaminases acting on dsRNA (ADAR) has become of increasing medical relevance, particularly because aberrant ADAR1 activity has been associated with autoimmunity and malignancies. However, the role of ADAR1 in dendritic cells (DC), representing critical professional APCs, is unknown. We have established conditional murine CD11c Cre-mediated ADAR1 gene ablation, which did not induce general apoptosis in CD11c+ cells but instead manifests in cell type-specific effects in DC subpopulations. Bone marrow-derived DC subset analysis revealed an incapacity to differentiate CD103 DC+ in both bulk bone marrow and purified pre-DC lineage progenitor assays. ADAR1 deficiency further resulted in a preferential systemic loss of CD8+/CD103+ DCs, revealing critical dependency on ADAR1, whereas other DC subpopulations were moderately affected or unaffected. Additionally, alveolar macrophages were depleted and dysfunctional, resembling pulmonary alveolar proteinosis. These results reveal an unrecognized role of ADAR1 in DC subset homeostasis and unveils the cell type-specific effects of RNA editing.
Asunto(s)
Adenosina Desaminasa/metabolismo , Células Dendríticas/inmunología , Homeostasis/inmunología , Macrófagos Alveolares/inmunología , Animales , Proliferación Celular , Células Dendríticas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Edición de ARN , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. RESULTS: To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected. CONCLUSIONS: In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.
Asunto(s)
Proteínas Arqueales , Exosomas , Sulfolobus solfataricus , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/genética , Exosomas/metabolismo , ARN/genética , Estabilidad del ARN , ARN de Archaea/genética , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismoRESUMEN
While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.We elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin A (CsA) and alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily available therapy in MERS-CoV infection.Calu-3 cells and primary human alveolar epithelial cells (hAECs) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nuclear factor of activated T-cells (NFATs) or mitogen-activated protein kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by quantitative real-time PCR and 50% tissue culture infective dose. Data were validated in a murine MERS-CoV infection model.Both CsA and ALV reduced MERS-CoV titres and viral RNA replication in Calu-3 cells and hAECs, improving epithelial integrity. While neither calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type III interferon (IFNλ) response and expression of antiviral genes. Downregulation of IRF1 or IFNλ increased MERS-CoV propagation in the presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication in vivo, correlating with elevated lung IFNλ levels and improved outcome.We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication in vitro and in vivo via upregulation of inflammatory antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate for treating MERS-CoV infection.
Asunto(s)
Infecciones por Coronavirus/prevención & control , Ciclofilinas/antagonistas & inhibidores , Ciclosporina/farmacología , Interferones/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Animales , Inhibidores de la Calcineurina/farmacología , Técnicas de Cultivo de Célula , Infecciones por Coronavirus/metabolismo , Modelos Animales de Enfermedad , Humanos , Factor 1 Regulador del Interferón/efectos de los fármacos , Factor 1 Regulador del Interferón/metabolismo , Interferones/efectos de los fármacos , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Replicación Viral/efectos de los fármacos , Interferón lambdaRESUMEN
During investigations of spoilage-associated meat microbiota, Pseudomonas isolates were found in two different laboratories showing highest similarities to Pseudomonas lactis DSM 29167T, Pseudomonas paralactis DSM 29164T and Pseudomonas azotoformans DSM 18862T based on 16S rRNA gene sequence comparisons. Phylogenetic analysis of the complete rpoB gene sequences of isolates B4-1T and SpeckC indicated a separate branch with 99.0 and 99.1â% identity, respectively, to their closest relative (P. lactis DSM 29167T). Further phenotypic and chemotaxonomic characterizations, as well as average nucleotide identity (ANIb) values obtained from the draft genomes, revealed that these isolates could be considered as representing a novel species, with ANIb values of around 94 and 90â% with their closest relatives P. lactis and P. paralactis. Other related species showed ANIb values below 90â%, including Pseudomonas libanensis DSM 17149T, Pseudomonas synxantha DSM 18928T, Pseudomonas orientalis DSM 17489T, Pseudomonas veronii DSM 11331T and P. azotoformans DSM 18862T. Genome-to-genome distance calculations between B4-1T and its closest relative, P. lactis DSM 29167T, showed 62.6â% relatedness. The G+C contents of B4-1T and SpeckC were 59.8 and 59.9 mol%, respectively. The major cellular lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q9. Based on these data, the new species Pseudomonas carnis sp. nov. is proposed, the type strain is B4-1T (=DSM 107652T=LMG 30892T); a second strain is SpeckC (=DSM 107651=LMG 30893).
Asunto(s)
Microbiología de Alimentos , Carne/microbiología , Filogenia , Pseudomonas/clasificación , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Alemania , Hibridación de Ácido Nucleico , Fosfolípidos/química , Aves de Corral , Pseudomonas/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Porcinos , Ubiquinona/químicaRESUMEN
Two Pseudomonas isolates derived independently from raw refrigerated processing meat of bovine origin intended for the manufacture of Bologna-type cooked sausage could be distinguished from other known species in subsequent phylogenetic analyses. Comparison of the complete rpoB gene sequences in combination with nearly complete 16S rRNA gene sequences revealed a separate branch within the Pseudomonas fragi group. In further analyses, comprising phenotypic and chemotaxonomic characterization as well as average nucleotide identity (ANI) values obtained from the draft genome assemblies, the two isolates could be distinguished from all so far published closely related species. The closest relative was P. fragi DSM 3456T with ANI values of about 90.2â%. Other close Pseudomonas neighbours were P. psychrophila DSM 17535T (86.5â%), P. deceptionensis DSM 26521T (86.4â%), P. versuta DSM 101070T (83.8â%), P. taetrolens DSM 21104T (83.2â%), P. weihenstephanensis DSM 29166T (82.3â%), P. helleri DSM 29165T (82.7â%) and P. lundensis DSM 6252T (81.9â%). The G+C contents of isolates TH39T and TH26 were both 58.2 mol%. The major cellular lipids of strain TH39T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q9 with small amounts of Q8. Based on these data, the isolates TH39T and TH26 (=DSM 107389=LMG 30831) represent a novel species within the genus Pseudomonas , for which the name Pseudomonas bubulae sp. nov. is proposed. The type strain is TH39T (=DSM 107390T=LMG 30830T).
Asunto(s)
Filogenia , Pseudomonas/clasificación , Carne Roja/microbiología , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , Bovinos , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Hibridación de Ácido Nucleico , Fosfolípidos/química , Pseudomonas/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ubiquinona/químicaRESUMEN
On the basis of two other publications (Yarza et al. 2013; Nemec et al. 2019) and on the basis of resequencing of the 16S rRNA gene of Prolinoborus fasciculus CIP 103579T it is concluded that Prolinoborus fasciculus CIP 103579T, which is the only available strain of the species from culture collections, does not conform to the original description given by Pot et al. (1992). The strain investigated is a member of the genus Acinetobacter within the Moraxellaceae, a family of the Gammaproteobacteria and not a member of the Betaproteobacteria as originally proposed. Prolinoborus fasciculus CIP 103579T shared 99.8â% 16S rRNA gene sequence similarity with Acinetobacter lwoffii DSM 2403T. The two strains clustered together by rpoB- and core genome-based phylogenetic analyses and shared an average nucleotide identity of 96.47% (reciprocal, 96.56â%) and a digital genome distance calculation (GGDC) value of 66.9â%. Furthermore, the two strains shared matrix-assisted laser desorption/ionization time of flight MS profiles to a high extent and showed highly similar cellular fatty acid profiles and physiological substrate utilization patterns. It is proposed that the Judicial commission consider (1) that the strain currently deposited as CIP 103579 be recognized as a member of Acinetobacter lwoffii; (2) placing Prolinoborus fasciculus (Pot et al. 1992) on the list of rejected names if a suitable replacement strain, or a neotype strain cannot be found within 2 years of publication of this request; and (3) place the genus name Prolinoborus (Pot et al. 1992) on the list of rejected names [Recommendation 20D (3) of the Code].