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Sweet-tasting proteins (SPs) are proteins of plant origin initially isolated from tropical fruits. They are thousands of times sweeter than sucrose and most artificial sweeteners. SPs are a class of proteins capable of causing a sweet taste sensation in humans when interacting with the T1R2/T1R3 receptor. SP thaumatin has already been introduced in the food industry in some countries. Other SPs, such as monellin and brazzein, are promising products. An important stage in researching SPs, in addition to confirming the absence of toxicity, mutagenicity, oncogenicity, and allergenic effects, is studying their influence on gut microbiota. In this paper we describe changes in the composition of rat gut microbiota after six months of consuming one of two recombinant SPs-brazzein or monellin. A full length 16S gene sequencing method was used for DNA library barcoding. The MaAsLin2 analysis results showed noticeable fluctuations in the relative abundances of Anaerocella delicata in brazzein-fed rat microbiota, and of Anaerutruncus rubiinfantis in monellin-fed rat microbiota, which, however, did not exceed the standard deviation. The sucrose-fed group was associated with an increase in the relative abundance of Faecalibaculum rodentium, which may contribute to obesity. Overall, prolonged consumption of the sweet proteins brazzein and monellin did not significantly change rat microbiota and did not result in the appearance of opportunistic microbiota. This provides additional evidence for the safety of these potential sweeteners.
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The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.
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Luminiscencia , Plantas , Animales , MamíferosRESUMEN
The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.
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Eucariontes , Luminiscencia , Animales , MamíferosRESUMEN
MOTIVATION: The Oxford Nanopore technology has a great potential for the analysis of methylated motifs in genomes, including whole-genome methylome profiling. However, we found that there are no methylation motifs detection algorithms, which would be sensitive enough and return deterministic results. Thus, the MEME suit does not extract all Helicobacter pylori methylation sites de novo even using the iterative approach implemented in the most up-to-date methylation analysis tool Nanodisco. RESULTS: We present Snapper, a new highly sensitive approach, to extract methylation motif sequences based on a greedy motif selection algorithm. Snapper does not require manual control during the enrichment process and has enrichment sensitivity higher than MEME coupled with Tombo or Nanodisco instruments that was demonstrated on H.pylori strain J99 studied earlier by the PacBio technology and on four external datasets representing different bacterial species. We used Snapper to characterize the total methylome of a new H.pylori strain A45. At least four methylation sites that have not been described for H.pylori earlier were revealed. We experimentally confirmed the presence of a new CCAG-specific methyltransferase and inferred a gene encoding a new CCAAK-specific methyltransferase. AVAILABILITY AND IMPLEMENTATION: Snapper is implemented using Python and is freely available as a pip package named "snapper-ont." Also, Snapper and the demo dataset are available in Zenodo (10.5281/zenodo.10117651).
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Genoma Bacteriano , Nanoporos , Metilación de ADN , Metiltransferasas/genética , Metiltransferasas/metabolismo , Algoritmos , Análisis de Secuencia de ADN/métodos , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Aphelids are a holomycotan group, represented exclusively by parasitoids infecting algae. They form a sister lineage to Fungi in the phylogenetic tree and represent a key group for reconstruction of the evolution of Holomycota and for analysis of the origin of Fungi. The newly assembled genome of Aphelidium insullamus (Holomycota, Aphelida) with a total length of 18.9 Mb, 7820 protein-coding genes and a GC percentage of 52.05% was obtained by a hybrid assembly based on Oxford Nanopore long reads and Illumina paired reads. In order to trace the origin and the evolution of fungal osmotrophy and its presence or absence in Aphelida, we analyzed the set of main fungal transmembrane transporters, which are proteins of the Major Facilitator superfamily (MFS), in the predicted aphelid proteomes. This search has shown an absence of a specific fungal protein family Drug:H+ antiporters-2 (DAH-2) and specific fungal orthologs of the sugar porters (SP) family, and the presence of common opisthokont's orthologs of the SP family in four aphelid genomes. The repertoire of SP orthologs in aphelids turned out to be less diverse than in free-living opisthokonts, and one of the most limited among opisthokonts. We argue that aphelids do not show signs of similarity with fungi in terms of their osmotrophic abilities, despite the sister relationships of these groups. Moreover, the osmotrophic abilities of aphelids appear to be reduced in comparison with free-living unicellular opisthokonts. Therefore, we assume that the evolution of fungi-specific traits began after the separation of fungal and aphelid lineages, and there are no essential reasons to consider aphelids as a prototype of the fungal ancestor.
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Bacteriophages are often considered as possible agents of biological control of unwanted bacterial populations in medicine, agriculture and food industry. Although the virulent phages can efficiently kill the infected host cells but at the population level phage attack not always leads to the host population collapse but may result in establishment of a more or less stable co-existence. The mechanism of the long-term stabilization of the mixed phage-host cultures is poorly understood. Here we describe bacteriophages VyarbaL and Hena2, the members of the Molineuxvirinae and the Ounavirinae subfamilies, respectively, that are able to form the pseudolysogenic associations (PA) with their host Erwinia amylovora 1/79Sm on solid media. These PAs were stable through multiple passages. The phenomenon of the PA formation between a bacterial culture and bacteriophages decreases the effectiveness of bacteriophage-mediated biological control agents based on lytic bacteriophages.
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Bacteriófagos , Erwinia amylovora , Humanos , Myoviridae , Bacterias , Enfermedades de las Plantas/microbiologíaRESUMEN
Background: RAPID ALKALINIZATION FACTOR (RALFs) are cysteine-rich peptides that regulate multiple physiological processes in plants. This peptide family has considerably expanded during land plant evolution, but the role of ancient RALFs in modulating stress responses is unknown.Results: Here, we used the moss Physcomitrium patens as a model to gain insight into the role of RALF peptides in the coordination of plant growth and stress response in non-vascular plants. The quantitative proteomic analysis revealed concerted downregulation of M6 metalloprotease and some membrane proteins, including those involved in stress response, in PpRALF1, 2 and 3 knockout (KO) lines. The subsequent analysis revealed the role of PpRALF3 in growth regulation under abiotic and biotic stress conditions, implying the importance of RALFs in responding to various adverse conditions in bryophytes. We found that knockout of the PpRALF2 and PpRALF3 genes resulted in increased resistance to bacterial and fungal phytopathogens, Pectobacterium carotovorum and Fusarium solani, suggesting the role of these peptides in negative regulation of the immune response in P. patens. Comparing the transcriptomes of PpRALF3 KO and wild-type plants infected by F. solani showed that the regulation of genes in the phenylpropanoid pathway and those involved in cell wall modification and biogenesis was different in these two genotypes. Conclusion: Thus, our study sheds light on the function of the previously uncharacterized PpRALF3 peptide and gives a clue to the ancestral functions of RALF peptides in plant stress response.
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G-quadruplexes (G4s) are gaining increasing attention as possible regulators of chromatin packaging, and robust approaches to their studies in pseudo-native context are much needed. Here, we designed a simple in vitro model of G4-prone genomic DNA and employed it to elucidate the impact of G4s and G4-stabilizing ligands on nucleosome occupancy. We obtained two 226-bp dsDNA constructs composed of the strong nucleosome positioning sequence and an internucleosomal DNA-imitating tail. The tail was G4-free in the control construct and harbored a "strong" (stable) G4 motif in the construct of interest. An additional "weak" (semi-stable) G4 motif was found within the canonical nucleosome positioning sequence. Both G4s were confirmed by optical methods and 1H NMR spectroscopy. Electrophoretic mobility assays showed that the weak G4 motif did not obstruct nucleosome assembly, while the strong G4 motif in the tail sequence diminished nucleosome yield. Atomic force microscopy data and molecular modeling confirmed that the strong G4 was maintained in the tail of the correctly assembled nucleosome structure. Using both in vitro and in silico models, we probed three known G4 ligands and detected nucleosome-disrupting effects of the least selective ligand. Our results are in line with the negative correlation between stable G4s and nucleosome density, support G4 tolerance between regularly positioned nucleosomes, and highlight the importance of considering chromatin context when targeting genomic G4s.
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Cromatina , G-Cuádruplex , Cromatina/genética , Nucleosomas , Ligandos , ADN/químicaRESUMEN
The complete genomes of the new Erwinia amylovora bacteriophages Loshitsa2 and Micant are 43,092 bp and 43,028 bp long, respectively, encode 51 putative proteins, and have two tRNA genes. Comparative analysis with representatives of the class Caudoviricetes suggests that bacteriophages Loshitsa2 and Micant are related to LIMElight bacteriophage belonging to the family Autographiviridae and could be proposed to be members of a novel subfamily.
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Bacteriófagos , Erwinia amylovora , Erwinia amylovora/genética , Bacteriófagos/genética , Enfermedades de las PlantasRESUMEN
The bacterium Streptomyces sp. KMM 9044 from a sample of marine sediment collected in the northwestern part of the Sea of Japan produces highly chlorinated depsiheptapeptides streptocinnamides A (1) and B (2), representatives of a new structural group of antibiotics. The structures of 1 and 2 were determined using nuclear magnetic resonance and mass spectrometry studies and confirmed by a series of chemical transformations. Streptocinnamide A potently inhibits Micrococcus sp. KMM 1467, Arthrobacter sp. ATCC 21022, and Mycobacterium smegmatis MC2 155.
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Depsipéptidos , Streptomyces , Antibacterianos/farmacología , Depsipéptidos/química , Sedimentos Geológicos/microbiología , Japón , Filogenia , Streptomyces/químicaRESUMEN
We report the spontaneous formation of DNA-DNA junctions in solution in the absence of proteins visualised using atomic force microscopy. The synapsis position fits with potential G-quadruplex (G4) sites. In contrast to the Holliday structure, these conjugates have an affinity for G4 antibodies. Molecular modelling was used to elucidate the possible G4/IM-synaptic complex structures. Our results indicate a new role of the intermolecular noncanonical structures in chromatin architecture and genomic rearrangement.
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Nonribosomal peptides are a class of secondary metabolites synthesized by multimodular enzymes named nonribosomal peptide synthetases and mainly produced by bacteria and fungi. NMR, LC-MS/MS and other analytical methods allow to determine a peptide structure precisely, but it is often not a trivial task to find natural producers of them. There are cases when potential producers should be found among hundreds of strains, for instance, when analyzing metagenomic data. We have developed BioCAT, a tool designed for finding biosynthetic gene clusters which may produce a given nonribosomal peptide when the structure of an interesting nonribosomal peptide has already been found. BioCAT unites the antiSMASH software and the rBAN retrosynthesis tool but some improvements were added to both gene cluster and peptide structure analysis. The main feature of the method is an implementation of a position-specific score matrix to store specificities of nonribosomal peptide synthetase modules, which has increased the alignment sensitivity in comparison with more strict approaches developed earlier. We tested the method on a manually curated nonribosomal peptide producers database and compared it with competing tools GARLIC and Nerpa. Finally, we showed the method's applicability on several external examples.
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The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.
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Animales Salvajes/microbiología , Antibacterianos/administración & dosificación , Bacillus pumilus/química , Escherichia coli/crecimiento & desarrollo , Microbiota , Probióticos/administración & dosificación , Staphylococcus aureus/crecimiento & desarrollo , Animales , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/efectos de los fármacos , Genoma Bacteriano , Metaboloma , Familia de Multigenes , Probióticos/aislamiento & purificación , Staphylococcus aureus/efectos de los fármacosRESUMEN
The SARS-CoV-2 pandemic is a big challenge for humanity. The COVID-19 severity differs significantly from patient to patient, and it is important to study the factors protecting from severe forms of the disease. Respiratory microbiota may influence the patient's susceptibility to infection and disease severity due to its ability to modulate the immune system response of the host organism. This data article describes the microbiome dataset from the upper respiratory tract of SARS-CoV-2 positive patients from Russia. This dataset reports the microbial community profile of 335 human nasopharyngeal swabs collected between 2020-05 and 2021-03 during the first and the second epidemic waves. Samples were collected from both inpatients and outpatients in 4 cities of the Russian Federation (Moscow, Kazan, Irkutsk, Nizhny Novgorod) and sequenced using the 16S rRNA gene amplicon sequencing of V3-V4 region. Data contains information about the patient such as age, sex, hospitalization status, percent of damaged lung tissue, oxygen saturation (SpO2), respiratory rate, need for supplemental oxygen, chest computer tomography severity score, SARS-CoV-2 lineage, and also information about smoking and comorbidities. The amplicon sequencing data were deposited at NCBI SRA as BioProject PRJNA751478.
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The imbalance of the renin-angiotensin system is currently considered as a potentially important factor of the pathogenesis of COVID-19 disease. It has been shown previously in the murine model, that the expression of angiotensin-converting enzyme 2 (ACE2) on the cell surface is downregulated in response to the infection by SARS-CoV virus or recombinant spike protein (S protein) alone. In the case of natural infection, circulation of the S protein in a soluble form is unlikely. However, in SARS-CoV-2, a large fraction of S protein trimers is pre-processed during virion morphogenesis due to the presence of furin protease cleavage site between the S1 and S2 subunits. Therefore, S protein transition into the fusion conformation may be accompanied by the separation of the S1 subunits carrying the receptor-binding domains from the membrane-bound S2 subunits. The fate of the S1 particles shed due to the spontaneous "firing" of some S protein trimers exposed on the virions and on the surface of infected cells has been never investigated. We hypothesize that the soluble S1 subunits of the SARS-CoV-2 S protein shed from the infected cells and from the virions in vivo may bind to the ACE2 and downregulate cell surface expression of this protein. The decrease in the ACE2 activity on the background of constant or increased ACE activity in the lungs may lead to the prevalence of angiotensin II effects over those of angiotensin (1-7), thus promoting thrombosis, inflammation, and pulmonary damage. This hypothesis also suggests the association between less pronounced shedding of the S1 particles reported for the S protein carrying the D614G mutation (vs. the wild type D614 protein), and lack of increased severity of the COVID-19 infection caused by the mutant (D614G) SARS-CoV-2 strain, despite its higher infectivity and higher in vivo viral load.
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COVID-19/virología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/metabolismo , COVID-19/patología , Humanos , Ratones , Modelos Moleculares , Mutación , Multimerización de Proteína , Subunidades de Proteína , Sistema Renina-Angiotensina , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Antimicrobial peptides (AMPs) are considered a promising new class of anti-infectious agents. This study reports new antimicrobial peptides derived from the Hirudo medicinalis microbiome identified by a computational analysis method applied to the H. medicinalis metagenome. The identified AMPs possess a strong antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC range: 5.3 to 22.4 µM), including Staphylococcus haemolyticus, an opportunistic coagulase-negative pathogen. The secondary structure analysis of peptides via CD spectroscopy showed that all the AMPs except pept_352 have mostly disordered structures that do not change under different conditions. For peptide pept_352, the α-helical content increases in the membrane environment. The examination of the mechanism of action of peptides suggests that peptide pept_352 exhibits a direct membranolytic activity. Furthermore, the cytotoxicity assay demonstrated that the nontoxic peptide pept_1545 is a promising candidate for drug development. Overall, the analysis method implemented in the study may serve as an effective tool for the identification of new AMPs.
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Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Descubrimiento de Drogas/métodos , Hirudo medicinalis/metabolismo , Hirudo medicinalis/microbiología , Microbiota/fisiología , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , Fibroblastos/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Metagenoma , Ratones , Pruebas de Sensibilidad Microbiana , Conformación Proteica en Hélice alfaRESUMEN
Feae's viper Azemipos feae belongs to the Azemiopinae subfamily of the Viperidae family. The effects of Viperidae venoms are mostly coagulopathic with limited neurotoxicity manifested by phospholipases A2. From A. feae venom, we have earlier isolated azemiopsin, a novel neurotoxin inhibiting the nicotinic acetylcholine receptor. To characterize other A. feae toxins, we applied label-free quantitative proteomics, which revealed 120 unique proteins, the most abundant being serine proteinases and phospholipases A2. In total, toxins representing 14 families were identified, among which bradykinin-potentiating peptides with unique amino acid sequences possessed biological activity in vivo. The proteomic analysis revealed also basal (commonly known as non-conventional) three-finger toxins belonging to the group of those possessing neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venom. In parallel, the transcriptomic analysis of venom gland performed by Illumina next-generation sequencing further revealed 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae, which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.
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An amendment to this paper has been published and can be accessed via the original article.
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BACKGROUND: Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. RESULTS: We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. CONCLUSIONS: Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.
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Genoma , Hirudo medicinalis/genética , Proteínas y Péptidos Salivales/genética , Animales , Anticoagulantes/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hirudo medicinalis/metabolismo , Sanguijuelas/clasificación , Sanguijuelas/genética , Sanguijuelas/metabolismo , Proteómica , Saliva/metabolismo , Proteínas y Péptidos Salivales/metabolismoRESUMEN
Here, we report the draft genome sequences of the green sulfur bacterium Chlorobium phaeovibrioides strains GrTcv12 and PhvTcv-s14, isolated from the chemocline zone from meromictic Lake Trekhtzvetnoe, separated from the White Sea, in Russia. This is the first report showing the presence of plasmids containing antiphage systems in the Chlorobium sp. genome.