2,411 research outputs found

    Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family.

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    BackgroundIt is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5'-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.ResultsPhylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5'-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.ConclusionsLike the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5'-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious

    ReporTree: a surveillance-oriented tool to strengthen the linkage between pathogen genetic clusters and epidemiological data

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    OHEJP Project: BeONE https://doi.org/10.1186/s13073-023-01196-1 This repository contains the Open Access PDF file of the article: Mixão, V., Pinto, M., Sobral, D. et al. ReporTree: a surveillance-oriented tool to strengthen the linkage between pathogen genetic clusters and epidemiological data. Genome Med 15, 43 (2023). ----------------------------------- Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. ----------------------------------- Acknowledgements The authors thank Dr. Holger Brendebach, Dr. Carlus Deneke, and Dr. Simon Tausch from the German Federal Institute for Risk Assessment for their support during the genome assembly of the samples used in ReporTree benchmarking and Dr. João André Carriço for the productive discussions throughout ReporTree development. We would also like to thank the National Distributed Computing Infrastructure of Portugal (INCD) for providing the necessary resources to run the genome assemblies. INCD was funded by FCT and FEDER under the project 22153-01/SAICT/2016. Funding This work was supported by funding from the European Union’s Horizon 2020 Research and Innovation program under grant agreement No 773830: One Health European Joint Programme (2020–2022) and by national funds through FCT—Foundation for Science and Technology, I.P., in the frame of Individual CEEC 2022.00851.CEECIND/CP1748/CT0001 (2023 onwards)

    Incorporating molecular data in fungal systematics: a guide for aspiring researchers

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    The last twenty years have witnessed molecular data emerge as a primary research instrument in most branches of mycology. Fungal systematics, taxonomy, and ecology have all seen tremendous progress and have undergone rapid, far-reaching changes as disciplines in the wake of continual improvement in DNA sequencing technology. A taxonomic study that draws from molecular data involves a long series of steps, ranging from taxon sampling through the various laboratory procedures and data analysis to the publication process. All steps are important and influence the results and the way they are perceived by the scientific community. The present paper provides a reflective overview of all major steps in such a project with the purpose to assist research students about to begin their first study using DNA-based methods. We also take the opportunity to discuss the role of taxonomy in biology and the life sciences in general in the light of molecular data. While the best way to learn molecular methods is to work side by side with someone experienced, we hope that the present paper will serve to lower the learning threshold for the reader.Comment: Submitted to Current Research in Environmental and Applied Mycology - comments most welcom

    Genomic and Experimental Evidence for Multiple Metabolic Functions in the RidA/YjgF/YER057c/UK114 (Rid) Protein Family

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    BACKGROUND: It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5\u27-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions. RESULTS: Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5\u27-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate. CONCLUSIONS: Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5\u27-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious

    Trypanosome diversity in wildlife species from the Serengeti and Luangwa Valley ecosystems

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    <p>Background: The importance of wildlife as reservoirs of African trypanosomes pathogenic to man and livestock is well recognised. While new species of trypanosomes and their variants have been identified in tsetse populations, our knowledge of trypanosome species that are circulating in wildlife populations and their genetic diversity is limited.</p> <p>Methodology/Principal Findings: Molecular phylogenetic methods were used to examine the genetic diversity and species composition of trypanosomes circulating in wildlife from two ecosystems that exhibit high host species diversity: the Serengeti in Tanzania and the Luangwa Valley in Zambia. Phylogenetic relationships were assessed by alignment of partial 18S, 5.8S and 28S trypanosomal nuclear ribosomal DNA array sequences within the Trypanosomatidae and using ITS1, 5.8S and ITS2 for more detailed analysis of the T. vivax clade. In addition to Trypanosoma brucei, T. congolense, T. simiae, T. simiae (Tsavo), T. godfreyi and T. theileri, three variants of T. vivax were identified from three different wildlife species within one ecosystem, including sequences from trypanosomes from a giraffe and a waterbuck that differed from all published sequences and from each other, and did not amplify with conventional primers for T. vivax.</p> <p>Conclusions/Significance: Wildlife carries a wide range of trypanosome species. The failure of the diverse T. vivax in this study to amplify with conventional primers suggests that T. vivax may have been under-diagnosed in Tanzania. Since conventional species-specific primers may not amplify all trypanosomes of interest, the use of ITS PCR primers followed by sequencing is a valuable approach to investigate diversity of trypanosome infections in wildlife; amplification of sequences outside the T. brucei clade raises concerns regarding ITS primer specificity for wildlife samples if sequence confirmation is not also undertaken.</p&gt

    2b-RAD genotyping for population genomic studies of Chagas disease vectors: Rhodnius ecuadoriensis in Ecuador

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    Background: Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. Methodology/Principal findings: The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. Conclusions/Significance: Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment. Author summary: Understanding Chagas disease vector (triatomine) population dispersal is key for the design of control measures tailored for the epidemiological situation of a particular region. In Ecuador, Rhodnius ecuadoriensis is a cause of concern for Chagas disease transmission, since it is widely distributed from the central coast to southern Ecuador. Here, a genome-wide sequencing (2b-RAD) approach was performed in 20 specimens from four communities from Manabí (central coast) and Loja (southern) provinces of Ecuador, and the effectiveness of three type IIB restriction enzymes was assessed. The findings of this study show that this genotyping methodology is cost effective in R. ecuadoriensis and likely in other triatomine species. In addition, preliminary population genomic analysis results detected a signal of population structure among geographically distinct communities and genetic variability within communities. As such, 2b-RAD shows significant promise as a relatively low-tech solution for determination of vector population genomics, dynamics, and spread

    Isolierung und Charakterisierung von Myxobakterien und anderen seltenen Meeresbakterien und deren Multilocus-Sequenzanalyse der indonesischen Biodiversität

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    Indonesia is a country with high potential in biodiversity exploration, especially for microorganisms. This study used samples collected from the islands of Java, Sulawesi, and Bali under the Germany-Indonesia Antiinfectives Cooperation (GINAICO) project. We have isolated 59 strains of gliding bacteria, 98% of which are myxobacteria while the remaining 2% is made up of other gliding bacteria. In this research, we have identified a rare myxobacterium and a novel non-myxobacterial strain from our selected samples. Finally, we confirmed the effectiveness of housekeeping genes as a core method in analysing the degree of relatedness between different strains. The strain Soce1964KM was found to be closest to the rare genus Sorangium and strain 1932KM was identified as a novel genus in the family Flammeovirgaceae. Both results are according to the analysis of its 16S rRNA gene sequence and whole-genome sequencing analysis for strain 1932KM. We also performed screening for antiinfectives against Escherichia coli, Candida albicans and Staphylococcus aureus. Strain Soce1964KM has the highest activity against Staphylococcus aureus and Candida albicans. Analysis of the methanol extract of Soce1964KM through the chromatogram from High-Pressure Liquid Chromatography (HPLC) and High-Resolution Electrospray Ionisation Mass Spectrometry (HRESIMS)–in addition to the database in Myxobase–showed that the extract contains a known compound, namely Disorazol. We found 1932KM to be a novel strain after an in-depth examination of this strain’s phenotype and genotype as part of a polyphasic taxonomy analysis. Also, we conducted a gene cluster analysis and bioassay against pathogenic bacteria and statistical analysis of 16S rRNA gene sequences from this strain with the statistical tool PAST. Bioinformatic tools, such as Prokka, RAST, AntiSmash, NP.searcher, PubChem, SMART and Molinspiration, were used for an in-depth analysis of the whole-genome sequence. Strain 1932KM has also obtained a deposition number from the Indonesian Culture Collections, namely InaCC B1242 with the proposed name of Balibacter flavus gen.nov.sp.nov. Meanwhile, we used the housekeeping genes pgm, pyrG and rpoB to analyse the relatedness between the genera Corallococcus and Myxococcus. These housekeeping genes were used in this study as the core to analyse the similarities between the isolated strains and the type strains. The analysis involved the reconstruction of a phylogenetic tree and its polymorphisms. Bioinformatics tools supported these analyses. These analyses showed that the method is suitable for preliminary analysis in the search for novel species and/or subspecies.Indonesien ist ein Land mit hohem Potenzial für die Erforschung der biologischen Vielfalt, insbesondere für Mikroorganismen. In dieser Studie wurden Proben verwendet, die auf den Inseln Java, Sulawesi, und Bali im Rahmen des Projekts GINAICO (Deutschland-Indonesien Antiinfectives Cooperation) gesammelt wurden. Wir haben 59 Stämme gleitender Bakterien isoliert, von denen 98% Myxobakterien sind, während die restlichen 2% aus anderen gleitenden Bakterien bestehen. In dieser Studie haben wir aus unseren ausgewählten Proben ein seltenes Myxobakterium und einen neuartigen nicht-myxobakteriellen Stamm identifiziert. Schließlich bestätigten wir die Wirksamkeit von Housekeeping-Genen als Kernmethode bei der Analyse des Grads der Verwandtschaft zwischen verschiedenen Stämmen. Es wurde festgestellt, dass der Stamm Soce1964KM der seltenen Gattung Sorangium am nächsten kommt, und der Stamm 1932KM wurde als neuartige Gattung in der Familie der Flammeovirgaceae identifiziert. Beide Ergebnisse entsprechen der Analyse seiner 16S-rRNA-Gensequenz und der Gesamtgenomsequenzierungsanalyse für den Stamm 1932KM. Wir führten auch ein Screening auf Antiinfektiva gegen Escherichia coli, Candida albicans und Staphylococcus aureus durch. Der Stamm Soce1964KM hat die höchste Aktivität gegen Staphylococcus aureus und Candida albicans. Die Analyse des Methanolextrakts von Soce1964KM mittels Chromatogramms aus Hochdruckflüssigchromatographie (HPLC) und hochauflösender Elektrospray-Ionisationsmassenspektrometrie (HRESIMS) - zusätzlich zur Datenbank in Myxobase - zeigte, dass der Extrakt eine bekannte Verbindung enthält, nämlich Disorazol. Nach einer eingehenden Untersuchung des Phänotyps und Genotyps dieses Stammes im Rahmen einer mehrphasigen Taxonomieanalyse stellten wir fest, dass 1932KM ein neuartiger Stamm ist. Außerdem führten wir mit dem statistischen Tool PAST eine Genclusteranalyse und einen Bioassay gegen pathogene Bakterien sowie eine statistische Analyse der 16S-rRNA-Gensequenzen dieses Stammes durch. Bioinformatische Werkzeuge wie Prokka, RAST, AntiSmash, NP.searcher, PubChem, SMART und Molinspiration wurden für eine eingehende Analyse der gesamten Genomsequenz verwendet. Der Stamm 1932KM hat auch eine Hinterlegungsnummer aus den indonesischen Kultursammlungen erhalten, nämlich InaCC B1242 mit dem vorgeschlagenen Namen Balibacter flavus gen.nov.sp.nov. In der Zwischenzeit haben wir die Housekeeping-Gene pgm, pyrG und rpoB verwendet, um die Verwandtschaft zwischen den Gattungen Corallococcus und Myxococcus zu analysieren. Diese Housekeeping-Gene wurden in dieser Studie als Kern verwendet, um die Ähnlichkeiten zwischen den isolierten Stämmen und den Typstämmen zu analysieren. Die Analyse umfasste die Rekonstruktion eines phylogenetischen Baums und seiner Polymorphismen. Bioinformatik-Tools unterstützten diese Analysen. Diese Untersuchungen zeigten, dass die Methode für eine vorläufige Analyse bei der Suche nach neuen Arten und / oder Unterarten geeignet ist

    Contrasting patterns of selection between MHC I and II across populations of Humboldt and Magellanic penguins

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    Indexación: Web of ScienceThe evolutionary and adaptive potential of populations or species facing an emerging infectious disease depends on their genetic diversity in genes, such as the major histocompatibility complex (MHC). In birds, MHC class I deals predominantly with intracellular infections (e.g., viruses) and MHC class II with extracellular infections (e.g., bacteria). Therefore, patterns of MHC I and II diversity may differ between species and across populations of species depending on the relative effect of local and global environmental selective pressures, genetic drift, and gene flow. We hypothesize that high gene flow among populations of Humboldt and Magellanic penguins limits local adaptation in MHC I and MHC II, and signatures of selection differ between markers, locations, and species. We evaluated the MHC I and II diversity using 454 next-generation sequencing of 100 Humboldt and 75 Magellanic penguins from seven different breeding colonies. Higher genetic diversity was observed in MHC I than MHC II for both species, explained by more than one MHC I loci identified. Large population sizes, high gene flow, and/or similar selection pressures maintain diversity but limit local adaptation in MHC I. A pattern of isolation by distance was observed for MHC II for Humboldt penguin suggesting local adaptation, mainly on the northernmost studied locality. Furthermore, trans species alleles were found due to a recent speciation for the genus or convergent evolution. High MHC I and MHC II gene diversity described is extremely advantageous for the long term survival of the species.http://onlinelibrary.wiley.com/doi/10.1002/ece3.2502/epd
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