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1.
PLoS One ; 15(10): e0231562, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33006967

RESUMO

One of the adaptive strategies for the constantly changing conditions of the environment utilized in bacterial cells involves the condensation of DNA in complex with the DNA-binding protein, Dps. With the use of electron microscopy and electron tomography, we observed several morphologically different types of DNA condensation in dormant Escherichia coli cells, namely: nanocrystalline, liquid crystalline, and the folded nucleosome-like. We confirmed the presence of both Dps and DNA in all of the ordered structures using EDX analysis. The comparison of EDX spectra obtained for the three different ordered structures revealed that in nanocrystalline formation the majority of the Dps protein is tightly bound to nucleoid DNA. The dps-null cells contained only one type of condensed DNA structure, liquid crystalline, thus, differing from those with Dps. The results obtained here shed some light on the phenomenon of DNA condensation in dormant prokaryotic cells and on the general problem of developing a response to stress. We demonstrated that the population of dormant cells is structurally heterogeneous, allowing them to respond flexibly to environmental changes. It increases the ability of the whole bacterial population to survive under extreme stress conditions.


Assuntos
DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/crescimento & desenvolvimento , Empacotamento do DNA , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Microscopia Eletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Estresse Fisiológico
2.
Nucleic Acids Res ; 48(19): 11016-11029, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33035310

RESUMO

Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing an open structure, but how the initiator participates in this process and the contributions of the several binding sites remain unclear. Here, we show that the initiator protein of Vibrio cholerae specific to chromosome 2 (Chr2) also has single-strand binding activity in the AT-rich region of its origin. Binding is strand specific, depends on repeats of the sequence 5'ATCA and is greatly stabilized in vitro by specific double-stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single- and double-stranded sites. An IHF site lies between these two kinds of sites in the Chr2 origin and an IHF-induced looping out of the intervening DNA mediates their interaction. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin.


Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Vibrio cholerae/genética , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Origem de Replicação
3.
Nat Commun ; 11(1): 4947, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009392

RESUMO

Pseudomonas syringae is a Gram-negative and model pathogenic bacterium that causes plant diseases worldwide. Here, we set out to identify binding motifs for all 301 annotated transcription factors (TFs) of P. syringae using HT-SELEX. We successfully identify binding motifs for 100 TFs. We map functional interactions between the TFs and their targets in virulence-associated pathways, and validate many of these interactions and functions using additional methods such as ChIP-seq, electrophoretic mobility shift assay (EMSA), RT-qPCR, and reporter assays. Our work identifies 25 virulence-associated master regulators, 14 of which had not been characterized as TFs before.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Pseudomonas syringae/metabolismo , Fatores de Transcrição/metabolismo , Sistemas de Secreção Bacterianos , Sítios de Ligação , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Multimerização Proteica , Pseudomonas syringae/patogenicidade , Reprodutibilidade dos Testes , Técnica de Seleção de Aptâmeros , Virulência
4.
BMC Bioinformatics ; 21(1): 415, 2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-32962628

RESUMO

BACKGROUND: In silico promoter prediction represents an important challenge in bioinformatics as it provides a first-line approach to identifying regulatory elements to support wet-lab experiments. Historically, available promoter prediction software have focused on sigma factor-associated promoters in the model organism E. coli. As a consequence, traditional promoter predictors yield suboptimal predictions when applied to other prokaryotic genera, such as Pseudomonas, a Gram-negative bacterium of crucial medical and biotechnological importance. RESULTS: We developed SAPPHIRE, a promoter predictor for σ70 promoters in Pseudomonas. This promoter prediction relies on an artificial neural network that evaluates sequences on their similarity to the - 35 and - 10 boxes of σ70 promoters found experimentally in P. aeruginosa and P. putida. SAPPHIRE currently outperforms established predictive software when classifying Pseudomonas σ70 promoters and was built to allow further expansion in the future. CONCLUSIONS: SAPPHIRE is the first predictive tool for bacterial σ70 promoters in Pseudomonas. SAPPHIRE is free, publicly available and can be accessed online at www.biosapphire.com . Alternatively, users can download the tool as a Python 3 script for local application from this site.


Assuntos
Biologia Computacional/métodos , Redes Neurais de Computação , Regiões Promotoras Genéticas , Pseudomonas/genética , Fator sigma/metabolismo , DNA Bacteriano/metabolismo , Software
5.
Adv Exp Med Biol ; 1267: 45-58, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894476

RESUMO

In this chapter, we will focus on ParABS: an apparently simple, three-component system, required for the segregation of bacterial chromosomes and plasmids. We will specifically describe how biophysical measurements combined with physical modeling advanced our understanding of the mechanism of ParABS-mediated complex assembly, segregation and positioning.


Assuntos
Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , Posicionamento Cromossômico , DNA Bacteriano/metabolismo , Plasmídeos/metabolismo
6.
Int J Food Microbiol ; 335: 108891, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-32977153

RESUMO

Antimicrobial peptides are being explored for use as food preservatives to prevent foodborne diseases. In this study, bioinformatics tools were used to screen potential antimicrobial amino acid sequences from the whey acidic protein (WAP) of large yellow croaker (Larimichthys crocea). A novel antimicrobial peptide, designated as LCWAP, was identified and its antimicrobial effect and mechanism of action on Staphylococcus aureus was explored. The minimal inhibitory concentration (MIC) of LCWAP on S. aureus was 15.6 µg/mL. Transmission electron microscopy and laser confocal microscopy revealed that LCWAP kills bacteria by aggregating on the cell surface, destroying the integrity of bacterial cell membrane and resulting in the leakage of intracellular solutes. Moreover, peptide LCWAP inhibit biofilm formation, at concentrations of 1-1/16 × MIC, with biofilm formation found to decrease by 94.3%-13.7% upon LCWAP treatment. The ability of peptide LCWAP to bind bacteria DNA was revealed using electrophoresis analysis and ultraviolet absorption spectroscopy, with peptide LCWAP/DNA weight ratios of 125/1, and 17.3% decrease in the absorption peak of LCWAP. Furthermore, LCWAP had no cytotoxic effects on normal human hepatocytes, although it had strong inhibitory effect on S. aureus growth in milk.


Assuntos
Antibacterianos/farmacologia , Proteínas do Leite/farmacologia , Leite/microbiologia , Perciformes , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Conservantes de Alimentos/química , Conservantes de Alimentos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Proteínas do Leite/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Staphylococcus aureus/crescimento & desenvolvimento
7.
Nat Commun ; 11(1): 3888, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753666

RESUMO

First proposed as antimicrobial agents, histones were later recognized for their role in condensing chromosomes. Histone antimicrobial activity has been reported in innate immune responses. However, how histones kill bacteria has remained elusive. The co-localization of histones with antimicrobial peptides (AMPs) in immune cells suggests that histones may be part of a larger antimicrobial mechanism in vivo. Here we report that histone H2A enters E. coli and S. aureus through membrane pores formed by the AMPs LL-37 and magainin-2. H2A enhances AMP-induced pores, depolarizes the bacterial membrane potential, and impairs membrane recovery. Inside the cytoplasm, H2A reorganizes bacterial chromosomal DNA and inhibits global transcription. Whereas bacteria recover from the pore-forming effects of LL-37, the concomitant effects of H2A and LL-37 are irrecoverable. Their combination constitutes a positive feedback loop that exponentially amplifies their antimicrobial activities, causing antimicrobial synergy. More generally, treatment with H2A and the pore-forming antibiotic polymyxin B completely eradicates bacterial growth.


Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Estruturas Cromossômicas/efeitos dos fármacos , Histonas/metabolismo , Prótons , Animais , Estruturas Cromossômicas/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Sinergismo Farmacológico , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Imunidade Inata , Mamíferos , Polimixina B/farmacologia , Análise de Sequência de RNA , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(36): 21889-21895, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32820079

RESUMO

DNA glycosylase is responsible for repairing DNA damage to maintain the genome stability and integrity. However, how glycosylase can efficiently and accurately recognize DNA lesions across the enormous DNA genome remains elusive. It has been hypothesized that glycosylase translocates along the DNA by alternating between a fast but low-accuracy diffusion mode and a slow but high-accuracy mode when searching for DNA lesions. However, the slow mode has not been successfully characterized due to the limitation in the spatial and temporal resolutions of current experimental techniques. Using a newly developed scanning fluorescence resonance energy transfer (FRET)-fluorescence correlation spectroscopy (FCS) platform, we were able to observe both slow and fast modes of glycosylase AlkD translocating on double-stranded DNA (dsDNA), reaching the temporal resolution of microsecond and spatial resolution of subnanometer. The underlying molecular mechanism of the slow mode was further elucidated by Markov state model built from extensive all-atom molecular dynamics simulations. We found that in the slow mode, AlkD follows an asymmetric diffusion pathway, i.e., rotation followed by translation. Furthermore, the essential role of Y27 in AlkD diffusion dynamics was identified both experimentally and computationally. Our results provided mechanistic insights on how conformational dynamics of AlkD-dsDNA complex coordinate different diffusion modes to accomplish the search for DNA lesions with high efficiency and accuracy. We anticipate that the mechanism adopted by AlkD to search for DNA lesions could be a general one utilized by other glycosylases and DNA binding proteins.


Assuntos
Bacillus cereus/genética , Proteínas de Bactérias/química , DNA Glicosilases/química , Bacillus cereus/química , Bacillus cereus/enzimologia , Bacillus cereus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Cadeias de Markov , Simulação de Dinâmica Molecular , Espectrometria de Fluorescência , Especificidade por Substrato
9.
BMC Infect Dis ; 20(1): 587, 2020 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-32770954

RESUMO

BACKGROUND: Tuberculosis (TB) is transmitted in bioaerosols containing Mycobacterium tuberculosis (Mtb). Despite being central to ongoing TB transmission, no routine diagnostic assay exists to measure Mtb in bioaerosols. Furthermore, published studies of Mtb in bioaerosol samples have been limited to individuals with sputum-positive pulmonary TB. Notably, TB diagnosis is based on clinical symptoms and sputum laboratory findings. This is despite the fact that approximately half of all patients commencing TB treatment are sputum-negative, resulting in a high proportion of presumptive treatments. Here, we propose to use a sensitive air sampling protocol to investigate the prevalence of Mtb-containing bioaerosols in both sputum-positive and sputum-negative TB suspects, at the same time evaluating the potential to identify unrecognized transmitters of TB. METHODS: Our parallel-group design will identify viable Mtb in bioaerosols produced by individuals attending a TB clinic in South Africa. Sampling will be performed on eligible individuals presenting with symptoms indicative of TB and repeated at 14 days if initially positive. Participants will be prospectively classified into three distinct groups based on National TB Control Program (NTBCP) criteria: Group A, TB notification with sputum-based laboratory confirmation; Group B, TB notification with empiric diagnosis; and Group C, individuals not notified. Group C individuals with detectable Mtb bioaerosol will be monitored until resolution of clinical and laboratory status. Collection of bioaerosol specimens will be via two consecutive sampling modalities: (1) direct sampling following a specific respiratory manoeuvre; and (2) indirect sampling during passive respiratory activity. Bioaerosol specimens will be analyzed for viable Mtb using DMN-trehalose staining and live-cell fluorescence microscopy. Mtb genomes and mycobacterial and host lipids will be detected using droplet digital PCR and mass spectrometry analyses, respectively. The primary objective is to determine the prevalence of Mtb bioaerosols in all TB clinic attendees and in each of the groups. Secondary objectives are to investigate differences in prevalence of Mtb bioaerosol by HIV status and current isoniazid preventive therapy (IPT) use; we will also determine the impact of anti-TB chemotherapy on Mtb-containing bioaerosol production. DISCUSSION: Respiratory bioaerosol has a potential role in non-invasive TB diagnosis, infectivity measurement and treatment monitoring. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04241809 . Date of Registration: 27/1/2020.


Assuntos
Aerossóis/análise , Manejo de Espécimes/métodos , Tuberculose Pulmonar/diagnóstico , Adulto , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase , África do Sul , Escarro/microbiologia
10.
PLoS One ; 15(8): e0237466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790762

RESUMO

In the past decade, two leptospirosis outbreaks occurred among strawberry harvesters in Germany, with 13, and 45 reported cases respectively. In both outbreaks, common voles (Microtus arvalis) infected with Leptospira kischneri serovar Grippotyphosa were identified as the most likely outbreak source. In an univariate analysis, eating unwashed strawberries was identified as one of the risk factors associated with Leptospira infection. The aim of this study was to evaluate the survival time of L. kirschneri serovar Grippotyphosa on strawberries under varying conditions. Strawberries were spiked with 5x109 of both a laboratory reference strain (strain Moskva V) and an outbreak field strain (94-6/2007) of L. kirschneri serovar Grippotyphosa sequence type 110. Survival times were investigated in a fully crossed design with three incubation times (2h, 4h, 6h and 8h) and three temperatures (15°C, 21°C and 25°C) with three replicated for each condition. A wash protocol was developed and recovered Leptospira were determined by qPCR, dark field microscopy and culturing. Viable L. kirschneri of both the reference strain and the field strain were identified in all samples at 25°C and an incubation time of 2h, but only 1/9 (11%) and 4/9 (44%) of the samples incubated at 15°C were positive, respectively. Both reference and field strain were viable only in 2/9 (22%) at 25° after 6h. After an 8h incubation, viable Leptospira could not be identified on the surface of the strawberries or within the fruit for any of the tested conditions. Based on these results, the exposure risk of consumers to viable Leptospira spp. through the consumption of strawberries bought at the retail level is most likely very low. However, there is a potential risk of Leptospira infection by consumption of strawberries on pick-your-own farms.


Assuntos
Fragaria/microbiologia , Leptospira/fisiologia , DNA Bacteriano/metabolismo , Frutas/microbiologia , Alemanha/epidemiologia , Humanos , Leptospira/genética , Leptospirose/epidemiologia , Leptospirose/patologia , Microscopia , Reação em Cadeia da Polimerase em Tempo Real , Sorogrupo , Temperatura , Fatores de Tempo
11.
Mol Cell ; 79(5): 797-811.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32750314

RESUMO

Pausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a "stepping" mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a "scrunching" mechanism, has not. We report a method that directly defines the RNAP-active-center position relative to DNA with single-nucleotide resolution (XACT-seq; "crosslink-between-active-center-and-template sequencing"). We apply this method to detect and quantify pausing in initial transcription at 411 (∼4,000,000) promoter sequences in vivo in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4 to 5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard coded by sequence elements similar to those for transcription-elongation pausing.


Assuntos
DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Domínio Catalítico , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transcrição Genética
12.
PLoS One ; 15(8): e0237135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822422

RESUMO

DNA-binding Transcription Factors (TFs) play a central role in regulation of gene expression in prokaryotic organisms, and similarities at the sequence level have been reported. These proteins are predicted with different abundances as a consequence of genome size, where small organisms contain a low proportion of TFs and large genomes contain a high proportion of TFs. In this work, we analyzed a collection of 668 experimentally validated TFs across 30 different species from diverse taxonomical classes, including Escherichia coli K-12, Bacillus subtilis 168, Corynebacterium glutamicum, and Streptomyces coelicolor, among others. This collection of TFs, together with 111 hidden Markov model profiles associated with DNA-binding TFs collected from diverse databases such as PFAM and DBD, was used to identify the repertoire of proteins putatively devoted to gene regulation in 1321 representative genomes of Archaea and Bacteria. The predicted regulatory proteins were posteriorly analyzed in terms of their genomic context, allowing the prediction of functions for TFs and their neighbor genes, such as genes involved in virulence, enzymatic functions, phosphorylation mechanisms, and antibiotic resistance. The functional analysis associated with PFAM groups showed diverse functional categories were significantly enriched in the collection of TFs and the proteins encoded by the neighbor genes, in particular, small-molecule binding and amino acid transmembrane transporter activities associated with the LysR family and proteins devoted to cellular aromatic compound metabolic processes or responses to drugs, stress, or abiotic stimuli in the MarR family. We consider that with the increasing data derived from new technologies, novel TFs can be identified and help improve the predictions for this class of proteins in complete genomes. The complete collection of experimentally characterized and predicted TFs is available at http://web.pcyt.unam.mx/EntrafDB/.


Assuntos
Archaea/genética , Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli K12/genética , Fatores de Transcrição/genética , Archaea/patogenicidade , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Arqueal/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli K12/patogenicidade , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Genoma Arqueal , Genoma Bacteriano , Ligação Proteica , Fatores de Transcrição/metabolismo , Virulência/genética
13.
Nucleic Acids Res ; 48(17): 9571-9588, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32813023

RESUMO

Iron is essential for all bacteria. In most bacteria, intracellular iron homeostasis is tightly regulated by the ferric uptake regulator Fur. However, how Fur activates the iron-uptake system during iron deficiency is not fully elucidated. In this study, we found that YdiV, the flagella gene inhibitor, is involved in iron homeostasis in Escherichia coli. Iron deficiency triggers overexpression of YdiV. High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner. Thus, the cooperation of YdiV, SlyD and Fur activates the gene expression of iron-uptake systems under conditions of iron deficiency. Bacterial invasion assays also demonstrated that both ydiV and slyD are necessary for the survival and growth of uropathogenic E. coli in bladder epithelial cells. This reveals a mechanism where YdiV not only represses flagella expression to make E. coli invisible to the host immune system, but it also promotes iron acquisition to help E. coli overcome host nutritional immunity.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Repressoras/metabolismo , Escherichia coli Uropatogênica/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Linhagem Celular , DNA Bacteriano/metabolismo , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Homeostase , Humanos , Peptidilprolil Isomerase/genética , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Bexiga Urinária/microbiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Escherichia coli Uropatogênica/metabolismo
14.
Mol Cell ; 79(3): 416-424.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645367

RESUMO

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
15.
PLoS One ; 15(7): e0235703, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678857

RESUMO

INTRODUCTION: Diagnosis of pertussis is challenging especially in infants. Most low and middle-income countries (LMIC) lack resources for laboratory confirmation, relying largely on clinical diagnosis alone for both case management and surveillance. This necessitates robust clinical case definitions. OBJECTIVES: This study assesses the accuracy of clinical case definitions with and without lymphocytosis in diagnosing pertussis in children with severe lower respiratory tract infection (LRTI) in a LMIC setting. METHODS: Children hospitalized with severe LRTI in a South African hospital were prospectively enrolled and evaluated for pertussis using PCR on respiratory samples. Clinical signs and differential white cell counts were recorded. Sensitivity and specificity of pertussis clinical diagnosis using WHO and Global Pertussis Initiative (GPI) criteria; and with addition of lymphocytosis were assessed with PCR as the reference standard. RESULTS: 458 children <10 years were enrolled. Bordetella pertussis infection was confirmed in 32 (7.0%). For WHO criteria, sensitivity was 78.1% (95% CI 60.7-89.2%) and specificity 15.5% (95% CI 12.4-19.3%); for GPI sensitivity was 34.4% (95% CI 20.1-52.1) and specificity 64.8% (95% CI 60.1-69.2%). Area under the curve (AUC) on receiver operating character (ROC) analysis was 0.58 (95% CI 0.46-0.70 for WHO criteria, and 0.72 (95% CI 0.56-0.88) for GPI with highest likelihood ratios of 5.33 and 4.42 respectively. Diagnostic accuracy was highest between five and seven days of symptoms for both criteria. Lymphocytosis had sensitivity of 31.3% (95% CI 17.5-49.3%) and specificity of 70.7% (95% CI 66.1-74.8%) and showed a marginal impact on improving clinical criteria. CONCLUSION: Clinical criteria lack accuracy for diagnosis and surveillance of pertussis. Non-outbreak settings should consider shorter durations in clinical criteria. New recommendations still fall short of what is required for a viable clinical screening test which means the need to improve access to laboratory diagnostic support remains crucial.


Assuntos
Infecções Respiratórias/diagnóstico , Coqueluche/diagnóstico , Área Sob a Curva , Bordetella pertussis/genética , Bordetella pertussis/isolamento & purificação , Criança , Pré-Escolar , DNA Bacteriano/metabolismo , Feminino , Humanos , Lactente , Linfocitose/diagnóstico , Masculino , Reação em Cadeia da Polimerase , Curva ROC , Infecções Respiratórias/microbiologia , Sensibilidade e Especificidade , Coqueluche/microbiologia
16.
Proc Natl Acad Sci U S A ; 117(31): 18540-18549, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675239

RESUMO

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imagem Individual de Molécula , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
17.
Nat Commun ; 11(1): 3576, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681021

RESUMO

CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , DNA Bacteriano/metabolismo , Streptococcus pyogenes/enzimologia , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Cinética , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo
18.
PLoS One ; 15(6): e0234751, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555719

RESUMO

Perinatal factors can shape fecal microbiome patterns among pregnant women and their infants. However, there is scarce information about the effect of maternal demographics and perinatal exposures on antibiotic resistance genes (ARG) and mobile genetic element (MGE) patterns in pregnant women and infants. We examined fecal samples from pregnant women during their third trimester of pregnancy (n = 51) and 6-month-old infants (n = 40). Of the 91 participants, 72 represented 36 maternal-infant dyads, 15 were additional pregnant women, and 4 were additional infants. We assessed the effects of demographics, pre-pregnancy BMI, smoking and parity in the pregnancy resistome and the effects of demographics, delivery mode, feeding habits and prenatal antibiotic treatment on the infancy resistome. ARG and MGE richness and abundance were assessed using a SmartChip qPCR-array. Alpha diversity (Shannon and Inverse Simpson index) and beta diversity (Sorensen and Bray-Curtis index) were calculated. The Wilcoxon and the Kruskal non-parametric test were used for comparisons. There is a high variability in shared resistome patterns between pregnant women and their infants. An average of 29% of ARG and 24% of MGE were shared within dyads. Infants had significantly greater abundance and higher diversity of ARG and MGE compared to pregnant women. Pregnancy and infancy samples differed in ARG and MGE gene composition and structure. Composition of the fecal resistome was significantly associated with race in pregnant women, with non-white women having different patterns than white women, and, in infants, with extent of solid food consumption. Our data showed that the pregnancy and infancy resistome had different structure and composition patterns, with maternal race and infant solid food consumption as possible contributors to ARG. By characterizing resistome patterns, our results can inform the mechanism of antibiotic resistome development in pregnant women and their infants.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Fezes/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Antibacterianos/uso terapêutico , Bactérias/genética , Bactérias/isolamento & purificação , Índice de Massa Corporal , Aleitamento Materno , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Feminino , Humanos , Lactente , Paridade , Gravidez , Terceiro Trimestre da Gravidez , Análise de Componente Principal , Fatores de Risco , Fatores Sexuais , Fumar
19.
BMC Infect Dis ; 20(1): 356, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32517758

RESUMO

BACKGROUND: Vancomycin-resistant Enterococcus spp. (VRE) have spread all over the world. The present study aims to investigate the species distribution, specimen type and susceptibilities of Enterococcal species collected from Nanjing Drum Tower Hospital from 2013 to 2018. Additionally, distribution of VRE and prevalence of van gene among VRE isolates were also analyzed. METHODS: The susceptibilities of 3913 Enterococcus isolates were retrospectively investigated. Among these strains, 60 VRE strains were further anazlyed in this study. The minimum inhibitory concentrations (MICs) of the VRE strains towards vancomycin, teicoplanin and linezolid were determined by E-test. Polymerase chain reaction (PCR) and DNA sequencing were used to investigate the prevalence of van genes among VRE. Furthermore, the sequence types (STs) of VRE strains were explored by multi-locus sequence typing (MLST). RESULTS: Among the 3913 enterococci isolates, Enterococcus faecalis (n = 1870, 47.8%) and Enterococcus faecium (1738, 44.4%) were the main isolates. These Enterococcus strains were mainly isolated from urine (n = 1673, 42.8%), followed by secretions (n = 583, 14.9%) and ascites (n = 554, 14.2%). VRE displayed a decreasing trend year by year. Molecular analysis revealed that 49 out of 60 VRE isolates carried vanA gene, 10 carried vanM, and 1 carried both vanA and vanM genes. Sixteen distinct STs were identified among the 58 VREM, with ST78 (n = 16), ST192 (n = 8) and ST570 (n = 7) being the most dominant ones. CONCLUSIONS: E. faecalis and E. faecium were the major enterococci strains which are the main pathogens of urinary traction infections; vanA and vanM were the main determinants conferring resistance to vancomycin; ST78, ST192 and ST570 were the leading STs of VREM which displayed a decreasing trend of prevalence year by year.


Assuntos
Enterococcus/isolamento & purificação , Infecções por Bactérias Gram-Positivas/microbiologia , Enterococos Resistentes à Vancomicina/isolamento & purificação , Antibacterianos/farmacologia , China , DNA Bacteriano/metabolismo , Enterococcus/efeitos dos fármacos , Enterococcus/genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Infecções por Bactérias Gram-Positivas/diagnóstico , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Estudos Retrospectivos , Centros de Atenção Terciária , Vancomicina/farmacologia , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/genética
20.
PLoS One ; 15(6): e0234119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492051

RESUMO

Recently, a new rapid assay for the detection of tcdB gene of Clostridioides difficile was developed using the GENECUBE. The assay can directly detect the tcdB gene from stool samples without a purification in approximately 35 minutes with a few minutes of preparation process. We performed a prospective comparative study of the performance of the assay at eight institutions in Japan. Fresh residual stool samples (Bristol stool scale ≥5) were used and comparisons were performed with the BD MAX Cdiff assay and toxigenic cultures. For the evaluation of 383 stool samples compared with the BD MAX Cdiff assay, the sensitivity, and specificity of the two assays was 99.0% (379/383), 98.1% (52/53), 99.1% (327/330), respectively. In the comparison with toxigenic culture, the total, sensitivity, and specificity were 96.6% (370/383), 85.0% (51/60), and 98.8% (319/323), respectively. The current investigation indicated the GENECUBE Clostridioides difficile assay has equivalent performance with the BD MAX Cdiff assay for the detection of tcdB gene of C. difficile.


Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridium difficile/genética , Fezes/microbiologia , Infecções por Clostridium/diagnóstico , Clostridium difficile/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enterotoxinas/genética , Humanos , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Proteínas Repressoras/genética , Sensibilidade e Especificidade
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