Topoisomerase II regulates yeast genes with singular chromatin architectures

Eukaryotic topoisomerase II (topo II) is the essential decatenase of newly replicated chromosomes and the main relaxase of nucleosomal DNA. Apart from these general tasks, topo II participates in more specialized functions. In mammals, topo IIa interacts with specific RNA polymerases and chromatin-remodeling complexes, whereas topo IIb regulates developmental genes in conjunction with chromatin remodeling and heterochromatin transitions. Here we show that in budding yeast, topo II regulates the expression of specific gene subsets. To uncover this, we carried out a genomic transcription run-on shortly after the thermal inactivation of topo II. We identified a modest number of genes not involved in the general stress response but strictly dependent on topo II. These genes present distinctive functional and structural traits in comparison with the genome average. Yeast topo II is a positive regulator of genes with well-defined promoter architecture that associates to chromatin remodeling complexes; it is a negative regulator of genes extremely hypo-acetylated with complex promoters and undefined nucleosome positioning, many of which are involved in polyamine transport. These findings indicate that yeast topo II operates on singular chromatin architectures to activate or repress DNA transcription and that this activity produces functional responses to ensure chromatin stability

FunOMIC: Pipeline with built-in fungal taxonomic and functional databases for human mycobiome profiling

Fungal databases; Taxonomy and functions; Inter-kingdom interactionsBases de datos de hongos; Taxonomía y funciones; Interacciones entre reinosBases de dades de fongs; Taxonomia i funcions; Interaccions entre regnesWhile analysis of the bacterial microbiome has become routine, that of the fungal microbiome is still hampered by the lack of robust databases and bioinformatic pipelines. Here, we present FunOMIC, a pipeline with built-in taxonomic (1.6 million marker genes) and functional (3.4 million non-redundant fungal proteins) databases for the identification of fungi. Applied to more than 2,600 human metagenomic samples, the tool revealed fungal species associated with geography, body sites, and diseases. Correlation network analysis provided new insights into inter-kingdom interactions. With this pipeline and two of the most comprehensive fungal databases, we foresee a fast-growing resource for mycobiome studies.This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Action, Innovative Training Network [grant number 812969] and by the Instituto de Salud Carlos III /FEDER, a government agency (Grant No: PI17/00614; PI20/00130)

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

Peer reviewedPublisher PD

GSR-DB : a manually curated and optimized taxonomical database for 16S rRNA amplicon analysis

Taxonomic assignments of microorganisms have long been hindered by inconsistent nomenclature and annotation issues in existing databases like SILVA, Greengenes, Greengenes2, Genome Taxonomy Database, or Ribosomal Database Project. To overcome these issues, we created Greengenes-SILVA-RDP database (GSR-DB), accurate and comprehensive taxonomic annotations of 16S amplicon data. Unlike previous approaches, our innovative pipeline includes a unique taxonomy unification step, ensuring consistent and reliable annotations. Our evaluation analyses showed that GSR-DB outperforms existing databases in providing species-level resolution, especially based on mock-community analysis, making it a game-changer for microbiome studies. Moreover, GSR-DB is designed to be accessible to researchers with limited computational resources, making it a powerful tool for scientists across the board. Available for full-length 16S sequences and commonly used hypervariable regions, including V4, V1-V3, V3-V4, and V3-V5, GSR-DB is a go-to database for robust and accurate microbial taxonomy analysis

Shotgun metagenomics reveals interkingdom association between intestinal bacteria and fungi involving competition for nutrients

Comprehensive database; Diet; MicrobiomeBase de datos integral; Dieta; MicrobiomaBase de dades integral; Dieta; MicrobiomaBackground The accuracy of internal-transcribed-spacer (ITS) and shotgun metagenomics has not been robustly evaluated, and the effect of diet on the composition and function of the bacterial and fungal gut microbiome in a longitudinal setting has been poorly investigated. Here we compared two approaches to study the fungal community (ITS and shotgun metagenomics), proposed an enrichment protocol to perform a reliable mycobiome analysis using a comprehensive in-house fungal database, and correlated dietary data with both bacterial and fungal communities. Results We found that shotgun DNA sequencing after a new enrichment protocol combined with the most comprehensive and novel fungal databases provided a cost-effective approach to perform gut mycobiome profiling at the species level and to integrate bacterial and fungal community analyses in fecal samples. The mycobiome was significantly more variable than the bacterial community at the compositional and functional levels. Notably, we showed that microbial diversity, composition, and functions were associated with habitual diet composition instead of driven by global dietary changes. Our study indicates a potential competitive inter-kingdom interaction between bacteria and fungi for food foraging. Conclusion Together, our present work proposes an efficient workflow to study the human gut microbiome integrating robustly fungal, bacterial, and dietary data. These findings will further advance our knowledge of the interaction between gut bacteria and fungi and pave the way for future investigations in human mycobiome.This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Action, Innovative Training Network [grant number 812969]

GSR-DB: a manually curated and optimized taxonomical database for 16S rRNA amplicon analysis

Database; Microbiome; TaxonomyBase de datos; Microbioma; TaxonomíaBase de dades; Microbioma; TaxonomiaAmplicon-based 16S ribosomal RNA sequencing remains a widely used method to profile microbial communities, especially in low biomass samples, due to its cost-effectiveness and low-complexity approach. Reference databases are a mainstay for taxonomic assignments, which typically rely on popular databases such as SILVA, Greengenes, Genome Taxonomy Database (GTDB), or Ribosomal Database Project (RDP). However, the inconsistency of the nomenclature across databases and the presence of shortcomings in the annotation of these databases are limiting the resolution of the analysis. To overcome these limitations, we created the GSR database (Greengenes, SILVA, and RDP database), an integrated and manually curated database for bacterial and archaeal 16S amplicon taxonomy analysis. Unlike previous integration approaches, this database creation pipeline includes a taxonomy unification step to ensure consistency in taxonomical annotations. The database was validated with three mock communities, two real data sets, and a 10-fold cross-validation method and compared with existing 16S databases such as Greengenes, Greengenes 2, GTDB, ITGDB, SILVA, RDP, and MetaSquare. Results showed that the GSR database enhances taxonomical annotations of 16S sequences, outperforming current 16S databases at the species level, based on the evaluation of the mock communities. This was confirmed by the 10-fold cross-validation, except for Greengenes 2. The GSR database is available for full-length 16S sequences and the most commonly used hypervariable regions: V4, V1–V3, V3–V4, and V3–V5. IMPORTANCE Taxonomic assignments of microorganisms have long been hindered by inconsistent nomenclature and annotation issues in existing databases like SILVA, Greengenes, Greengenes2, Genome Taxonomy Database, or Ribosomal Database Project. To overcome these issues, we created Greengenes-SILVA-RDP database (GSR-DB), accurate and comprehensive taxonomic annotations of 16S amplicon data. Unlike previous approaches, our innovative pipeline includes a unique taxonomy unification step, ensuring consistent and reliable annotations. Our evaluation analyses showed that GSR-DB outperforms existing databases in providing species-level resolution, especially based on mock-community analysis, making it a game-changer for microbiome studies. Moreover, GSR-DB is designed to be accessible to researchers with limited computational resources, making it a powerful tool for scientists across the board. Available for full-length 16S sequences and commonly used hypervariable regions, including V4, V1–V3, V3–V4, and V3–V5, GSR-DB is a go-to database for robust and accurate microbial taxonomy analysis.This work was supported by the Instituto de Salud Carlos III/FEDER (PI20/00130). Funding for open access charge was provided by Instituto de Salud Carlos III/FEDER

Lectin-like bacteriocins from pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins

Intercontinental Gut Microbiome Variances in IBD

The development of biomarkers for inflammatory bowel disease (IBD) diagnosis would be relevant in a generalized context. However, intercontinental investigation on these microbial biomarkers remains scarce. We examined taxonomic microbiome variations in IBD using published DNA shotgun metagenomic data. For this purpose, we used sequenced data from our previous Spanish Crohn's disease (CD) and ulcerative colitis (UC) cohort, downloaded sequence data from a Chinese CD cohort, and downloaded taxonomic and functional profiling tables from a USA CD and UC cohort. At the global level, geographical location and disease phenotype were the main explanatory covariates of microbiome variations. In healthy controls (HC) and UC, geography turned out to be the most important factor, while disease intestinal location was the most important one in CD. Disease severity correlated with lower alpha-diversity in UC but not in CD. Across geography, alpha-diversity was significantly different independently of health status, except for CD. Despite recruitment from different countries and with different disease severity scores, CD patients may harbor a very similar microbial taxonomic profile. Our study pointed out that geographic location, disease activity status, and other environmental factors are important contributing factors in microbiota changes in IBD. We therefore strongly recommend taking these factors into consideration for future IBD studies to obtain globally valid and reproducible biomarker

Structural and Functional Alterations in the Microbial Community and Immunological Consequences in a Mouse Model of Antibiotic-Induced Dysbiosis

The aim of this study was to establish continuous therapeutic-dose ampicillin (CTDA)-induced dysbiosis in a mouse model, mimicking typical adult exposure, with a view to using this to assess its impact on gut microbiota, intestinal metabolites and host immune responses. Mice were exposed to ampicillin for 14 days and antibiotic-induced dysbiosis was evaluated by alteration of microbiota and gut permeability. The cecal index was increased in the CTDA group, and the gut permeability indicated by fluorescent dextran, endotoxin and D-Lactate in the serum was significantly increased after antibiotic use. The tight-junction proteins ZO-1 and occludin in the colon were reduced to half the control level in CTDA. We found that alpha-diversity was significantly decreased in mice receiving CTDA, and microbial community structure was altered compared with the control. Key taxa were identified as CTDA-specific, and the relative abundance of Enterococcus and Klebsiella was particularly enriched while Lachnospiraceae, Coprobacillus and Dorea were depleted after antibiotic treatment. In particular, a significant increase in succinate and a reduction in butyrate was detected in CTDA mice, and the triggering of NF-κB enhancement reflected that the host immune response was influenced by ampicillin use. The observed perturbation of the microbiota was accompanied by modulation of inflammatory state; this included increase in interferon-γ and RegIIIγ, and a decrease in secretory IgA in the colon mucosa. This study allowed us to identify the key taxa associated with an ampicillin-induced state of dysbiosis in mice and to characterize the microbial communities via molecular profiling. Thus, this work describes the bacterial ecology of antibiotic exposure model in combination with host physiological characteristics at a detailed level of microbial taxa

The levamisole sensitive nicotinic acetylcholine receptor of the potato cyst nematode Globodera pallida

The potato cyst nematode Globodera pallida costs the UK potato industry over £50 million per annum. In order to invade a host plant, the infective J2 stage must hatch from eggs within the soil and migrate towards the root system. Orthologues of Caenorhabditis elegans genes involved in neurotransmission were identified in the G. pallida and G. rostochiensis genome assemblies. The complement of cys loop ligand gated ion channel genes was distinct compared to C. elegans and other parasitic nematodes. Orthologues of genes encoding subunits which comprise the C. elegans levamisole sensitive nicotinic acetylcholine receptor (cel-lev 1, cel-lev 8, cel-unc 29, cel-unc 63 and cel-unc 38) were searched for, and cel-lev 1 and cel-lev 8 orthologues were absent in both Globodera spp. Two orthologues were identified for cel-unc 29 and cel-unc 38. This suggested that the composition of the G. pallida L nAChR may differ. The use of C. elegans as a heterologous system to study the expression pattern of G. pallida nAChR genes was explored. GFP expressing lines were created using promoter regions of gpa acr 2 and gpa unc 63. Expression was observed in the ventral nerve cord and nerve ring for pgpa-acr 2. Expression of pgpa-unc 63 was variable, but was found in the head and tail region and along the ventral side of the body. The impact of this distinct complement of cys loop subunits on anthelmintic sensitivity was demonstrated by the increased resistance of both G. pallida and G. rostochiensis J2s to levamisole. The EC50 of G. pallida and G. rostochiensis was 19.7 mM and 5.6 mM respectively, compared to the EC50 of 9 µM for C. elegans, representing a 500 – 2000 fold increase in levamisole resistance. This increased resistance to levamisole was associated with an orthologue of cel-unc 38 identified in G. pallida, gpa unc 38.1. Rescue of C. elegans unc 38(x20) mutants with gpa unc 38.1 restores normal movement suggesting a functional reconstitution of the L nAChR, but full sensitivity to levamisole is not restored. Gpa unc 38.1 was expressed with the remaining four subunits from C. elegans in Xenopus oocytes to produce a chimeric receptor. The EC50 of the response to acetylcholine and levamisole of the chimeric receptor and the native receptor was comparable and had similar opening responses to different agonists. Chimeric genes were created to analyse key motifs in gpa unc 38.1 that may affect receptor function and levamisole sensitivity. Gpa unc 38.1 was necessary for structural reformation of the receptor, but not acetylcholine binding. Removal or addition of a loop B glutamate residue, previously associated with levamisole sensitivity of Cel UNC 38, did not affect levamisole sensitivity of Cel-UNC 38 or Gpa UNC 38.1. An amino acid change (I>M) in TM2 of Cel-UNC 38 increased levamisole sensitivity and basal thrashing rate. The reciprocal change (M>I) in Gpa UNC 38.1 comprised basal thrashing rescue. The basis of increased levamisole resistance of gpa unc 38.1 was not identified, as all gpa unc 38.1 chimeric genes retained a higher resistance to levamisole than cel-unc 38. This works reveals that the nAChRs of plant parasitic nematodes have distinct pharmacological characteristics