RESUMEN
Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.
Asunto(s)
Proteínas Asociadas a CRISPR/ultraestructura , Sistemas CRISPR-Cas/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Proteínas Bacterianas , Proteínas Asociadas a CRISPR/química , ADN/química , Desoxirribonucleasas/metabolismo , Endorribonucleasas/metabolismo , Modelos Moleculares , ARN/química , ARN Bacteriano/química , ARN Guía de Kinetoplastida/química , Ribonucleasas/metabolismoRESUMEN
Viruses compete with each other for limited cellular resources, and some deliver defense mechanisms that protect the host from competing genetic parasites1. PARIS is a defense system, often encoded in viral genomes, that is composed of a 55 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB)2. However, the mechanism by which AriA and AriB function in phage defense is unknown. Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that is not cleaved by PARIS, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids3.
RESUMEN
CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut1-3. The genomes of all crAss-like phages encode a large virion-packaged protein2,4 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)5. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference6,7, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus8.
Asunto(s)
Bacteriófagos/clasificación , Bacteriófagos/enzimología , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Flavobacteriaceae/virología , Bacteriófagos/genética , Dominio Catalítico , Sistema Libre de Células , Cristalografía por Rayos X , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/genética , ARN Polimerasas Dirigidas por ADN/genética , Evolución Molecular , Regulación Viral de la Expresión Génica , Genes Virales/genética , Modelos Biológicos , Modelos Moleculares , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Interferencia de ARN , Transcripción GenéticaRESUMEN
The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Bacteriocinas/biosíntesis , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Complejos Multienzimáticos/metabolismo , Ribosomas/enzimología , Inhibidores de Topoisomerasa II/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriocinas/química , Bacteriocinas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Ribosomas/efectos de los fármacos , Ribosomas/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Difracción de Rayos XRESUMEN
Bacterial defence systems are tightly regulated to avoid autoimmunity. In Type I restriction-modification (R-M) systems, a specific mechanism called restriction alleviation (RA) controls the activity of the restriction module. In the case of the Escherichia coli Type I R-M system EcoKI, RA proceeds through ClpXP-mediated proteolysis of restriction complexes bound to non-methylated sites that appear after replication or reparation of host DNA. Here, we show that RA is also induced in the presence of plasmids carrying EcoKI recognition sites, a phenomenon we refer to as plasmid-induced RA. Further, we show that the anti-restriction behavior of plasmid-borne non-conjugative transposons such as Tn5053, previously attributed to their ardD loci, is due to plasmid-induced RA. Plasmids carrying both EcoKI and Chi sites induce RA in RecA- and RecBCD-dependent manner. However, inactivation of both RecA and RecBCD restores RA, indicating that there exists an alternative, RecA-independent, homologous recombination pathway that is blocked in the presence of RecBCD. Indeed, plasmid-induced RA in a RecBCD-deficient background does not depend on the presence of Chi sites. We propose that processing of random dsDNA breaks in plasmid DNA via homologous recombination generates non-methylated EcoKI sites, which attract EcoKI restriction complexes channeling them for ClpXP-mediated proteolysis.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Plásmidos , Rec A Recombinasas , Plásmidos/genética , Escherichia coli/genética , Rec A Recombinasas/metabolismo , Rec A Recombinasas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Recombinación Genética , Desoxirribonucleasas de Localización Especificada Tipo I/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo I/genética , Endopeptidasa Clp/metabolismo , Endopeptidasa Clp/genética , Exodesoxirribonucleasa V/metabolismo , Exodesoxirribonucleasa V/genética , ADN Bacteriano/metabolismo , Elementos Transponibles de ADN/genética , Enzimas de Restricción del ADN , Proteínas de Unión al ADNRESUMEN
A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.
Asunto(s)
Emparejamiento Base , Escherichia coli , Fluoruros , Conformación de Ácido Nucleico , Riboswitch , Transcripción Genética , Riboswitch/genética , Fluoruros/química , Escherichia coli/genética , Simulación de Dinámica Molecular , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Pliegue del ARN , Magnesio/química , Secuencia de Bases , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Thermus/genética , Thermus/enzimologíaRESUMEN
CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy and microfluidics analyses we demonstrate that plasmids can persist for multiple generations in some Escherichia coli cell lineages at conditions of continuous targeting by the type I-E CRISPR-Cas system. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to continuous maintenance of mobile genetic elements in some cells, which leads to diversification of phenotypes in the entire community and allows rapid changes in the population structure to meet the demands of a changing environment.
Asunto(s)
Sistemas CRISPR-Cas , Escherichia coli , Secuencias Repetitivas Esparcidas , Plásmidos , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Escherichia coli/genética , Interacción Gen-Ambiente , Secuencias Repetitivas Esparcidas/genética , Modelos Genéticos , Plásmidos/genéticaRESUMEN
Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.
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Antibacterianos , Bacteriocinas , Antibacterianos/farmacología , Bacteriocinas/farmacología , Bacteriocinas/química , Péptidos/farmacología , Péptidos/química , BacteriasRESUMEN
In this issue of Molecular Cell, Patterson et al. (2016) reveal a functional link between the CRISPR-Cas and quorum sensing systems, which could allow bacteria to activate the potentially lethal adaptive immunity when they need it most: in high-density cultures, when bacteriophage infections can be most detrimental.
Asunto(s)
Bacterias/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Inmunidad Adaptativa , Bacteriófagos/genética , Sistemas CRISPR-Cas , Percepción de QuorumRESUMEN
The choice of guide RNA (gRNA) for CRISPR-based gene targeting is an essential step in gene editing applications, but the prediction of gRNA specificity remains challenging. Lack of transparency and focus on point estimates of efficiency disregarding the information on possible error sources in the model limit the power of existing Deep Learning-based methods. To overcome these problems, we present a new approach, a hybrid of Capsule Networks and Gaussian Processes. Our method predicts the cleavage efficiency of a gRNA with a corresponding confidence interval, which allows the user to incorporate information regarding possible model errors into the experimental design. We provide the first utilization of uncertainty estimation in computational gRNA design, which is a critical step toward accurate decision-making for future CRISPR applications. The proposed solution demonstrates acceptable confidence intervals for most test sets and shows regression quality similar to existing models. We introduce a set of criteria for gRNA selection based on off-target cleavage efficiency and its variance and present a collection of pre-computed gRNAs for human chromosome 22. Using Neural Network Interpretation methods, we show that our model rediscovers an established biological factor underlying cleavage efficiency, the importance of the seed region in gRNA.
Asunto(s)
Sistemas CRISPR-Cas , Aprendizaje Profundo , Edición Génica , Marcación de Gen , ARN Guía de Kinetoplastida/genética , Algoritmos , Edición Génica/métodos , Marcación de Gen/métodos , Genómica/métodos , Humanos , Redes Neurales de la Computación , Reproducibilidad de los ResultadosRESUMEN
The action of Type II restriction-modification (RM) systems depends on restriction endonuclease (REase), which cleaves foreign DNA at specific sites, and methyltransferase (MTase), which protects host genome from restriction by methylating the same sites. We here show that protection from phage infection increases as the copy number of plasmids carrying the Type II RM Esp1396I system is increased. However, since increased plasmid copy number leads to both increased absolute intracellular RM enzyme levels and to a decreased MTase/REase ratio, it is impossible to determine which factor determines resistance/susceptibility to infection. By controlled expression of individual Esp1396I MTase or REase genes in cells carrying the Esp1396I system, we show that a shift in the MTase to REase ratio caused by overproduction of MTase or REase leads, respectively, to decreased or increased protection from infection. Consistently, due to stochastic variation of MTase and REase amount in individual cells, bacterial cells that are productively infected by bacteriophage have significantly higher MTase to REase ratios than cells that ward off the infection. Our results suggest that cells with transiently increased MTase to REase ratio at the time of infection serve as entry points for unmodified phage DNA into protected bacterial populations.
Asunto(s)
Bacteriófagos , Enzimas de Restricción del ADN/genética , Bacteriófagos/genética , Metiltransferasas , Enzimas de Restricción-Modificación del ADN/genética , ADNRESUMEN
CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.
Asunto(s)
Acidaminococcus , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Acidaminococcus/genética , Acidaminococcus/metabolismo , División del ADN , Francisella/genética , Francisella/metabolismo , Edición GénicaRESUMEN
Type II toxin-antitoxin (TA) systems are two-gene modules widely distributed among prokaryotes. GNAT toxins associated with the DUF1778 antitoxins represent a large family of type II TAs. GNAT toxins inhibit cell growth by disrupting translation via acetylation of aminoacyl-tRNAs. In this work, we explored the evolutionary trajectory of GNAT toxins. Using LC/MS detection of acetylated aminoacyl-tRNAs combined with ribosome profiling, we systematically investigated the in vivo substrate specificity of an array of diverse GNAT toxins. Our functional data show that the majority of GNAT toxins are specific to Gly-tRNA isoacceptors. However, the phylogenetic analysis shows that the ancestor of GNAT toxins was likely a relaxed specificity enzyme capable of acetylating multiple elongator tRNAs. Together, our data provide a remarkable snapshot of the evolution of substrate specificity.
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Antitoxinas , Toxinas Bacterianas , Sistemas Toxina-Antitoxina , Antitoxinas/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Filogenia , ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/genética , Sistemas Toxina-Antitoxina/genéticaRESUMEN
For Type I CRISPR-Cas systems, a mode of CRISPR adaptation named priming has been described. Priming allows specific and highly efficient acquisition of new spacers from DNA recognized (primed) by the Cascade-crRNA (CRISPR RNA) effector complex. Recognition of the priming protospacer by Cascade-crRNA serves as a signal for engaging the Cas3 nuclease-helicase required for both interference and primed adaptation, suggesting the existence of a primed adaptation complex (PAC) containing the Cas1-Cas2 adaptation integrase and Cas3. To detect this complex in vivo, we here performed chromatin immunoprecipitation with Cas3-specific and Cas1-specific antibodies using cells undergoing primed adaptation. We found that prespacers are bound by both Cas1 (presumably, as part of the Cas1-Cas2 integrase) and Cas3, implying direct physical association of the interference and adaptation machineries as part of PAC.
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ADN Helicasas/metabolismo , Endonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sistemas CRISPR-CasRESUMEN
Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.
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Bacteriófagos , Bacteriófagos/fisiología , Filogenia , Evolución Biológica , Bacterias , InglaterraRESUMEN
Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.
Asunto(s)
Bacterias , Proteínas Bacterianas , Sistemas CRISPR-Cas/fisiología , Evolución Molecular , ARN Bacteriano , Ribonucleasas , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Estructura Terciaria de Proteína , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Recombinación Genética/fisiología , Ribonucleasas/genética , Ribonucleasas/metabolismoRESUMEN
Conventional "bulk" PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.
Asunto(s)
Emulsiones/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa/métodos , ADN/genética , Cartilla de ADN/genética , Biblioteca de Genes , Genoma/genética , Humanos , Modelos Teóricos , Técnicas de Amplificación de Ácido Nucleico/métodos , Moldes GenéticosRESUMEN
Commensal bacteriocin-producing Escherichia coli are of interest for possible use as probiotics to selectively control the spread of pathogenic bacteria. Here, we evaluated the biosafety and efficacy of two new bacteriocin-producing E. coli strains, Q5 (VKM B-3706D) and C41 (VKM B-3707D), isolated from healthy farm animals. The genomes of both strains were sequenced, and genes responsible for the antagonistic and colonization abilities of each strain were identified. In vitro studies have shown that both strains were medium-adhesive and demonstrated antagonistic activity against most enteropathogens tested. Oral administration of 5 × 108 to 5 × 1010 colony-forming units of both strains to rats with drinking water did not cause any disease symptoms or side effects. Short-term (5 days) oral administration of both strains protected rats from colonization and pathogenic effects of a toxigenic beta-lactam-resistant strain of E. coli C55 and helped preserve intestinal homeostasis. Taken together, these in silico, in vitro, and in vivo data indicate that both strains (and especially E. coli Q5) can be potentially used for the prevention of colibacillosis in farm animals.
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Bacteriocinas , Infecciones por Escherichia coli , Probióticos , Animales , Ratas , Escherichia coli , Administración Oral , Infecciones por Escherichia coli/prevención & control , Animales Domésticos , Bacteriocinas/farmacología , Probióticos/farmacologíaRESUMEN
Extreme Antarctic conditions provide one of the closest analogues of extraterrestrial environments. Since air and snow samples, especially from polar regions, yield DNA amounts in the lower picogram range, binning of prokaryotic genomes is challenging and renders studying the dispersal of biological entities across these environments difficult. Here, we hypothesized that dispersal of host-associated bacteriophages (adsorbed, replicating, or prophages) across the Antarctic continent can be tracked via their genetic signatures, aiding our understanding of virus and host dispersal across long distances. Phage genome fragments (PGFs) reconstructed from surface snow metagenomes of three Antarctic stations were assigned to four host genomes, mainly Betaproteobacteria, including Ralstonia spp. We reconstructed the complete genome of a temperate phage with nearly complete alignment to a prophage in the reference genome of Ralstonia pickettii 12D. PGFs from different stations were related to each other at the genus level and matched similar hosts. Metagenomic read mapping and nucleotide polymorphism analysis revealed a wide dispersal of highly identical PGFs, 13 of which were detected in seawater from the Western Antarctic Peninsula at a distance of 5,338 km from the snow sampling stations. Our results suggest that host-associated phages, especially of Ralstonia sp., disperse over long distances despite the harsh conditions of the Antarctic continent. Given that 14 phages associated with two R. pickettii draft genomes isolated from space equipment were identified, we conclude that Ralstonia phages are ideal mobile genetic elements to track dispersal and contamination in ecosystems relevant for astrobiology. IMPORTANCE Host-associated phages of the bacterium Ralstonia identified in snow samples can be used to track microbial dispersal over thousands of kilometers across the Antarctic continent, which functions as an extraterrestrial analogue because of its harsh environmental conditions. Due to the presence of these bacteria carrying genome-integrated prophages on space-related equipment and the potential for dispersal of host-associated phages demonstrated here, our work has implications for planetary protection, a discipline in astrobiology interested in preventing contamination of celestial bodies with alien biomolecules or forms of life.
Asunto(s)
Bacteriófagos , Regiones Antárticas , Bacteriófagos/genética , Ecosistema , Metagenómica , Profagos/genéticaRESUMEN
BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction-modification systems, is also active against BREX. In contrast to the wild-type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R-M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.