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1.
Nat Commun ; 11(1): 6151, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262347

RESUMO

Fluorescence microscopy enables spatial and temporal measurements of live cells and cellular communities. However, this potential has not yet been fully realized for investigations of individual cell behaviors and phenotypic changes in dense, three-dimensional (3D) bacterial biofilms. Accurate cell detection and cellular shape measurement in densely packed biofilms are challenging because of the limited resolution and low signal to background ratios (SBRs) in fluorescence microscopy images. In this work, we present Bacterial Cell Morphometry 3D (BCM3D), an image analysis workflow that combines deep learning with mathematical image analysis to accurately segment and classify single bacterial cells in 3D fluorescence images. In BCM3D, deep convolutional neural networks (CNNs) are trained using simulated biofilm images with experimentally realistic SBRs, cell densities, labeling methods, and cell shapes. We systematically evaluate the segmentation accuracy of BCM3D using both simulated and experimental images. Compared to state-of-the-art bacterial cell segmentation approaches, BCM3D consistently achieves higher segmentation accuracy and further enables automated morphometric cell classifications in multi-population biofilms.


Assuntos
Bactérias/citologia , Biofilmes , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Bactérias/química , Bactérias/crescimento & desenvolvimento , Fenômenos Fisiológicos Bacterianos , Aprendizado Profundo , Análise de Célula Única/métodos
2.
Nucleic Acids Res ; 48(22): 12566-12576, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33245763

RESUMO

Metabolic labeling of RNAs with noncanonical nucleosides that are chemically active, followed by chemoselective conjugation with imaging probes or enrichment tags, has emerged as a powerful method for studying RNA transcription and degradation in eukaryotes. However, metabolic RNA labeling is not applicable for prokaryotes, in which the complexity and distinctness of gene regulation largely remain to be explored. Here, we report 2'-deoxy-2'-azidoguanosine (AzG) as a noncanonical nucleoside compatible with metabolic labeling of bacterial RNAs. With AzG, we develop AIR-seq (azidonucleoside-incorporated RNA sequencing), which enables genome-wide analysis of transcription upon heat stress in Escherichia coli. Furthermore, AIR-seq coupled with pulse-chase labeling allows for global analysis of bacterial RNA degradation. Finally, we demonstrate that RNAs of mouse gut microbiotas can be metabolically labeled with AzG in living animals. The AzG-enabled metabolic RNA labeling should find broad applications in studying RNA biology in various bacterial species.


Assuntos
Bactérias/metabolismo , RNA/metabolismo , Análise de Sequência de RNA/métodos , Coloração e Rotulagem , Animais , Bactérias/química , Genoma/genética , Células HeLa , Humanos , Camundongos , Nucleosídeos/metabolismo , RNA/química , RNA/isolamento & purificação , Sondas RNA/química , Sondas RNA/metabolismo , Estabilidade de RNA/genética
3.
PLoS One ; 15(10): e0241183, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33112901

RESUMO

Most literature exploring the biological effects of ocean acidification (OA) has focused on macroscopic organisms and far less is known about how marine microbial communities will respond. Studies of OA and microbial community composition and diversity have examined communities from a limited number of ocean regions where the ambient pH is near or above the global average. At San Juan Island (Salish Sea), a region that experiences naturally low pH (average = 7.8), the picoplankton (cell diameter is 0.2-2µm) community was predicted to show no response to experimental acidification in a three-week mesocosm experiment. Filtered seawater mesocosms were maintained via semicontinuous culturing. Three control mesocosms were maintained at pH 8.05 and three acidified mesocosms were maintained at pH 7.60. Total bacteria was quantified daily with a flow cytometer. Microbial communities were sampled every two days via filtration followed by DNA extraction, 16S rRNA amplification, and MiSeq sequencing. There was no significant difference in total bacteria between pH treatments throughout the experiment. Acidification significantly reduced Shannon's diversity over time. During the final week of the experiment, acidification resulted in a significant decrease in Shannon's diversity, Faith's phylogenetic distance, and Pielous's Evenness. ANCOM results revealed four bacterial ASVs (amplicon sequence variants), in families Flavobaceriaceae and Hyphomonadaceae that significantly decreased in relative frequency under acidification and two bacterial ASVs, in families Flavobacteriaceae and Alteromonadaceae, that significantly increased under acidification. This is the first OA study on the microbial community of the Salish Sea, a nutrient rich, low pH region, and the first of its kind to report a decrease in both picoplankton richness and evenness with acidification. These findings demonstrate that marine microbial communities that naturally experience acidic conditions are still sensitive to acidification.


Assuntos
Ácidos/farmacologia , Bactérias/classificação , Biodiversidade , Dióxido de Carbono/análise , Microbiota/efeitos dos fármacos , Água do Mar/análise , Ácidos/química , Bactérias/química , Bactérias/efeitos dos fármacos , Bactérias/genética , Concentração de Íons de Hidrogênio , RNA Ribossômico 16S/genética , Água do Mar/química , Água do Mar/microbiologia
4.
Nat Commun ; 11(1): 5328, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087704

RESUMO

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 105 cells are analysed one-by-one with microfluidic impedance cytometry for 2-3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species.


Assuntos
Testes de Sensibilidade Microbiana/métodos , Bactérias/química , Bactérias/efeitos dos fármacos , Fenômenos Biofísicos , Farmacorresistência Bacteriana , Impedância Elétrica , Desenho de Equipamento , Humanos , Klebsiella pneumoniae/química , Klebsiella pneumoniae/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Meropeném/administração & dosagem , Testes de Sensibilidade Microbiana/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos
5.
Nat Protoc ; 15(11): 3663-3677, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33005038

RESUMO

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.


Assuntos
Celulose/química , Ácidos Nucleicos/isolamento & purificação , Animais , Bactérias/química , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Plantas/química , Fatores de Tempo , Vírus/química
6.
J Vis Exp ; (163)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-33016949

RESUMO

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20-100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.


Assuntos
Bactérias/química , Bactérias/classificação , Microscopia de Força Atômica/métodos , Espectrofotometria Infravermelho/métodos , Bactérias/ultraestrutura , Humanos
7.
Proc Natl Acad Sci U S A ; 117(40): 24720-24728, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32934140

RESUMO

Landscape-scale reconstructions of ancient environments within the cradle of humanity may reveal insights into the relationship between early hominins and the changing resources around them. Many studies of Olduvai Gorge during Pliocene-Pleistocene times have revealed the presence of precession-driven wet-dry cycles atop a general aridification trend, though may underestimate the impact of local-scale conditions on early hominins, who likely experienced a varied and more dynamic landscape. Fossil lipid biomarkers from ancient plants and microbes encode information about their surroundings via their molecular structures and composition, and thus can shed light on past environments. Here, we employ fossil lipid biomarkers to study the paleolandscape at Olduvai Gorge at the emergence of the Acheulean technology, 1.7 Ma, through the Lower Augitic Sandstones layer. In the context of the expansion of savanna grasslands, our results represent a resource-rich mosaic ecosystem populated by groundwater-fed rivers, aquatic plants, angiosperm shrublands, and edible plants. Evidence of a geothermally active landscape is reported via an unusual biomarker distribution consistent with the presence of hydrothermal features seen today at Yellowstone National Park. The study of hydrothermalism in ancient settings and its impact on hominin evolution has not been addressed before, although the association of thermal springs in the proximity of archaeological sites documented here can also be found at other localities. The hydrothermal features and resources present at Olduvai Gorge may have allowed early hominins to thermally process edible plants and meat, supporting the possibility of a prefire stage of human evolution.


Assuntos
Bactérias/química , Hominidae/fisiologia , Microbiota , Animais , Arqueologia , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Evolução Biológica , Biomarcadores/análise , Ecossistema , Paleontologia
8.
Anal Chem ; 92(19): 13396-13404, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32867467

RESUMO

Rapid, accurate, reliable, and risk-free tracking of pathogenic microorganisms at the single-cell level is critical to achieve efficient source control and prevent outbreaks of microbial infectious diseases. For the first time, we report a promising approach for integrating the concepts of a remarkably large Stokes shift and dual-recognition into a single matrix to develop a pathogenic microorganism stimuli-responsive ratiometric fluorescent nanoprobe with speed, cost efficiency, stability, ultrahigh specificity, and sensitivity. As a proof-of-concept, we selected the Gram-positive bacterium Staphylococcus aureus (S. aureus) as the target analyte model, which easily bound to its recognition aptamer and the broad-spectrum glycopeptide antibiotic vancomycin (Van). To improve the specificity and short sample-to-answer time, we employed classic noncovalent π-π stacking interactions as a driving force to trigger the binding of Van and aptamer dual-functionalized near-infrared (NIR) fluorescent Apt-Van-QDs to the surface of an unreported blue fluorescent π-rich electronic carbon nanoparticles (CNPs), achieving S. aureus stimuli-responsive ratiometric nanoprobe Apt-Van-QDs@CNPs. In the assembly of Apt-Van-QDs@CNPs, the blue CNPs (energy donor) and NIR Apt-Van-QDs (energy acceptor) became close to allow the fluorescence resonance energy transfer (FRET) process, leading to a remarkable blue fluorescence quenching for the CNPs at ∼465 nm and a clear NIR fluorescence enhancement for Apt-Van-QDs at ∼725 nm. In the presence of S. aureus, the FRET process from CNPs to Apt-Van-QDs was disrupted, causing the nanoprobe Apt-Van-QDs@CNPs to display a ratiometric fluorescent response to S. aureus, which exhibited a large Stokes shift of ∼260 nm and rapid sample-to-answer detection time (∼30.0 min). As expected, the nanoprobe Apt-Van-QDs@CNPs showed an ultrahigh specificity for ratiometric fluorescence detection of S. aureus with a good detection limit of 1.0 CFU/mL, allowing the assay at single-cell level. Moreover, we also carried out the precise analysis of S. aureus in actual samples with acceptable results. We believe that this work offers new insight into the rational design of efficient ratiometric nanoprobes for rapid on-site accurate screening of pathogenic microorganisms at the single-cell level in the early stages, especially during the worldwide spread of COVID-19 today.


Assuntos
Bactérias/química , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/síntese química , Nanotecnologia/métodos , Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos , Infecções por Coronavirus/complicações , Infecções por Coronavirus/microbiologia , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Microbiologia de Alimentos/métodos , Humanos , Nanopartículas , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/microbiologia , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Vancomicina/farmacologia
9.
PLoS One ; 15(8): e0237748, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866195

RESUMO

Soil microbiota are considered a source of undiscovered bioactive compounds, yet cultivation of most bacteria within a sample remains generally unsuccessful. Two main reasons behind the unculturability of bacteria are the presence of cells in a viable but not culturable state (such as dormant cells) and the failure to provide the necessary growth requirements in vitro (leading to the classification of some bacterial taxa as yet-to-be-cultured). The present work focuses on the development of a single procedure that helps distinguish between both phenomena of unculturability based on viability staining coupled with flow cytometry and fluorescence-activated cell sorting. In the selected soil sample, the success rate of cultured bacteria was doubled by selecting viable and metabolically active bacteria. It was determined that most of the uncultured fraction was not dormant or dead but likely required different growth conditions. It was also determined that the staining process introduced changes in the taxonomic composition of the outgrown bacterial biomass, which should be considered for further developments. This research shows the potential of flow cytometry and fluorescence-activated cell sorting applied to soil samples to improve the success rate of bacterial cultivation by estimating the proportion of dormant and yet-to-be-cultured bacteria and by directly excluding dormant cells from being inoculated into growth media.


Assuntos
Bactérias/crescimento & desenvolvimento , Técnicas Microbiológicas/métodos , Microbiota/fisiologia , Microbiologia do Solo , Bactérias/química , Bactérias/genética , Biomassa , Separação Celular/métodos , Meios de Cultura , DNA Bacteriano/isolamento & purificação , Estudos de Viabilidade , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Coloração e Rotulagem/métodos
10.
PLoS Comput Biol ; 16(9): e1008159, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925923

RESUMO

Intracellular spatial heterogeneity is frequently observed in bacteria, where the chromosome occupies part of the cell's volume and a circuit's DNA often localizes within the cell. How this heterogeneity affects core processes and genetic circuits is still poorly understood. In fact, commonly used ordinary differential equation (ODE) models of genetic circuits assume a well-mixed ensemble of molecules and, as such, do not capture spatial aspects. Reaction-diffusion partial differential equation (PDE) models have been only occasionally used since they are difficult to integrate and do not provide mechanistic understanding of the effects of spatial heterogeneity. In this paper, we derive a reduced ODE model that captures spatial effects, yet has the same dimension as commonly used well-mixed models. In particular, the only difference with respect to a well-mixed ODE model is that the association rate constant of binding reactions is multiplied by a coefficient, which we refer to as the binding correction factor (BCF). The BCF depends on the size of interacting molecules and on their location when fixed in space and it is equal to unity in a well-mixed ODE model. The BCF can be used to investigate how spatial heterogeneity affects the behavior of core processes and genetic circuits. Specifically, our reduced model indicates that transcription and its regulation are more effective for genes located at the cell poles than for genes located on the chromosome. The extent of these effects depends on the value of the BCF, which we found to be close to unity. For translation, the value of the BCF is always greater than unity, it increases with mRNA size, and, with biologically relevant parameters, is substantially larger than unity. Our model has broad validity, has the same dimension as a well-mixed model, yet it incorporates spatial heterogeneity. This simple-to-use model can be used to both analyze and design genetic circuits while accounting for spatial intracellular effects.


Assuntos
Bactérias , Redes Reguladoras de Genes/genética , Genes Bacterianos/genética , Modelos Biológicos , Bactérias/química , Bactérias/citologia , Bactérias/genética , Biologia Computacional , Difusão , Espaço Intracelular/química , Espaço Intracelular/genética
11.
J Food Sci ; 85(10): 3467-3477, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32885423

RESUMO

The research aimed to generate an early warning system highlighting in real-time bacterial contamination of meat matrices and providing information which could support companies in accepting or rejecting batches. Current microorganisms' detection methods rely on techniques (plate counting), which provide retrospective values for microbial contamination. The purpose of this research was to evaluate the ability of the headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC/MS) methodologies to detect volatile organic carbons (VOCs), which may be associated to a peculiar microbiological contamination of food. The disposal of fast headspace gas chromatography-mass spectrometry (HS-SPME-GC/MS) able to accurately and rapidly (30 min per sample) detect pathogens in raw meat could replace the traditional and time-consuming (3 to 4 days) standardized microbiological analysis required by regulations. Experiments focused on qualitative and quantitative evaluations of VOCs produced by Salmonella Typhimurium, Campylobacter jejuni, and Staphylococcus aureus in different types of raw meat (beef, pork, chicken). HS-SPME-GC/MS allowed to use smaller sample volumes compared to traditional methods with no sample processing and the potentiality for its application on various food matrices for the detection of a wide variety of pathogens. Data analysis showed the identification of unique VOCs' profiles being possible markers of meat contamination due to their association to specific pathogens. The identification of VOCs markers in association to selected bacterial pathogens and their metabolites could support the rapid determination of specific meat samples contamination. Further research is required to outline-specific metabolic profiles for each microorganism responsible of meat contamination and prevent false positives.


Assuntos
Bactérias/metabolismo , Contaminação de Alimentos/análise , Carne/microbiologia , Compostos Orgânicos Voláteis/química , Animais , Bactérias/química , Biomarcadores/análise , Bovinos , Galinhas , Cromatografia Gasosa-Espectrometria de Massas/métodos , Carne/análise , Metaboloma , Metabolômica , Microextração em Fase Sólida/métodos , Suínos , Compostos Orgânicos Voláteis/metabolismo
12.
Nature ; 586(7829): 429-433, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32877915

RESUMO

Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP-AMP signalling during viral infection and anti-tumour immunity1-5. STING shares no structural homology with other known signalling proteins6-9, which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD+ cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIR-STING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGAS-STING pathway in prokaryotic defence against bacteriophages.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Evolução Molecular , Proteínas de Membrana , Sistemas do Segundo Mensageiro , Animais , Bactérias/química , Bactérias/virologia , Proteínas de Bactérias/química , Bacteriófagos , Cristalografia por Raios X , GMP Cíclico/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , NAD/metabolismo , Nucleotidiltransferases/metabolismo
13.
Mol Pharmacol ; 98(4): 343-349, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32764096

RESUMO

For decades, traditional drug discovery has used natural product and synthetic chemistry approaches to generate libraries of compounds, with some ending as promising drug candidates. A complementary approach has been to adopt the concept of biomimicry of natural products and metabolites so as to improve multiple drug-like features of the parent molecule. In this effort, promiscuous and weak interactions between ligands and receptors are often ignored in a drug discovery process. In this Emerging Concepts article, we highlight microbial metabolite mimicry, whereby parent metabolites have weak interactions with their receptors that then have led to discrete examples of more potent and effective drug-like molecules. We show specific examples of parent-metabolite mimics with potent effects in vitro and in vivo. Furthermore, we show examples of emerging microbial ligand-receptor interactions and provide a context in which these ligands could be improved as potential drugs. A balanced conceptual advance is provided in which we also acknowledge potential pitfalls-hyperstimulation of finely balanced receptor-ligand interactions could also be detrimental. However, with balance, we provide examples of where this emerging concept needs to be tested. SIGNIFICANCE STATEMENT: Microbial metabolite mimicry is a novel way to expand on the chemical repertoire of future drugs. The emerging concept is now explained using specific examples of the discovery of therapeutic leads from microbial metabolites.


Assuntos
Bactérias/química , Produtos Biológicos/química , Indóis/farmacologia , Descoberta de Drogas , Humanos , Indóis/química , Ligantes , Mimetismo Molecular
14.
BMC Bioinformatics ; 21(1): 284, 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32631215

RESUMO

BACKGROUND: The European Community has adopted very restrictive policies regarding the dissemination and use of genetically modified organisms (GMOs). In fact, a maximum threshold of 0.9% of contaminating GMOs is tolerated for a "GMO-free" label. In recent years, imports of undescribed GMOs have been detected. Their sequences are not described and therefore not detectable by conventional approaches, such as PCR. RESULTS: We developed DUGMO, a bioinformatics pipeline for the detection of genetically modified (GM) bacteria, including unknown GM bacteria, based on Illumina paired-end sequencing data. The method is currently focused on the detection of GM bacteria with - possibly partial - transgenes in pure bacterial samples. In the preliminary steps, coding sequences (CDSs) are aligned through two successive BLASTN against the host pangenome with relevant tuned parameters to discriminate CDSs belonging to the wild type genome (wgCDS) from potential GM coding sequences (pgmCDSs). Then, Bray-Curtis distances are calculated between the wgCDS and each pgmCDS, based on the difference of genomic vocabulary. Finally, two machine learning methods, namely the Random Forest and Generalized Linear Model, are carried out to target true GM CDS(s), based on six variables including Bray-Curtis distances and GC content. Tests carried out on a GM Bacillus subtilis showed 25 positive CDSs corresponding to the chloramphenicol resistance gene and CDSs of the inserted plasmids. On a wild type B. subtilis, no false positive sequences were detected. CONCLUSION: DUGMO detects exogenous CDS, truncated, fused or highly mutated wild CDSs in high-throughput sequencing data, and was shown to be efficient at detecting GM sequences, but it might also be employed for the identification of recent horizontal gene transfers.


Assuntos
Bactérias/química , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Organismos Geneticamente Modificados/genética , Reação em Cadeia da Polimerase/métodos , Humanos
16.
J Appl Microbiol ; 129(6): 1441-1457, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32627318

RESUMO

Symbiotic relationships between corals and their associated micro-organisms are essential to maintain host homeostasis. Coral-associated bacteria (CAB) can have different beneficial roles in the coral metaorganism, such as metabolizing essential nutrients for the coral host and protecting the coral from pathogens. Many CAB exert these functions via secondary metabolites, which include antibacterial, antifouling, antitumour, antiparasitic and antiviral compounds. This review describes how analysis of CAB has led to the discovery of secondary metabolites with potential biotechnological applications. The most commonly found types of secondary metabolites, antimicrobial and antibiofilm compounds, are emphasized and described. Recently developed methods that can be applied to enhance the culturing of CAB from shallow-water reefs and the less-studied deep-sea coral reefs are also discussed. Last, we suggest how the combined use of meta-omics and innovative growth-diffusion techniques can vastly improve the discovery of novel compounds in coral environments.


Assuntos
Antozoários/microbiologia , Bactérias/química , Produtos Biológicos/metabolismo , Animais , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Técnicas Bacteriológicas , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Biotecnologia , Recifes de Corais , Genômica , Simbiose
17.
J Food Sci ; 85(7): 2153-2163, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32572986

RESUMO

The application of bacterial cellulose (BC) as a wrapping material for vacuum-packaged beef was studied and compared against unwrapped beef for up to 3 weeks. The impact of BC wrap on the weight loss, purge accumulation, and drip loss were assessed along with low-field nuclear magnetic resonance, physicochemical, microbiological, and sensorial evaluations. The BC wrap significantly (P < 0.05) reduced purge accumulation in vacuum packages which was confirmed by an increased swelling ratio and scanning electron microscopy images. Colorimetric measurements showed significantly (P < 0.05) increased redness and yellowness values in wrapped samples compared to unwrapped samples. BC wrap did not affect pH, tenderness, and odor of meat, but significantly (P < 0.05) increased lipid oxidation, and numbers of lactic acid bacteria and Brochothrix thermosphacta counts. This study shows that BC wrap has potential as a purge absorbent in vacuum packaged meat. PRACTICAL APPLICATION: Bacteria cellulose has good water holding capacity that can be utilized to absorb purge exudate from beef. It helps to improve the appearance and consequently consumer acceptance of vacuum packed beef.


Assuntos
Celulose/química , Embalagem de Alimentos/métodos , Conservação de Alimentos/métodos , Carne/análise , Adsorção , Animais , Bactérias/química , Bactérias/metabolismo , Bovinos , Cor , Microbiologia de Alimentos , Embalagem de Alimentos/instrumentação , Conservação de Alimentos/instrumentação , Humanos , Lactobacillales/crescimento & desenvolvimento , Carne/microbiologia , Paladar , Vácuo
18.
J Antibiot (Tokyo) ; 73(8): 504-525, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32507851

RESUMO

Marine-derived bacteria are a prolific source of a wide range of structurally diverse natural products. This review, dedicated to Professor William Fenical, begins by showcasing many seminal discoveries made at the University of California San Diego from marine-derived actinomycetes. Discussed early on is the 20-year journey of discovery and advancement of the seminal actinomycetes natural product salinosporamide A into Phase III anticancer clinical trials. There are many fascinating parallels discussed that were gleaned from the comparative literature of marine sponge, tunicate, and bacteria-derived natural products. Identifying bacterial biosynthetic machinery housed in sponge and tunicate holobionts through both culture-independent and culture-dependent approaches is another important and expanding subject that is analyzed. Work reviewed herein also evaluates the hypotheses that many marine invertebrate-derived natural products are biosynthesised by associated or symbiotic bacteria. The insights and outcomes from metagenomic sequencing and synthetic biology to expand molecule discovery continue to provide exciting outcomes and they are predicted to be the source of the next generation of novel marine natural product chemical scaffolds.


Assuntos
Organismos Aquáticos/química , Bactérias/química , Produtos Biológicos/química , Poríferos/química , Urocordados/química , Animais , Metagenômica/métodos
19.
Prog Lipid Res ; 79: 101047, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32540152

RESUMO

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3), arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5-n3) are nutritionally important for humans and animals. De novo biosynthesis of these fatty acids mainly occurs in microorganisms and goes through either an aerobic pathway catalyzed by type I/II fatty acid synthase, desaturases and elongases or an anaerobic pathway catalyzed by a polyunsaturated fatty acid synthase. After synthesis, VLCPUFAs must be incorporated into glycerolipids for storage through acyl assembly processes. Understanding the mechanisms for the biosynthesis of VLCPUFAs and their incorporation into glycerolipids is important not only for developing a renewable, sustainable and environment-friendly source of these fatty acids in microorganisms, but also, for designing effective strategies for metabolic engineering of these fatty acids in heterologous systems. This review highlights recent findings which have increased our understanding of biosynthesis of VLCPUFAs and their incorporation into glycerolipids in microorganisms. Future directions in improving the production of VLCPUFAs in native microbial producers are also discussed along with transgenic production of these fatty acids in oleaginous microorganisms and oilseed crops for food and feed uses.


Assuntos
Bactérias/metabolismo , Ácidos Graxos Insaturados/biossíntese , Fungos/metabolismo , Microalgas/metabolismo , Parasitos/metabolismo , Animais , Bactérias/química , Ácidos Graxos Insaturados/química , Fungos/química , Humanos , Microalgas/química , Parasitos/química
20.
Anal Chem ; 92(14): 9699-9705, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32441935

RESUMO

A novel coronavirus (SARS-CoV-2) was recently identified in patients with acute respiratory disease and spread quickly worldwide. A specific and rapid diagnostic method is important for early identification. The reverse-transcription recombinase-aided amplification (RT-RAA) assay is a rapid detection method for several pathogens. Assays were performed within 5-15 min as a one-step single tube reaction at 39 °C. In this study, we established two RT-RAA assays for the S and orf1ab gene of SARS-CoV-2 using clinical specimens for validation. The analytical sensitivity of the RT-RAA assay was 10 copies for the S and one copy for the orf1ab gene per reaction. Cross-reactions were not observed with any of the other respiratory pathogens. A 100% agreement between the RT-RAA and real-time PCR assays was accomplished after testing 120 respiratory specimens. These results demonstrate that the proposed RT-RAA assay will be beneficial as it is a faster, more sensitive, and more specific tool for the detection of SARS-CoV-2.


Assuntos
Betacoronavirus/química , Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase/métodos , Bactérias/química , Bactérias/genética , Reações Cruzadas , Sondas de DNA , Genes Virais , Humanos , Pandemias , Plasmídeos , Poliproteínas , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Proteínas Virais/genética , Vírus/química , Vírus/genética
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