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1.
Nat Commun ; 11(1): 1052, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103016

RESUMO

It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Fosfatos de Dinucleosídeos/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Capuzes de RNA/genética , RNA Polimerases Dirigidas por DNA/genética , Metilação , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Bacteriano/genética
2.
Nat Commun ; 11(1): 450, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974350

RESUMO

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Transcrição Genética , Carbocianinas/química , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/química , Regiões Promotoras Genéticas , Fator sigma/química , Fator sigma/genética , Fator sigma/metabolismo , Imagem Individual de Molécula , Regiões Terminadoras Genéticas , Iniciação da Transcrição Genética , Terminação da Transcrição Genética
3.
Nat Commun ; 11(1): 448, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974358

RESUMO

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Transcrição Genética , Trifosfato de Adenosina/genética , Citidina Trifosfato/genética , DNA/genética , DNA/metabolismo , DNA Antissenso/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Imagem Individual de Molécula
4.
Nat Commun ; 11(1): 281, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941912

RESUMO

Yersinia pestis is transmitted from fleas to rodents when the bacterium develops an extensive biofilm in the foregut of a flea, starving it into a feeding frenzy, or, alternatively, during a brief period directly after feeding on a bacteremic host. These two transmission modes are in a trade-off regulated by the amount of biofilm produced by the bacterium. Here by investigating 446 global isolated Y. pestis genomes, including 78 newly sequenced isolates sampled over 40 years from a plague focus in China, we provide evidence for strong selection pressures on the RNA polymerase ω-subunit encoding gene rpoZ. We demonstrate that rpoZ variants have an increased rate of biofilm production in vitro, and that they evolve in the ecosystem during colder and drier periods. Our results support the notion that the bacterium is constantly adapting-through extended phenotype changes in the fleas-in response to climate-driven changes in the niche.


Assuntos
Proteínas de Bactérias/genética , Peste/microbiologia , Sifonápteros/microbiologia , Yersinia pestis/fisiologia , Animais , Biofilmes , Evolução Biológica , China , Clima , RNA Polimerases Dirigidas por DNA/genética , Reservatórios de Doenças , Ecossistema , Infestações por Pulgas , Variação Genética , Genoma Bacteriano , Interações Hospedeiro-Parasita , Interações Hospedeiro-Patógeno , Marmota/parasitologia , Fenótipo , Filogenia , Sciuridae/parasitologia , Seleção Genética , Sifonápteros/fisiologia , Yersinia pestis/genética
5.
Proc Natl Acad Sci U S A ; 117(6): 3185-3191, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31992637

RESUMO

A fundamental feature of life is that ribosomes read the genetic code in messenger RNA (mRNA) as triplets of nucleotides in a single reading frame. Mutations that shift the reading frame generally cause gene inactivation and in essential genes cause loss of viability. Here we report and characterize a +1-nt frameshift mutation, centrally located in rpoB, an essential gene encoding the beta-subunit of RNA polymerase. Mutant Escherichia coli carrying this mutation are viable and highly resistant to rifampicin. Genetic and proteomic experiments reveal a very high rate (5%) of spontaneous frameshift suppression occurring on a heptanucleotide sequence downstream of the mutation. Production of active protein is stimulated to 61-71% of wild-type level by a feedback mechanism increasing translation initiation. The phenomenon described here could have broad significance for predictions of phenotype from genotype. Several frameshift mutations have been reported in rpoB in rifampicin-resistant clinical isolates of Mycobacterium tuberculosis (Mtb). These mutations have never been experimentally validated, and no mechanisms of action have been proposed. This work shows that frameshift mutations in rpoB can be a mutational mechanism generating antibiotic resistance. Our analysis further suggests that genetic elements supporting productive frameshifting could rapidly evolve de novo, even in essential genes.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Proteínas de Escherichia coli/genética , Mutação da Fase de Leitura/genética , Genes Essenciais/genética , Escherichia coli/efeitos dos fármacos , Evolução Molecular , Rifampina/farmacologia
6.
Science ; 367(6474): 200-204, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31919223

RESUMO

Drug combinations are widely used in clinical practice to prevent the evolution of resistance. However, little is known about the effect of tolerance, a different mode of survival, on the efficacy of drug combinations for preventing the evolution of resistance. In this work, we monitored Staphylococcus aureus strains evolving in patients under treatment. We detected the rapid emergence of tolerance mutations, followed by the emergence of resistance, despite the combination treatment. Evolution experiments on the clinical strains in vitro revealed a new way by which tolerance promotes the evolution of resistance under combination treatments. Further experiments under different antibiotic classes reveal the generality of the effect. We conclude that tolerance is an important factor to consider in designing combination treatments that prevent the evolution of resistance.


Assuntos
Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Evolução Molecular , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/microbiologia , Antibacterianos/uso terapêutico , RNA Polimerases Dirigidas por DNA/genética , Daptomicina/farmacologia , Daptomicina/uso terapêutico , Quimioterapia Combinada , Humanos , Testes de Sensibilidade Microbiana , Mutação , Polimorfismo de Nucleotídeo Único , Rifampina/farmacologia , Rifampina/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Vancomicina/farmacologia , Vancomicina/uso terapêutico
7.
Nat Commun ; 11(1): 368, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953395

RESUMO

The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities - nucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (POD) and C-terminal domain (PCTD) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (PNTD) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.


Assuntos
Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , RNA Replicase/química , Vírus Sincicial Respiratório Humano/enzimologia , Proteínas Virais/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Fosfoproteínas/química , Conformação Proteica , Domínios Proteicos , RNA Replicase/genética , RNA Replicase/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Estruturas Virais
8.
Nucleic Acids Res ; 48(5): e28, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980824

RESUMO

We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive ß-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased ß-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.


Assuntos
Bioensaio , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Transcrição Genética , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Sistemas CRISPR-Cas , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Repressores Lac/deficiência , Repressores Lac/genética , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
9.
Nucleic Acids Res ; 48(4): 2144-2155, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31965171

RESUMO

Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak-trigger-freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.


Assuntos
Carbono-Nitrogênio Ligases/química , RNA Polimerases Dirigidas por DNA/química , RNA Bacteriano/química , Transcrição Genética , Bacillus subtilis/química , Bacillus subtilis/genética , Carbono-Nitrogênio Ligases/genética , Cristalografia por Raios X , DNA/química , DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Fator sigma/química , Fator sigma/genética , Uridina Trifosfato/química , Uridina Trifosfato/genética
10.
Arch Virol ; 165(1): 21-31, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31624917

RESUMO

To obtain insight into the sequence diversity of strawberry latent ringspot virus (SLRSV), isolates from collections and diagnostic samples were sequenced by high-throughput sequencing. For five SLRSV isolates, the complete genome sequences were determined, and for 18 other isolates nearly complete genome sequences were determined. The sequence data were analysed in relation to sequences of SLRSV and related virus isolates available in the NCBI GenBank database. The genome sequences were annotated, and sequences of the protease-polymerase (Pro-Pol) region and coat proteins (CPs) (large and small CP together) were used for phylogenetic analysis. The amino acid sequences of the Pro-Pol region were very similar, whereas the nucleotide sequences of this region were more variable. The amino acid sequences of the CPs were less similar, which was corroborated by the results of a serological comparison performed using antisera raised against different isolates of SLRSV. Based on these results, we propose that SLRSV and related unassigned viruses be assigned to a new genus within the family Secoviridae, named "Stralarivirus". Based on the phylogenetic analysis, this genus should include at least three viruses, i.e., SLRSV-A, SLRSV-B and lychnis mottle virus. The newly generated sequence data provide a basis for designing molecular tests to screen for SLRSV.


Assuntos
Fragaria/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Secoviridae/classificação , Análise de Sequência de RNA/métodos , Proteínas do Capsídeo/genética , RNA Polimerases Dirigidas por DNA/genética , Variação Genética , Anotação de Sequência Molecular , Peptídeo Hidrolases/genética , Filogenia , Vírus de Plantas/classificação , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , RNA Viral/genética , Secoviridae/genética , Secoviridae/isolamento & purificação
11.
Proc Natl Acad Sci U S A ; 117(2): 1181-1190, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879355

RESUMO

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5' hammerhead and 3' ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(-)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.


Assuntos
DNA Complementar/genética , Tospovirus/genética , Tospovirus/fisiologia , Tospovirus/patogenicidade , RNA Polimerases Dirigidas por DNA/genética , Vírus Delta da Hepatite/genética , Proteínas do Nucleocapsídeo/genética , Doenças das Plantas/virologia , RNA Catalítico/genética , RNA Viral/genética , Replicon , Tabaco/virologia , Proteínas Virais/genética , Vírion/genética , Vírion/metabolismo , Replicação Viral
12.
Int J Food Microbiol ; 312: 108374, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31669765

RESUMO

Salmonella enterica outbreaks in sprouts originate from contaminated seeds; conventional prevention technologies have been reported from many research institutes. In this study, we applied a biological control approach to inhibit S. enterica growth using the seed-dwelling non-antagonistic bacteria. We isolated non-antibacterial seed-dwelling bacteria from vegetable sprouts. A total of 206 bacteria exhibiting non-antibacterial activity against S. enterica were subjected to alfalfa sprout development tests. Eight isolates exhibiting no deleterious effect on the growth of alfalfa sprouts were tested for S. enterica growth inhibition on alfalfa seeds and sprouts, and an isolate EUS78 was finally selected for further investigation. Based on 16S rRNA, gyrB, and rpoB gene sequence analyses, strain EUS78 was identified as Erwinia persicina. In population competition, the S. enterica population increased by >3 log CFU/g after 6 days of alfalfa sprout growth, whereas S. enterica growth was significantly inhibited by treatment with EUS78 (P < .05). This effect of S. enterica growth inhibition by EUS78 was sustained until the end of the alfalfa sprout harvest. Overall, bacterial strain EUS78 significantly reduced S. enterica growth on alfalfa sprouts in a manner consistent with competitive exclusion. These findings led us to monitor EUS78 behavior on seeds during early sprout development using fluorescence and scanning electron microscopy. Strain EUS78 initially colonized alfalfa sprout seed coat edges, cotyledons, and finally root surfaces during early sprout germination. As alfalfa sprouts grew, EUS78 bacterial cells established colonies on newly emerged plant tissues such as root tips. The results of this study suggest that strain EUS78 has potential as a biological control agent to inhibit S. enterica contamination in the sprout food industry.


Assuntos
Antibiose/fisiologia , Agentes de Controle Biológico , Erwinia/fisiologia , Medicago sativa/microbiologia , Salmonella enterica/crescimento & desenvolvimento , Sementes/microbiologia , DNA Girase/genética , RNA Polimerases Dirigidas por DNA/genética , Erwinia/genética , Microbiologia de Alimentos , Indústria de Processamento de Alimentos , Germinação/fisiologia , Medicago sativa/química , RNA Ribossômico 16S/genética , Verduras/microbiologia
13.
BMC Infect Dis ; 19(1): 1047, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31823734

RESUMO

BACKGROUND: Molecular tests can allow the rapid detection of tuberculosis (TB) and multidrug-resistant TB (MDR-TB). TB-SPRINT 59-Plex Beamedex® is a microbead-based assay developed for the simultaneous spoligotyping and detection of MDR-TB. The accuracy and cost evaluation of new assays and technologies are of great importance for their routine use in clinics and in research laboratories. The aim of this study was to evaluate the performance of TB-SPRINT at three laboratory research centers in Brazil and calculate its mean cost (MC) and activity-based costing (ABC). METHODS: TB-SPRINT data were compared with the phenotypic and genotypic profiles obtained using Bactec™ MGIT™ 960 system and Genotype® MTBDRplus, respectively. RESULTS: Compared with MGIT, the accuracies of TB-SPRINT for the detection of rifampicin and isoniazid resistance ranged from 81 to 92% and 91.3 to 93.9%, respectively. Compared with MTBDRplus, the accuracies of TB-SPRINT for rifampicin and isoniazid were 99 and 94.2%, respectively. Moreover, the MC and ABC of TB-SPRINT were USD 127.78 and USD 109.94, respectively. CONCLUSION: TB-SPRINT showed good results for isoniazid and rifampicin resistance detection, but still needs improvement to achieve In Vitro Diagnostics standards.


Assuntos
Farmacorresistência Bacteriana , Citometria de Fluxo/métodos , Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Catalase/genética , Custos e Análise de Custo , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Citometria de Fluxo/economia , Genótipo , Humanos , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Regiões Promotoras Genéticas , Kit de Reagentes para Diagnóstico , Rifampina , Sensibilidade e Especificidade , Tuberculose/economia
14.
PLoS Negl Trop Dis ; 13(12): e0007990, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31877135

RESUMO

BACKGROUND: Leptospirosis is a widespread zoonotic disease that causes reproductive losses and/or hepatorenal failure in a number of animal species. Wild reservoirs of the disease, such as rodents, harbor the causative bacterium, Leptospira spp., in their kidneys and contaminate the environment by excreting infected urine. In this study, we tested small wild mammals, environmental water, and livestock in the Cumberland Gap region of southeastern Appalachia for the presence of pathogenic Leptospira or leptospiral antibodies. METHODS/RESULTS: Small wild mammals (n = 101) and environmental water samples (n = 89) were screened by a real time quantitative PCR that targets the pathogenic Leptospira-specific lipl32 gene. Kidneys from 63 small wild mammals (62.37%) and two water sources (2.25%) tested positive for leptospiral DNA. To identify the infecting leptospiral species in qPCR-positive water and kidney samples, a fragment of leptospiral rpoB gene was PCR amplified and sequenced. L. kirschneri and L. interrogans were the leptospiral species carried by small wild mammals. Furthermore, sera from livestock (n = 52; cattle and horses) were screened for leptospiral antibodies using microscopic agglutination test (MAT). Twenty sera (38.46%) from livestock had antibodies to one or more serovars of pathogenic Leptospira spp. CONCLUSIONS: In conclusion, results from our study show exposure to leptospiral infection in farm animals and the presence of this zoonotic pathogen in the environmental water and kidneys of a significant number of small wild mammals. The public health implications of these findings remain to be assessed.


Assuntos
Animais Domésticos , Leptospira/isolamento & purificação , Leptospirose/veterinária , Roedores , Microbiologia da Água , Animais , Região dos Apalaches/epidemiologia , Proteínas da Membrana Bacteriana Externa/genética , RNA Polimerases Dirigidas por DNA/genética , Rim/microbiologia , Leptospira/classificação , Leptospira/genética , Leptospirose/epidemiologia , Leptospirose/microbiologia , Lipoproteínas/genética , Reação em Cadeia da Polimerase em Tempo Real
15.
PLoS Genet ; 15(12): e1008492, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31887137

RESUMO

DNA cytosine methylation is an epigenetic mark associated with silencing of transposable elements (TEs) and heterochromatin formation. In plants, it occurs in three sequence contexts: CG, CHG, and CHH (where H is A, T, or C). The latter does not allow direct inheritance of methylation during DNA replication due to lack of symmetry, and methylation must therefore be re-established every cell generation. Genome-wide association studies (GWAS) have previously shown that CMT2 and NRPE1 are major determinants of genome-wide patterns of TE CHH methylation. Here we instead focus on CHH methylation of individual TEs and TE-families, allowing us to identify the pathways involved in CHH methylation simply from natural variation and confirm the associations by comparing them with mutant phenotypes. Methylation at TEs targeted by the RNA-directed DNA methylation (RdDM) pathway is unaffected by CMT2 variation, but is strongly affected by variation at NRPE1, which is largely responsible for the longitudinal cline in this phenotype. In contrast, CMT2-targeted TEs are affected by both loci, which jointly explain 7.3% of the phenotypic variation (13.2% of total genetic effects). There is no longitudinal pattern for this phenotype, however, because the geographic patterns appear to compensate for each other in a pattern suggestive of stabilizing selection.


Assuntos
Arabidopsis/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Elementos de DNA Transponíveis , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Arabidopsis/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Loci Gênicos , Variação Genética , Estudo de Associação Genômica Ampla , Fenótipo , Análise de Sequência de DNA
16.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(5): 629-634, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31762229

RESUMO

OBJECTIVE: To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53. METHODS: The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hygS were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants. RESULTS: The recombinant plasmid pJV53-SacB-hygS were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully. CONCLUSION: pJV53-SacB-hygS can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.


Assuntos
Farmacorresistência Bacteriana/genética , Recombinação Homóloga , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Plasmídeos/genética , Rifampina/farmacologia
17.
Int J Food Microbiol ; 311: 108356, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31670141

RESUMO

Different samples of three products including Bikalga and Soumbala from Burkina Faso (West Africa) and Ntoba Mbodi from Congo-Brazzaville (Central Africa) were evaluated. The bacteria (400) were phenotyped and genotypically characterized by Rep-PCR, PFGE, 16S rRNA and rpoB gene sequencing and spa typing. Their PFGE profiles were compared with those of 12,000 isolates in the Center for Disease Control (CDC, USA) database. They were screened for the production of enterotoxins, susceptibility to 19 antimicrobials, presence of 12 staphylococcal toxin and 38 AMR genes and the ability to transfer erythromycin and tetracycline resistance genes to Enterococcus faecalis JH2-2. Fifteen coagulase negative (CoNS) and positive (CoPS) species characterized by 25 Rep-PCR/PFGE clusters were identified: Staphylococcus arlettae, S. aureus, S. cohnii, S. epidermidis, S. gallinarum, S. haemolyticus, S. hominis, S. pasteuri, S. condimenti, S. piscifermentans, S. saprophyticus, S. sciuri, S. simulans, S. warneri and Macrococcus caseolyticus. Five species were specific to Soumbala, four to Bikalga and four to Ntoba Mbodi. Two clusters of S. gallinarum and three of S. sciuri were particular to Burkina Faso. The S. aureus isolates exhibited a spa type t355 and their PFGE profiles did not match any in the CDC database. Bacteria from the same cluster displayed similar AMR and toxin phenotypes and genotypes, whereas clusters peculiar to a product or a location generated distinct profiles. The toxin genes screened were not detected and the bacteria did not produce the staphylococcal enterotoxins A, B, C and D. AMR genes including blazA, cat501, dfr(A), dfr(G), mecA, mecA1, msr(A) and tet(K) were identified in CoNS and CoPS. Conjugation experiments produced JH2-2 isolates that acquired resistance to erythromycin and tetracycline, but no gene transfer was revealed by PCR. The investigation of the heterogeneity of Staphylococcus species from alkaline fermented foods, their relationship with clinical and environmental isolates and their safety in relation to antimicrobial resistance (AMR) and toxin production is anticipated to contribute to determining the importance of staphylococci in alkaline fermented foods, especially in relation to the safety of the consumers.


Assuntos
Enterotoxinas/genética , Staphylococcus , Antibacterianos/farmacologia , Burkina Faso , Coagulase/genética , Congo , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Eritromicina/farmacologia , Genótipo , Testes de Sensibilidade Microbiana , Fenótipo , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Staphylococcus/classificação , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Staphylococcus/isolamento & purificação , Tetraciclina/farmacologia
18.
Parasit Vectors ; 12(1): 495, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640746

RESUMO

BACKGROUND: Our study aimed to assess the diversity of the species of Anaplasmataceae in Senegal that infect animals and ticks in three areas: near Keur Momar Sarr (northern region), Dielmo and Diop (Sine Saloum, central region of Senegal), and in Casamance (southern region of Senegal). METHODS: A total of 204 ticks and 433 blood samples were collected from ruminants, horses, donkeys and dogs. Ticks were identified morphologically and by molecular characterization targeting the 12S rRNA gene. Molecular characterization of species of Anaplasmataceae infecting Senegalese ticks and animals was conducted using the 23S rRNA, 16S rRNA, rpoB and groEL genes. RESULTS: Ticks were identified as Rhipicephalus evertsi evertsi (84.3%), Hyalomma rufipes (8.3%), Hyalomma impeltatum (4.9%), R. bursa (1.5%) and R. muhsamae (0.9%). The overall prevalence of Anaplasmataceae infection in ticks was 0.9%, whereas 41.1% of the sampled animals were found infected by one of the species belonging to this family. We identified the pathogen Anaplasma ovis in 55.9% of sheep, A. marginale and A. centrale in 19.4% and 8.1%, respectively, of cattle, as well as a putative new species of Anaplasmataceae. Two Anaplasma species commonly infecting ruminants were identified. Anaplasma cf. platys, closely related to A. platys was identified in 19.8% of sheep, 27.7% of goats and 22.6% of cattle, whereas a putative new species, named here provisionally "Candidatus Anaplasma africae", was identified in 3.7% of sheep, 10.3% of goats and 8.1% of cattle. Ehrlichia canis and Anaplasma platys were identified only from dogs sampled in the Keur Momar Sarr area. Ehrlichia canis was identified in 18.8% of dogs and two R. e. evertsi ticks removed from the same sheep. Anaplasma platys was identified in 15.6% of dogs. Neither of the dogs sampled from Casamance region nor the horses and donkeys sampled from Keur Momar Sarr area were found infected by an Anaplasmataceae species. CONCLUSIONS: This study presents a summary of Anaplasmataceae species that infect animals and ticks in three areas from the northern, central and southern regions of Senegal. To our knowledge, our findings demonstrate for the first time the presence of multiple Anaplasmataceae species that infect ticks and domestic animals in Senegal. We recorded two potentially new species commonly infecting ruminants named here provisionally as Anaplasma cf. platys and "Candidatus Anaplasma africae". However, E. canis was the only species identified and amplified from ticks. None of the other Anaplasmataceae species identified in animals were identified in the tick species collected from animals.


Assuntos
Infecções por Anaplasmataceae/veterinária , Anaplasmataceae/classificação , Anaplasmataceae/genética , Animais Domésticos/microbiologia , Carrapatos/microbiologia , Infecções por Anaplasmataceae/microbiologia , Animais , Animais Domésticos/parasitologia , Bovinos , Chaperonina 60/genética , DNA Ribossômico/sangue , DNA Ribossômico/química , DNA Ribossômico/isolamento & purificação , RNA Polimerases Dirigidas por DNA/genética , Doenças do Cão/microbiologia , Doenças do Cão/parasitologia , Cães , Equidae/microbiologia , Equidae/parasitologia , Feminino , Variação Genética , Cabras , Doenças dos Cavalos/microbiologia , Doenças dos Cavalos/parasitologia , Cavalos , Masculino , Filogenia , RNA Ribossômico/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Ruminantes/microbiologia , Ruminantes/parasitologia , Senegal , Alinhamento de Sequência/veterinária , Ovinos , Infestações por Carrapato/complicações , Infestações por Carrapato/veterinária
19.
Nat Commun ; 10(1): 3916, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477705

RESUMO

Transcription by RNA polymerase V (Pol V) in plants is required for RNA-directed DNA methylation, leading to transcriptional gene silencing. Global chromatin association of Pol V requires components of the DDR complex DRD1, DMS3 and RDM1, but the assembly process of this complex and the underlying mechanism for Pol V recruitment remain unknown. Here we show that all DDR complex components co-localize with Pol V, and we report the cryoEM structures of two complexes associated with Pol V recruitment-DR (DMS3-RDM1) and DDR' (DMS3-RDM1-DRD1 peptide), at 3.6 Å and 3.5 Å resolution, respectively. RDM1 dimerization at the center frames the assembly of the entire complex and mediates interactions between DMS3 and DRD1 with a stoichiometry of 1 DRD1:4 DMS3:2 RDM1. DRD1 binding to the DR complex induces a drastic movement of a DMS3 coiled-coil helix bundle. We hypothesize that both complexes are functional intermediates that mediate Pol V recruitment.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA de Plantas/química , RNA de Plantas/genética
20.
Int J Mol Sci ; 20(18)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546962

RESUMO

RNA viruses are known to replicate by low fidelity polymerases and have high mutation rates whereby the resulting virus population tends to exist as a distribution of mutants. In this review, we aim to explore how genetic events such as spontaneous mutations could alter the genomic organization of RNA viruses in such a way that they impact virus replications and plaque morphology. The phenomenon of quasispecies within a viral population is also discussed to reflect virulence and its implications for RNA viruses. An understanding of how such events occur will provide further evidence about whether there are molecular determinants for plaque morphology of RNA viruses or whether different plaque phenotypes arise due to the presence of quasispecies within a population. Ultimately this review gives an insight into whether the intrinsically high error rates due to the low fidelity of RNA polymerases is responsible for the variation in plaque morphology and diversity in virulence. This can be a useful tool in characterizing mechanisms that facilitate virus adaptation and evolution.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Evolução Molecular , Quase-Espécies/genética , Vírus de RNA , Proteínas Virais/genética , Animais , Humanos , Vírus de RNA/genética , Vírus de RNA/patogenicidade , Virulência
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