Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we ...developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE–FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
The microbial community of the human gut has a crucial role in sustaining host homeostasis. High-throughput DNA sequencing has delineated the structural and functional configurations of gut ...metagenomes in world populations. The microbiota of the Russian population is of particular interest to researchers, because Russia encompasses a uniquely wide range of environmental conditions and ethnogeographical cohorts. Here we conduct a shotgun metagenomic analysis of gut microbiota samples from 96 healthy Russian adult subjects, which reveals novel microbial community structures. The communities from several rural regions display similarities within each region and are dominated by the bacterial taxa associated with the healthy gut. Functional analysis shows that the metabolic pathways exhibiting differential abundance in the novel types are primarily associated with the trade-off between the Bacteroidetes and Firmicutes phyla. The specific signatures of the Russian gut microbiota are likely linked to the host diet, cultural habits and socioeconomic status.
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.
Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today.
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.
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.
Antimicrobial peptides (AMPs) are considered a promising new class of anti-infectious agents. This study reports new antimicrobial peptides derived from the
microbiome identified by a computational ...analysis method applied to the
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
, 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.
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.
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.
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
resulted in repeated isolation of
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
,
, and
species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated
strains efficiently inhibit the growth of both Gram-positive
and Gram-negative
in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing
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.
Abstract
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).
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.
The first complete genome of the biotechnologically important species Sulfobacillus thermotolerans has been sequenced. Its 3 317 203-bp chromosome contains an 83 269-bp plasmid-like region, which ...carries heavy metal resistance determinants and the rusticyanin gene. Plasmid-mediated metal resistance is unusual for acidophilic chemolithotrophs. Moreover, most of their plasmids are cryptic and do not contribute to the phenotype of the host cells. A polyphosphate-based mechanism of metal resistance, which has been previously unknown in the genus Sulfobacillus or other Gram-positive chemolithotrophs, potentially operates in two Sulfobacillus species. The methylcitrate cycle typical for pathogens and identified in the genus Sulfobacillus for the first time can fulfill the energy and/or protective function in S. thermotolerans Kr1 and two other Sulfobacillus species, which have incomplete glyoxylate cycles. It is notable that the TCA cycle, disrupted in all Sulfobacillus isolates under optimal growth conditions, proved to be complete in the cells enduring temperature stress. An efficient antioxidant defense system gives S. thermotolerans another competitive advantage in the microbial communities inhabiting acidic metal-rich environments. The genomic comparisons revealed 80 unique genes in the strain Kr1, including those involved in lactose/galactose catabolism. The results provide new insights into metabolism and resistance mechanisms in the Sulfobacillus genus and other acidophiles.
PDF
