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1.
Nat Commun ; 11(1): 4379, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873785

RESUMO

The gut microbiome harbors a 'silent reservoir' of antibiotic resistance (AR) genes that is thought to contribute to the emergence of multidrug-resistant pathogens through horizontal gene transfer (HGT). To counteract the spread of AR, it is paramount to know which organisms harbor mobile AR genes and which organisms engage in HGT. Despite methods that characterize the overall abundance of AR genes in the gut, technological limitations of short-read sequencing have precluded linking bacterial taxa to specific mobile genetic elements (MGEs) encoding AR genes. Here, we apply Hi-C, a high-throughput, culture-independent method, to surveil the bacterial carriage of MGEs. We compare two healthy individuals with seven neutropenic patients undergoing hematopoietic stem cell transplantation, who receive multiple courses of antibiotics, and are acutely vulnerable to the threat of multidrug-resistant infections. We find distinct networks of HGT across individuals, though AR and mobile genes are associated with more diverse taxa within the neutropenic patients than the healthy subjects. Our data further suggest that HGT occurs frequently over a several-week period in both cohorts. Whereas most efforts to understand the spread of AR genes have focused on pathogenic species, our findings shed light on the role of the human gut microbiome in this process.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Microbioma Gastrointestinal/genética , Transferência Genética Horizontal , Genes Bacterianos/efeitos dos fármacos , Adulto , Idoso , Antibacterianos/uso terapêutico , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Microbioma Gastrointestinal/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequências Repetitivas Dispersas/efeitos dos fármacos , Pessoa de Meia-Idade
2.
Nat Commun ; 11(1): 3506, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665538

RESUMO

Acute myeloid leukemia (AML) is characterised by a series of genetic and epigenetic alterations that result in deregulation of transcriptional networks. One understudied source of transcriptional regulators are transposable elements (TEs), whose aberrant usage could contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to AML pathogenesis remain unexplored. Here we identify six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppresses cell growth by inducing apoptosis in leukemia cell lines. This work reveals that ERVs are a previously unappreciated source of AML enhancers that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.


Assuntos
Cromatina/metabolismo , Leucemia Mieloide Aguda/genética , Animais , Linhagem Celular , Cromatina/genética , Elementos de DNA Transponíveis/genética , Retrovirus Endógenos/genética , Epigênese Genética/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Genoma Humano/genética , Humanos , Sequências Repetitivas Dispersas/genética
3.
Nat Commun ; 11(1): 3363, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620750

RESUMO

Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.


Assuntos
Regulação Bacteriana da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Doenças Negligenciadas/imunologia , Orientia tsutsugamushi/genética , Tifo por Ácaros/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Genoma Bacteriano , Células Endoteliais da Veia Umbilical Humana , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Sequências Repetitivas Dispersas/genética , Camundongos , Doenças Negligenciadas/microbiologia , Orientia tsutsugamushi/imunologia , Orientia tsutsugamushi/patogenicidade , Proteômica , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , RNA-Seq , Tifo por Ácaros/microbiologia , Transcrição Genética , Sequenciamento Completo do Exoma
4.
PLoS One ; 15(7): e0236436, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32716946

RESUMO

Salmonella 4,[5],12:i:- are monophasic S. Typhimurium variants incapable of producing the second-phase flagellar antigen. They have emerged since the mid-1990s to become one of the most prevalent Salmonella serotypes causing human disease world-wide. Multiple genetic events associated with different genetic elements can result in the monophasic phenotype. Several jurisdictions have reported the emergence of a Salmonella 4,[5],12:i:- clone with SGI-4 and a genetic element (MREL) encoding a mercury resistance operon and antibiotic resistance loci that disrupts the second phase antigen region near the iroB locus in the Salmonella genome. We have sequenced 810 human and animal Canadian Salmonella 4,[5],12:i:- isolates and determined that isolates with SGI-4 and the mercury resistance element (MREL; also known as RR1&RR2) constitute several global clades containing various proportions of Canadian, US, and European isolates. Detailed analysis of the data provides a clearer picture of how these heavy metal elements interact with bacteria within the Salmonella population to produce the monophasic phenotype. Insertion of the MREL near iroB is associated with several deletions and rearrangements of the adjacent flaAB hin region, which may be useful for defining human case clusters that could represent outbreaks. Plasmids carrying genes encoding silver, copper, mercury, and antimicrobial resistance appear to be derived from IS26 mediated acquisition of these genes from genomes carrying SGI-4 and the MREL. Animal isolates with the mercury and As/Cu/Ag resistance elements are strongly associated with porcine sources in Canada as has been shown previously for other jurisdictions. The data acquired in these investigations, as well as from the extensive literature on the subject, may aid source attribution in outbreaks of the organism and interventions to decrease the prevalence of this clone and reduce its impact on human disease.


Assuntos
Metais Pesados/toxicidade , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Animais , Antígenos de Bactérias/genética , Sequência de Bases , Canadá , Variação Genética , Genoma Bacteriano , Genótipo , Humanos , Sequências Repetitivas Dispersas/genética , Fenótipo , Filogenia , Plasmídeos/genética , Salmonella typhimurium/isolamento & purificação , Suínos , Sintenia/genética
5.
Sci Total Environ ; 743: 140759, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659562

RESUMO

The abuse of heavy metals as feed additives in livestock is widespread and it might aggravate the spread of antibiotic resistance genes (ARGs) in the environment. However, the mechanisms that allow heavy metals to increase the transmission of ARGs in the environment remain unclear. Cu is the heavy metal present at the highest concentration in livestock manure, and thus Cu was selected to investigate the responses of ARGs to heavy metals. The effects of the microbial communities, mobile genetic elements (MGEs), and heavy metal resistance genes (HMRGs) on ARGs were determined in the presence of 75 and 227 mg L-1 Cu in a swine manure anaerobic digestion (AD) system. In the AD products, the presence of residual Cu (75 and 227 mg L-1) increased the total ARGs, HMRGs, and some MGEs, and the higher Cu selected more ARGs than the lower Cu treatment. The results demonstrated that Cu could promote the co-selection of HMRGs, ARGs, and MGEs. The different levels of Cu did not change the bacterial community composition, but they influenced the abundances of bacteria during AD. Network analysis showed that the presence of Cu increased the co-occurrence of specific bacteria containing ARGs, HMRGs, and MGEs. Furthermore, the co-occurrence of MGEs and ARGs increased greatly compared with that of HMRGs and ARGs. Therefore, compared HMRGs, the increased MGEs had the main effect on increasing of ARGs.


Assuntos
Cobre , Genes Bacterianos , Anaerobiose , Animais , Antibacterianos , Resistência Microbiana a Medicamentos , Sequências Repetitivas Dispersas , Esterco , Suínos
6.
Proc Natl Acad Sci U S A ; 117(27): 15755-15762, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571917

RESUMO

Mobile genetic elements (MGEs), such as plasmids, promote bacterial evolution through horizontal gene transfer (HGT). However, the rules governing the repertoire of traits encoded on MGEs remain unclear. In this study, we uncovered the central role of genetic dominance shaping genetic cargo in MGEs, using antibiotic resistance as a model system. MGEs are typically present in more than one copy per host bacterium, and as a consequence, genetic dominance favors the fixation of dominant mutations over recessive ones. In addition, genetic dominance also determines the phenotypic effects of horizontally acquired MGE-encoded genes, silencing recessive alleles if the recipient bacterium already carries a wild-type copy of the gene. The combination of these two effects governs the catalog of genes encoded on MGEs. Our results help to understand how MGEs evolve and spread, uncovering the neglected influence of genetic dominance on bacterial evolution. Moreover, our findings offer a framework to forecast the spread and evolvability of MGE-encoded genes, which encode traits of key human interest, such as virulence or antibiotic resistance.


Assuntos
Bactérias/genética , Evolução Molecular , Transferência Genética Horizontal/genética , Sequências Repetitivas Dispersas/genética , Farmacorresistência Bacteriana/genética , Humanos , Plasmídeos/genética , Virulência/genética
7.
PLoS Genet ; 16(6): e1008866, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32530914

RESUMO

Escherichia coli is mostly a commensal of birds and mammals, including humans, where it can act as an opportunistic pathogen. It is also found in water and sediments. We investigated the phylogeny, genetic diversification, and habitat-association of 1,294 isolates representative of the phylogenetic diversity of more than 5,000 isolates from the Australian continent. Since many previous studies focused on clinical isolates, we investigated mostly other isolates originating from humans, poultry, wild animals and water. These strains represent the species genetic diversity and reveal widespread associations between phylogroups and isolation sources. The analysis of strains from the same sequence types revealed very rapid change of gene repertoires in the very early stages of divergence, driven by the acquisition of many different types of mobile genetic elements. These elements also lead to rapid variations in genome size, even if few of their genes rise to high frequency in the species. Variations in genome size are associated with phylogroup and isolation sources, but the latter determine the number of MGEs, a marker of recent transfer, suggesting that gene flow reinforces the association of certain genetic backgrounds with specific habitats. After a while, the divergence of gene repertoires becomes linear with phylogenetic distance, presumably reflecting the continuous turnover of mobile element and the occasional acquisition of adaptive genes. Surprisingly, the phylogroups with smallest genomes have the highest rates of gene repertoire diversification and fewer but more diverse mobile genetic elements. This suggests that smaller genomes are associated with higher, not lower, turnover of genetic information. Many of these genomes are from freshwater isolates and have peculiar traits, including a specific capsule, suggesting adaptation to this environment. Altogether, these data contribute to explain why epidemiological clones tend to emerge from specific phylogenetic groups in the presence of pervasive horizontal gene transfer across the species.


Assuntos
Escherichia coli/genética , Evolução Molecular , Transferência Genética Horizontal , Variação Genética , Genoma Bacteriano/genética , Animais , Animais Selvagens/microbiologia , Austrália , Galinhas/microbiologia , Farmacorresistência Bacteriana/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Fezes/microbiologia , Água Doce/microbiologia , Tamanho do Genoma , Humanos , Doenças Inflamatórias Intestinais/microbiologia , Sequências Repetitivas Dispersas/genética , Mucosa Intestinal/microbiologia , Carne/microbiologia , Anotação de Sequência Molecular , Filogenia , Microbiologia do Solo , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
8.
Sci Total Environ ; 727: 138520, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32330714

RESUMO

Exposure of environmental bacteria to antibiotics may be increasing the global resistome. Antibiotic residues are entrained into agricultural soil through the application of animal and human wastes, and irrigation with reclaimed water. The impact of a mixture of three macrolide antibiotics on the abundance of selected genes associated with antibiotic resistance and genetic mobility were determined in a long-term field experiment undertaken in London, Canada. Replicated plots received annual applications of a mixture of erythromycin, clarithromycin and azithromycin every spring since 2010. Each antibiotic was added directly to the soil at a concentration of either 0.1 or 10 mg kg soil-1 and all plots were cropped to soybeans. By means of qPCR, no gene targets were enriched in soil exposed to the 0.1 mg kg soil-1 dose compared to untreated control. In contrast, the relative abundance of several gene targets including int1, sul2 and mphE increased significantly with the annual exposure to the 10 mg kg soil-1 dose. By means of high-throughput qPCR, numerous gene targets associated with resistance to aminoglycosides, sulfonamides, trimethoprim, streptomycin, quaternary ammonium chemicals as well as mobile genetic elements (tnpA, IS26 and IS6100) were detected in soil exposed to 10 mg kg soil-1, but not the lower dose. Overall, exposure of soil to macrolide antibiotics increased the relative abundance of numerous gene targets associated with resistance to macrolides and other antibiotics, and mobile genetic elements. This occurred at an exposure dose that is unrealistically high, but did not occur at the lower more realistic exposure dose.


Assuntos
Antibacterianos/farmacologia , Solo , Animais , Canadá , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Humanos , Sequências Repetitivas Dispersas , Londres , Macrolídeos , Microbiologia do Solo
9.
Braz J Infect Dis ; 24(3): 231-238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32325019

RESUMO

INTRODUCTION: Carbapenem resistance in members of order Enterobacterales is a growing public health problem causing high mortality in developing and industrialized countries. Its emergence and rapid propagation worldwide was due to both intercontinental spread of pandemic strains and horizontal dissemination via mobile genetic elements (MGE) such as plasmids and transposons. OBJECTIVE: To describe MGE carrying carbapenem resistance genes in Enterobacterales which have been reported in South America. SEARCH STRATEGY AND SELECTION CRITERIA: A search of the literature in English or Spanish published until 2019 in PubMed, Google Scholar, LILACS and SciELO databases was performed for studies of MGE in Enterobacterales reported in South American countries. RESULTS: Seven South American countries reported MGE related to carbapenemases. Carbapenemase-producing Klebsiella pneumoniae belonging to clonal complex 258 were the most prevalent pathogens reported; others carbapenemase-producing Enterobacterales such as Escherichia coli, Serratia marcescens, and Providencia rettgeri also have been reported. The MGE implicated in the spread of the most prevalent carbapenemase genes are Tn4401 and non-Tn4401 elements for blaKPC and ISAba125 for blaNDM, located in different plasmid incompatibility groups, i.e. L/M, A/C, FII and bacterial clones. CONCLUSION: This review indicates that, like in other parts of the world, the most commonly reported carbapenemases in Enterobacterales from South America are being disseminated through clones, plasmids, and transposons which have been previously reported in other parts of the world.


Assuntos
Proteínas de Bactérias/genética , beta-Lactamases/genética , Enterobacteriaceae , Sequências Repetitivas Dispersas , Klebsiella pneumoniae , Plasmídeos , América do Sul
10.
Ecotoxicol Environ Saf ; 197: 110612, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32302860

RESUMO

In this paper, we deciphered the core resistome disseminating from hospital wastewater to the aquatic environment by characterising the resistome, plasmidome, mobilome and virulome using metagenomic analysis. This study also elucidated different environmental resistome risks using shotgun-metagenomic assembly. The results showed that clinically relevant taxa were found in assessed matrices (Salmonella spp., Acinetobacter spp, Escherichia-Shigella spp., Pseudomonas spp., Staphylococcus spp. and Vibrio spp.). For the plasmidome, we found 249 core plasmidome sequences that were shared among all assessed matrices. The core mobilome of 2424 mobile genetic elements shared among all assessed matrices was found. Regarding the virulome, we found 148 core virulence factors shared among all assessed samples, and the core virulome content was consistently shared across the most abundant bacterial genera. Although influent of wastewater showed considerable higher relative bacterial abundance (P = 0.008), hospital wastewater showed significant higher environmental resistome risk scores against all other assessed matrices, with an average of 46.34% (P = 0.001). These results suggest hospital wastewater, effluent and sewage sludge should be subjected to stringent mitigating measures to minimise such dissemination.


Assuntos
Bactérias/genética , Bactérias/patogenicidade , Águas Residuárias/microbiologia , Poluentes da Água/toxicidade , Bactérias/isolamento & purificação , Farmacorresistência Bacteriana/genética , Hospitais , Humanos , Sequências Repetitivas Dispersas , Metagenômica , Plasmídeos/genética , Medição de Risco , Esgotos/microbiologia , Fatores de Virulência , Águas Residuárias/toxicidade
11.
Proc Natl Acad Sci U S A ; 117(11): 6264-6273, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32132208

RESUMO

Auxotrophies constrain the interactions of bacteria with their environment, but are often difficult to identify. Here, we develop an algorithm (AuxoFind) using genome-scale metabolic reconstruction to predict auxotrophies and apply it to a series of available genome sequences of over 1,300 Gram-negative strains. We identify 54 auxotrophs, along with the corresponding metabolic and genetic basis, using a pangenome approach, and highlight auxotrophies conferring a fitness advantage in vivo. We show that the metabolic basis of auxotrophy is species-dependent and varies with 1) pathway structure, 2) enzyme promiscuity, and 3) network redundancy. Various levels of complexity constitute the genetic basis, including 1) deleterious single-nucleotide polymorphisms (SNPs), in-frame indels, and deletions; 2) single/multigene deletion; and 3) movement of mobile genetic elements (including prophages) combined with genomic rearrangements. Fourteen out of 19 predictions agree with experimental evidence, with the remaining cases highlighting shortcomings of sequencing, assembly, annotation, and reconstruction that prevent predictions of auxotrophies. We thus develop a framework to identify the metabolic and genetic basis for auxotrophies in Gram-negatives.


Assuntos
Metabolismo Energético/genética , Genoma Bacteriano/fisiologia , Bactérias Gram-Negativas/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Modelos Biológicos , Algoritmos , Simulação por Computador , Genômica , Sequências Repetitivas Dispersas/genética , Redes e Vias Metabólicas/genética , Metabolômica , Nutrientes/metabolismo
12.
RNA ; 26(5): 664-673, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32127385

RESUMO

Group II introns are mobile genetic elements that perform both self-splicing and intron mobility reactions. These ribozymes are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Group IIC intron ribozymes have a minimal RNA architecture, and splice almost exclusively through hydrolysis in ribozyme reactions. Addition of the IEP allows the splicing reaction to form branched lariat RNPs capable of intron mobility. Here we examine ribozyme splicing, IEP-dependent splicing, and mobility reactions of a group IIC intron from the thermophilic bacterium Thermoanerobacter italicus (Ta.it.I1). We show that Ta.it.I1 is highly active for ribozyme activity, forming linear hydrolytic intron products. Addition of purified IEP switches activity to the canonical lariat forming splicing reaction. We demonstrate that the Ta.it.I1 group IIC intron coordinates the progression of the forward splicing reaction through a π-π' interaction between intron domains II and VI. We further show that branched splicing is supported in the absence of the IEP when the π-π' interaction is mutated. We also investigated the regulation of the two steps of reverse splicing during intron mobility into DNA substrates. Using a fluorescent mobility assay that simultaneously visualizes all steps of intron integration into DNA, we show that completion of reverse splicing is tightly coupled to cDNA synthesis regardless of mutation of the π-π' interaction.


Assuntos
Processamento de RNA/genética , RNA Catalítico/genética , RNA/genética , Ribonucleoproteínas/genética , Éxons/genética , Sequências Repetitivas Dispersas/genética , Íntrons/genética , Mutação/genética , Conformação de Ácido Nucleico , Ribonucleoproteínas/química
13.
Nucleic Acids Res ; 48(8): 4256-4273, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32187370

RESUMO

Repression of cellular reprogramming in germ cells is critical to maintaining cell fate and fertility. When germ cells mis-express somatic genes they can be directly converted into other cell types, resulting in loss of totipotency and reproductive potential. Identifying the molecular mechanisms that coordinate these cell fate decisions is an active area of investigation. Here we show that RNAi pathways play a key role in maintaining germline gene expression and totipotency after heat stress. By examining transcriptional changes that occur in mut-16 mutants, lacking a key protein in the RNAi pathway, at elevated temperature we found that genes normally expressed in the soma are mis-expressed in germ cells. Furthermore, these genes displayed increased chromatin accessibility in the germlines of mut-16 mutants at elevated temperature. These findings indicate that the RNAi pathway plays a key role in preventing aberrant expression of somatic genes in the germline during heat stress. This regulation occurs in part through the maintenance of germline chromatin, likely acting through the nuclear RNAi pathway. Identification of new pathways governing germ cell reprogramming is critical to understanding how cells maintain proper gene expression and may provide key insights into how cell identity is lost in some germ cell tumors.


Assuntos
Caenorhabditis elegans/genética , Células Germinativas/metabolismo , Resposta ao Choque Térmico/genética , Interferência de RNA , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Sequências Repetitivas Dispersas , Mutação , Oogênese/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Espermatogênese/genética , Regulação para Cima
14.
Nucleic Acids Res ; 48(6): e36, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32067044

RESUMO

Alu retrotransposons account for more than 10% of the human genome, and insertions of these elements create structural variants segregating in human populations. Such polymorphic Alus are powerful markers to understand population structure, and they represent variants that can greatly impact genome function, including gene expression. Accurate genotyping of Alus and other mobile elements has been challenging. Indeed, we found that Alu genotypes previously called for the 1000 Genomes Project are sometimes erroneous, which poses significant problems for phasing these insertions with other variants that comprise the haplotype. To ameliorate this issue, we introduce a new pipeline - TypeTE - which genotypes Alu insertions from whole-genome sequencing data. Starting from a list of polymorphic Alus, TypeTE identifies the hallmarks (poly-A tail and target site duplication) and orientation of Alu insertions using local re-assembly to reconstruct presence and absence alleles. Genotype likelihoods are then computed after re-mapping sequencing reads to the reconstructed alleles. Using a high-quality set of PCR-based genotyping of >200 loci, we show that TypeTE improves genotype accuracy from 83% to 92% in the 1000 Genomes dataset. TypeTE can be readily adapted to other retrotransposon families and brings a valuable toolbox addition for population genomics.


Assuntos
Sequências Repetitivas Dispersas/genética , Mutagênese Insercional/genética , Software , Sequenciamento Completo do Genoma/métodos , Bases de Dados Genéticas , Frequência do Gene/genética , Loci Gênicos , Genética Populacional , Genoma Humano , Genótipo , Humanos
15.
Nucleic Acids Res ; 48(7): 3906-3921, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32025726

RESUMO

RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site - contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.


Assuntos
Proteínas de Drosophila/metabolismo , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Argonauta/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Fertilidade/genética , Sequências Repetitivas Dispersas , Masculino , Mutação , Domínios Proteicos , RNA Helicases/metabolismo , Interferência de RNA , Transporte de RNA , RNA Antissenso , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/química , Ribonuclease III/metabolismo , Transcrição Genética
16.
Bioresour Technol ; 301: 122723, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31962245

RESUMO

During the municipal solid waste (MSW) composting, antibiotic resistance genes (ARGs) could be one of the concerns to hinder the application of MSW composting. However, the understanding of enrichment and dissemination of ARGs during the industrial-scale composting is still not clear. Hence, this study aimed to investigate the ARG distributions at different stages in an industrial-scale MSW composting plant. Seven target ARGs and four target mobile genetic elements (MGEs) and bacterial communities were investigated. The abundances of ARGs and MGEs increased during two aerobic thermophilic stages, but they decreased in most ARGs and MGEs after composting. Network analysis showed that potential host bacteria of ARGs were mainly Firmicutes and Actinobacteria. The reduction of potential host bacteria was important to remove ARGs. MGEs were an important factor hindering ARG removal. Water-extractable S and pH were two main physicochemical factors in the changes of microbial community and the abundance of ARGs.


Assuntos
Compostagem , Antibacterianos , Resistência Microbiana a Medicamentos , Genes Bacterianos , Sequências Repetitivas Dispersas , Esterco , Resíduos Sólidos
17.
Biochim Biophys Acta Gene Regul Mech ; 1863(2): 194489, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31935527

RESUMO

Bacteria exhibit an amazing diversity of mechanisms controlling gene expression to both maintain essential functions and modulate accessory functions in response to environmental cues. Over the years, it has become clear that bacterial regulation of gene expression is still far from fully understood. This review focuses on antisense RNAs (asRNAs), a class of RNA regulators defined by their location in cis and their perfect complementarity with their targets, as opposed to small RNAs (sRNAs) which act in trans with only short regions of complementarity. For a long time, only few functional asRNAs in bacteria were known and were almost exclusively found on mobile genetic elements (MGEs), thus, their importance among the other regulators was underestimated. However, the extensive application of transcriptomic approaches has revealed the ubiquity of asRNAs in bacteria. This review aims to present the landscape of studied asRNAs in bacteria by comparing 67 characterized asRNAs from both Gram-positive and Gram-negative bacteria. First we describe the inherent ambiguity in the existence of asRNAs in bacteria, second, we highlight their diversity and their involvement in all aspects of bacterial life. Finally we compare their location and potential mode of action toward their target between Gram-negative and Gram-positive bacteria and present tendencies and exceptions that could lead to a better understanding of asRNA functions.


Assuntos
Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , RNA Antissenso/fisiologia , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Sequências Repetitivas Dispersas , RNA Antissenso/metabolismo
18.
Nat Commun ; 11(1): 95, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911609

RESUMO

The flow of genetic material between bacteria is central to the adaptation and evolution of bacterial genomes. However, our knowledge about DNA transfer within complex microbiomes is lacking, with most studies of horizontal gene transfer (HGT) relying on bioinformatic analyses of genetic elements maintained on evolutionary timescales or experimental measurements of phenotypically trackable markers. Here, we utilize the CRISPR-Cas spacer acquisition process to detect DNA acquisition events from complex microbiota in real-time and at nucleotide resolution. In this system, an E. coli recording strain is exposed to a microbial sample and spacers are acquired from transferred plasmids and permanently stored in genomic CRISPR arrays. Sequencing and analysis of acquired spacers enables identification of the transferred plasmids. This approach allowed us to identify individual mobile elements without relying on phenotypic markers or post-transfer replication. We found that HGT into the recording strain in human clinical fecal samples can be extensive and is driven by different plasmid types, with the IncX type being the most actively transferred.


Assuntos
DNA Bacteriano/genética , Escherichia coli/genética , Microbioma Gastrointestinal , Sequências Repetitivas Dispersas , Sistemas CRISPR-Cas , Biologia Computacional , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Fezes/microbiologia , Transferência Genética Horizontal , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo
19.
Genome Biol ; 21(1): 16, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31973766

RESUMO

BACKGROUND: Transposable elements (TEs) make up half of mammalian genomes and shape genome regulation by harboring binding sites for regulatory factors. These include binding sites for architectural proteins, such as CTCF, RAD21, and SMC3, that are involved in tethering chromatin loops and marking domain boundaries. The 3D organization of the mammalian genome is intimately linked to its function and is remarkably conserved. However, the mechanisms by which these structural intricacies emerge and evolve have not been thoroughly probed. RESULTS: Here, we show that TEs contribute extensively to both the formation of species-specific loops in humans and mice through deposition of novel anchoring motifs, as well as to the maintenance of conserved loops across both species through CTCF binding site turnover. The latter function demonstrates the ability of TEs to contribute to genome plasticity and reinforce conserved genome architecture as redundant loop anchors. Deleting such candidate TEs in human cells leads to the collapse of conserved loop and domain structures. These TEs are also marked by reduced DNA methylation and bear mutational signatures of hypomethylation through evolutionary time. CONCLUSIONS: TEs have long been considered a source of genetic innovation. By examining their contribution to genome topology, we show that TEs can contribute to regulatory plasticity by inducing redundancy and potentiating genetic drift locally while conserving genome architecture globally, revealing a paradigm for defining regulatory conservation in the noncoding genome beyond classic sequence-level conservation.


Assuntos
Cromossomos de Mamíferos/química , Sequências Repetitivas Dispersas , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Cromatina/química , Humanos , Camundongos
20.
Sci Rep ; 10(1): 1392, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996747

RESUMO

Genotyping methods and genome sequencing are indispensable to reveal genomic structure of bacterial species displaying high level of genome plasticity. However, reconstruction of genome or assembly is not straightforward due to data complexity, including repeats, mobile and accessory genetic elements of bacterial genomes. Moreover, since the solution to this problem is strongly influenced by sequencing technology, bioinformatics pipelines, and selection criteria to assess assemblers, there is no systematic way to select a priori the optimal assembler and parameter settings. To assembly the genome of Pseudomonas aeruginosa strain AG1 (PaeAG1), short reads (Illumina) and long reads (Oxford Nanopore) sequencing data were used in 13 different non-hybrid and hybrid approaches. PaeAG1 is a multiresistant high-risk sequence type 111 (ST-111) clone that was isolated from a Costa Rican hospital and it was the first report of an isolate of P. aeruginosa carrying both blaVIM-2 and blaIMP-18 genes encoding for metallo-ß-lactamases (MBL) enzymes. To assess the assemblies, multiple metrics regard to contiguity, correctness and completeness (3C criterion, as we define here) were used for benchmarking the 13 approaches and select a definitive assembly. In addition, annotation was done to identify genes (coding and RNA regions) and to describe the genomic content of PaeAG1. Whereas long reads and hybrid approaches showed better performances in terms of contiguity, higher correctness and completeness metrics were obtained for short read only and hybrid approaches. A manually curated and polished hybrid assembly gave rise to a single circular sequence with 100% of core genes and known regions identified, >98% of reads mapped back, no gaps, and uniform coverage. The strategy followed to obtain this high-quality 3C assembly is detailed in the manuscript and we provide readers with an all-in-one script to replicate our results or to apply it to other troublesome cases. The final 3C assembly revealed that the PaeAG1 genome has 7,190,208 bp, a 65.7% GC content and 6,709 genes (6,620 coding sequences), many of which are included in multiple mobile genomic elements, such as 57 genomic islands, six prophages, and two complete integrons with blaVIM-2 and blaIMP-18 MBL genes. Up to 250 and 60 of the predicted genes are anticipated to play a role in virulence (adherence, quorum sensing and secretion) or antibiotic resistance (ß-lactamases, efflux pumps, etc). Altogether, the assembly and annotation of the PaeAG1 genome provide new perspectives to continue studying the genomic diversity and gene content of this important human pathogen.


Assuntos
Biologia Computacional/métodos , Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano/genética , Pseudomonas aeruginosa/genética , Análise de Sequência de DNA/métodos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Sequências Repetitivas Dispersas/genética , Anotação de Sequência Molecular , Pseudomonas aeruginosa/efeitos dos fármacos
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