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1.
Proc Natl Acad Sci U S A ; 116(28): 14222-14227, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239340

RESUMO

Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or "mating" channel, the channel-pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.


Assuntos
Membrana Celular/ultraestrutura , Escherichia coli/ultraestrutura , Fator F/ultraestrutura , Fímbrias Bacterianas/ultraestrutura , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Membrana Celular/genética , Conjugação Genética/genética , Microscopia Crioeletrônica , Citoplasma/genética , Citoplasma/ultraestrutura , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fator F/genética , Fímbrias Bacterianas/genética , Sistemas de Secreção Tipo IV/genética
2.
Science ; 364(6442): 778-782, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31123134

RESUMO

Drug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor-conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action.


Assuntos
Proteínas de Transporte/fisiologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Fator F/fisiologia , Antibacterianos/farmacologia , Antiporters/antagonistas & inibidores , Antiporters/biossíntese , Antiporters/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Conjugação Genética , DNA de Cadeia Simples , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fator F/genética , Microscopia , Biossíntese de Proteínas/efeitos dos fármacos , Tetraciclina/farmacologia
3.
Sci Total Environ ; 655: 263-272, 2019 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-30471594

RESUMO

Two groups of coliphages have been recently included in different water management policies as indicators of viral fecal pollution in water and food: somatic coliphages, which infect E. coli through cell wall receptors, and F-specific RNA coliphages, which infect through the F-pili. Somatic coliphages are more abundant in fecally contaminated waters, except reclaimed waters, those disinfected by UV irradiation, and some groundwater samples that show a higher level of F-specific coliphages. Somatic coliphages are morphologically similar to DNA enteric viruses while F-specific coliphages are similar to RNA viruses such as norovirus and hepatitis A viruses, which are the viral pathogens of concern in sewage. The use of strains sensitive to both types of phages has been proposed for total coliphage enumeration, thereby avoiding double analysis. The standardized methods available for coliphage detection are robust and cost-effective, but the introduction of ready-to-use methods would facilitate routine implementation in laboratories. The fastest available tool for somatic coliphage enumeration is the recently developed Bluephage, which uses a modified ß-glucuronide-overexpressing E. coli strain unable to take up the glucuronide substrate. The overexpressed enzyme accumulates inside the bacterial cells until released by phage-induced cell lysis, whereupon it encounters its substrate and the medium changes from yellow to blue. The present method uses E. coli strain CB12, sensitive to somatic coliphages and F-specific coliphages due to the expression of the F-pili. The Bluephage approach incorporating CB12 detects both types of coliphages in a time range of 1:30 to 4:00 h, as assayed with coliphages from raw sewage, river water, sludge and mussels. This strategy can be applied to obtain qualitative and quantitative results and is applicable to microplates as well as to large sample volumes (100 ml). Moreover it can provide monitoring of water bodies at real time, as for example for ambient recreational beach monitoring.


Assuntos
Colífagos/isolamento & purificação , Monitoramento Ambiental/métodos , Escherichia coli/virologia , Fator F/genética , Fezes/virologia , Água Doce/virologia , Microbiologia da Água/normas , Colífagos/genética , Escherichia coli/genética , Genes Bacterianos , Plasmídeos , Fagos RNA/isolamento & purificação
4.
J Bacteriol ; 201(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30322855

RESUMO

The F plasmid tra operon encodes most of the proteins required for bacterial conjugation. TraJ and ArcA are known activators of the tra operon promoter PY, which is subject to H-NS-mediated silencing. Donor ability and promoter activity assays indicated that PY is inactivated by silencers and requires both TraJ and ArcA for activation to support efficient F conjugation. The observed low-level, ArcA-independent F conjugation is caused by tra expression from upstream alternative promoters. Electrophoretic mobility shift assays showed that TraJ alone weakly binds to PY regulatory DNA; however, TraJ binding is significantly enhanced by ArcA binding to the same DNA, indicating cooperativity of the two proteins. Analysis of binding affinities between ArcA and various DNA fragments in the PY regulatory region defined a 22-bp tandem repeat sequence (from -76 to -55 of PY) sufficient for optimal ArcA binding, which is immediately upstream of the predicted TraJ-binding site (from -54 to -34). Deletion analysis of the PY promoter in strains deficient in TraJ, ArcA, and/or H-NS determined that sequences upstream of -103 are required by silencers including H-NS for PY silencing, whereas sequences downstream of -77 are targeted by TraJ and ArcA for activation. TraJ and ArcA appear not only to counteract PY silencers but also to directly activate PY in a cooperative manner. Our data reveal the cooperativity of TraJ and ArcA during PY activation and provide insights into the regulatory circuit controlling F-family plasmid-mediated bacterial conjugation.IMPORTANCE Conjugation is a major mechanism for dissemination of antibiotic resistance and virulence among bacterial populations. The tra operon in the F family of conjugative plasmids encodes most of the proteins involved in bacterial conjugation. This work reveals that activation of tra operon transcription requires two proteins, TraJ and ArcA, to bind cooperatively to adjacent sites immediately upstream of the major tra promoter PY The interaction of TraJ and ArcA with the tra operon not only relieves PY from silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Conjugação Genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Fator F , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Repressoras/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Deleção de Genes , Regiões Promotoras Genéticas , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Repressoras/genética
5.
Biochem Biophys Res Commun ; 503(4): 2386-2392, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-29966652

RESUMO

Bacterial conjugation, such as that mediated by the E. coli F plasmid, is a main mechanism driving bacterial evolution. Two important proteins required for F-pilus assembly and DNA transfer proficiency are TraW and TrbC. As members of a larger complex, these proteins assemble into a type IV secretion system and are essential components of pore formation and mating pair stabilization between the donor and the recipient cells. In the current report, we demonstrate the physical interaction of TraW and TrbC, show that TraW preferentially interacts with the N-terminal domain of TrbC, and that this interaction is important in restoring conjugation in traW/trbC knockouts.


Assuntos
Conjugação Genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Fator F/genética , Mapas de Interação de Proteínas , Sequência de Aminoácidos , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fator F/metabolismo , Técnicas de Inativação de Genes , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência
6.
PLoS One ; 13(7): e0200164, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29965999

RESUMO

Accumulated evidence has suggested associations between glucose abnormalities and insulin resistance with hepatitis C virus (HCV) and hepatitis B virus (HBV) infections. However, few studies have reported the effect of hepatitis virus infections on body composition. Our aim was to explore the association of hepatitis virus infections with percent body fat (PBF) in a cross-sectional analysis. A total of 69226 subjects obtained from the health examinations at Tri-Service General Hospital (TSGH) from 2010 to 2016 were enrolled in the study. Participants were divided into subgroups based on the presence of hepatitis B surface antigen (HBsAg) and anti-HCV. PBF was measured by bioelectrical impedance analysis (BIA). A multivariable linear regression model was applied to test the association of hepatitis virus infections with PBF and glycemic status. In male participants, hepatitis virus infections were closely associated with increased PBF, especially in those subjects with HCV/HBV coinfection. HCV/HBV coinfection was positively correlated with fasting plasma glucose and postprandial glucose while HCV and HBV mono-infection were not. The impact of hepatitis virus infection on increased PBF was observed in general population with gender difference. A further study on the treatment of hepatitis virus infection might help prevent the development of obesity-related diseases.


Assuntos
Tecido Adiposo/patologia , Coinfecção/patologia , Hepatite B/patologia , Hepatite C/patologia , Tecido Adiposo/diagnóstico por imagem , Adulto , Glicemia , Composição Corporal , Coinfecção/sangue , Coinfecção/diagnóstico por imagem , Coinfecção/epidemiologia , Estudos Transversais , Fator F , Feminino , Hepatite B/sangue , Hepatite B/diagnóstico por imagem , Hepatite B/epidemiologia , Antígenos de Superfície da Hepatite B/sangue , Hepatite C/sangue , Hepatite C/diagnóstico por imagem , Anticorpos Anti-Hepatite C/sangue , Humanos , Masculino , Pessoa de Meia-Idade
7.
EcoSal Plus ; 8(1)2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30022749

RESUMO

The F plasmid or F-factor is a large, 100-kbp, circular conjugative plasmid of Escherichia coli and was originally described as a vector for horizontal gene transfer and gene recombination in the late 1940s. Since then, F and related F-like plasmids have served as role models for bacterial conjugation. At present, more than 200 different F-like plasmids with highly related DNA transfer genes, including those for the assembly of a type IV secretion apparatus, are completely sequenced. They belong to the phylogenetically related MOBF12A group. F-like plasmids are present in enterobacterial hosts isolated from clinical as well as environmental samples all over the world. As conjugative plasmids, F-like plasmids carry genetic modules enabling plasmid replication, stable maintenance, and DNA transfer. In this plasmid backbone of approximately 60 kbp, the DNA transfer genes occupy the largest and mostly conserved part. Subgroups of MOBF12A plasmids can be defined based on the similarity of TraJ, a protein required for DNA transfer gene expression. In addition, F-like plasmids harbor accessory cargo genes, frequently embedded within transposons and/or integrons, which harness their host bacteria with antibiotic resistance and virulence genes, causing increasingly severe problems for the treatment of infectious diseases. Here, I focus on key genetic elements and their encoded proteins present on the F-factor and other typical F-like plasmids belonging to the MOBF12A group of conjugative plasmids.


Assuntos
Conjugação Genética/genética , Resistência Microbiana a Medicamentos/genética , Fator F/genética , Genes Bacterianos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Escherichia coli/genética , Transferência Genética Horizontal , Integrons/genética , Análise de Sequência de DNA , Sistemas de Secreção Tipo IV/genética , Virulência , Fatores de Virulência/genética
8.
Nucleic Acids Res ; 46(14): 6962-6973, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-29986051

RESUMO

Discontinuities in only a single strand of the DNA duplex occur frequently, as a result of DNA damage or as intermediates in essential nuclear processes and DNA repair. Nicks are the simplest of these lesions: they carry clean ends bearing 3'-hydroxyl groups that can undergo ligation or prime new DNA synthesis. In contrast, single-strand breaks also interrupt only one DNA strand, but they carry damaged ends that require clean-up before subsequent steps in repair. Despite their apparent simplicity, nicks can have significant consequences for genome stability. The availability of enzymes that can introduce a nick almost anywhere in a large genome now makes it possible to systematically analyze repair of nicks. Recent experiments demonstrate that nicks can initiate recombination via pathways distinct from those active at double-strand breaks (DSBs). Recombination at targeted DNA nicks can be very efficient, and because nicks are intrinsically less mutagenic than DSBs, nick-initiated gene correction is useful for genome engineering and gene therapy. This review revisits some physiological examples of recombination at nicks, and outlines experiments that have demonstrated that nicks initiate homology-directed repair by distinctive pathways, emphasizing research that has contributed to our current mechanistic understanding of recombination at nicks in mammalian cells.


Assuntos
Dano ao DNA , Reparo de DNA por Recombinação , Variação Antigênica , Quebras de DNA de Cadeia Simples , Replicação do DNA , Escherichia coli/genética , Fator F/genética , Proteínas de Fímbrias/genética , Quadruplex G , Conversão Gênica , Elementos Nucleotídeos Longos e Dispersos , Saccharomyces cerevisiae/genética
9.
Child Psychiatry Hum Dev ; 49(5): 757-765, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29508120

RESUMO

Since after the second world war there has been an increasing number of studies investigating secular changes in adolescent mental health. Although no general trends could be outlined, the majority of studies show at least partial deterioration of psychological wellbeing from year 2000 on. Our study adds to this knowledge by exploring changes in self-declared emotional and behavioral problems in Poland, which is a part of post-communist Europe. In this paper, we compared responses on the Youth Self-Report by Polish 16-year-olds from 2000 and those from 2011. Two independent samples consisted of 259 (year 2000) and 185 (year 2011) 16-year-olds of both genders, drawn from randomized, normative, school-based groups. We analyzed linear, ordinal and binary logistic regression models. The results revealed that teenagers from 2011 reported more self-rated internalizing and total problems. Social and thought problems also rose significantly. Gender related time trends hint at a male increase in externalizing, aggressive behaviors and anxiety/depression. Caseness rose significantly in most scales with female gender being an additional risk factor for internalizing and total problems. No reduction in self-reported emotional and behavioral problems was detected.


Assuntos
Sintomas Afetivos/psicologia , Saúde Mental/estatística & dados numéricos , Comportamento Problema/psicologia , Adolescente , Agressão , Estudos Transversais , Depressão/diagnóstico , Depressão/epidemiologia , Fator F , Feminino , Humanos , Masculino , Polônia/epidemiologia , Fatores de Risco , Autorrelato , Problemas Sociais/psicologia , Problemas Sociais/estatística & dados numéricos
10.
J Virol Methods ; 250: 25-28, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28939117

RESUMO

Somatic and F+ coliphages have been identified and validated as virus indicators of fecal contamination in ground water by US EPA and more recently they are being considered for use in managing both marine and fresh recreational water and wastewater discharges. Studies documenting their usefulness as viral indicator in reclaimed water sources in the USA are limited. However, simultaneous detection of both somatic and F+ coliphages on a single E. coli host is preferred over their separate analysis because both are abundant in wastewater, they may respond differently to wastewater reclamation treatment processes, and separate analysis for each group in separate host bacteria adds complexity and cost. In this study, a new total coliphage host (E. coli CB390, CECT9198) was evaluated for its ability to detect somatic, F+ coliphages, and total coliphages by US EPA Methods 1601 and 1602. No statistical difference was found in the detection coliphages in spiked phosphate buffered saline samples or in natural waters; additionally, no statistical difference was found between the detection of total coliphages by Methods 1601 and 1602.


Assuntos
Colífagos/isolamento & purificação , Escherichia coli/virologia , Águas Residuárias/virologia , Microbiologia da Água , Colífagos/genética , Escherichia coli/isolamento & purificação , Fator F , Fezes/virologia
11.
J Am Heart Assoc ; 6(7)2017 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-28724655

RESUMO

BACKGROUND: Despite higher thromboembolism risk, women with atrial fibrillation have lower oral anticoagulation (OAC) use compared to men. The influence of the CHA2DS2-VASc score or the introduction of non-vitamin K OACs on this relationship is not known. METHODS AND RESULTS: Using the PINNACLE National Cardiovascular Data Registry from 2008 to 2014, we compared the association of sex with OAC use (warfarin or non-vitamin K OACs) overall and by CHA2DS2-VASc score and examined temporal trends in OAC use by sex. Multivariable regression models assessed the association between sex and OAC use in those with CHA2DS2-VASc scores ≥2. Temporal analyses assessed changes in OAC use by sex over time. Of the 691 906 atrial fibrillation patients, 48.5% were women. Women were significantly less likely than men to use any OAC overall (56.7% versus 61.3%; P<0.001) and at all levels of CHA2DS2-VASc score (adjusted risk ratio 9% to 33% lower, all P<0.001). Compared to other thromboembolic risk factors, female sex was associated with lower use of OAC (risk ratio 0.90, 95%CI 0.90-0.91). Over time, non-vitamin K OAC use increased at a slightly higher rate in women (56.2% increase per year, 95%CI 54.6% to 57.9%) compared to men (53.6% increase per year, 95%CI 52.0% to 55.2%), yet women remained less likely to receive any OAC at all time points (P<0.001). CONCLUSIONS: Among patients with atrial fibrillation, women were significantly less likely to receive OAC at all levels of the CHA2DS2-VASc score. Despite increasing non-vitamin K OAC use, women had persistently lower rates of OAC use compared to men over time.


Assuntos
Anticoagulantes/administração & dosagem , Fibrilação Atrial/tratamento farmacológico , Disparidades em Assistência à Saúde/tendências , Padrões de Prática Médica/tendências , Acidente Vascular Cerebral/prevenção & controle , Tromboembolia/prevenção & controle , Varfarina/administração & dosagem , Administração Oral , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/complicações , Fibrilação Atrial/diagnóstico , Distribuição de Qui-Quadrado , Técnicas de Apoio para a Decisão , Fator F , Feminino , Hemorragia/induzido quimicamente , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Sistema de Registros , Fatores de Risco , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/etiologia , Tromboembolia/diagnóstico , Tromboembolia/etiologia , Estados Unidos
12.
Sleep Med ; 36: 133-140, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28735911

RESUMO

OBJECTIVE: Gender and racial/ethnic disparities in sleep duration are well documented among the U.S. adult population, but we know little about how these disparities are shaped during the early course of adult life, a period marked by substantial changes in social roles that can influence time for sleep. METHODS: Prospective data was used from the National Longitudinal Survey of Youth 1997 (NLSY97), a U.S.-based representative sample of persons born between 1980 and 1984, who were first interviewed in 1997. Sleep duration was assessed in 2002, 2007/2008, 2009, 2010, and 2011. Random-coefficient models were estimated to examine gender and racial/ethnic disparities in trajectories of sleep duration across early adulthood as a function of educational experiences, employment, and family relationships. RESULTS: Sleep duration declined during early adulthood. Women reported shorter sleep than men from age 18 to 22, but slept longer than men by age 28. Black Young adults reported sleep durations similar to those of White young adults until age 24, after which blacks slept less than whites. Educational experiences and employment characteristics reduced gender and racial/ethnic disparities, but family relationships exacerbated them. CONCLUSION: This study is the first to establish the emergence of gender and racial/ethnic disparities in sleep duration during early adulthood.


Assuntos
Disparidades nos Níveis de Saúde , Sono , Adolescente , Adulto , Envelhecimento , Grupos de Populações Continentais , Escolaridade , Emprego , Grupos Étnicos , Fator F , Família , Feminino , Seguimentos , Humanos , Estudos Longitudinais , Masculino , Estudos Prospectivos , Fatores de Tempo , Estados Unidos , Adulto Jovem
13.
J Mol Recognit ; 30(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28470740

RESUMO

An essential protein for bacterial growth, GTPase-Obg (Obg), is known to play an unknown but crucial role in stress response as its expression increases in Mycobacterium under stress conditions. It is well reported that Obg interacts with anti-sigma-F factor Usfx; however, a detailed analysis and structural characterization of their physical interaction remain undone. In view of above-mentioned points, this study was conceptualized for performing binding analysis and structural characterization of Obg-Usfx interaction. The binding studies were performed by surface plasmon resonance, while in silico docking analysis was done to identify crucial residues responsible for Obg-Usfx interaction. Surface plasmon resonance results clearly suggest that N-terminal and G domains of Obg mainly contribute to Usfx binding. Also, binding constants display strong affinity that was further evident by intermolecular hydrogen bonds and hydrophobic interactions in the predicted complex. Strong interaction between Obg and Usfx supports the view that Obg plays an important role in stress response, essentially required for Mycobacterium survival. As concluded by various studies that Obg is crucial for Mycobacterium survival under stress, this structural information may help us in designing novel and potential inhibitors against resistant Mycobacterium strains.


Assuntos
Proteínas de Bactérias/química , Fator F/química , Proteínas de Ligação ao GTP/química , Mycobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Cromatografia em Gel , Simulação por Computador , Sistemas Computacionais , Fator F/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica
14.
Mol Microbiol ; 104(6): 905-915, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28370625

RESUMO

Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base-pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base-pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones. Candidates are proteins with homology to FinO, a factor that promotes base pairing between the FinP antisense sRNA and the traJ mRNA to control F plasmid transfer. Recent papers have shown that the Salmonella enterica FinO-domain protein ProQ binds a large suite of sRNAs, including the RaiZ sRNA, which represses translation of the hupA mRNA, and the Legionella pneumophila protein RocC binds the RocR sRNA, which blocks expression of competence genes. Here we discuss what is known about FinO-domain structures, including the recently solved Escherichia coli ProQ structure, as well as the RNA binding properties of this family of proteins and evidence they act as chaperones. We compare these properties with those of Hfq. We further summarize what is known about the physiological roles of FinO-domain proteins and enumerate outstanding questions whose answers will establish whether they constitute a second major class of RNA chaperones.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Fator F , Chaperonas Moleculares/metabolismo , Conformação de Ácido Nucleico , Domínios Proteicos , RNA Antissenso/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/fisiologia , Relação Estrutura-Atividade
15.
Plasmid ; 91: 53-60, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28359666

RESUMO

The R1 antibiotic resistance plasmid, originally discovered in a clinical Salmonella isolate in London, 1963, has served for decades as a key model for understanding conjugative plasmids. Despite its scientific importance, a complete sequence of this plasmid has never been reported. We present the complete genome sequence of R1 along with a brief review of the current knowledge concerning its various genetic systems and a comparison to the F and R100 plasmids. R1 is 97,566 nucleotides long and contains 120 genes. The plasmid consists of a backbone largely similar to that of F and R100, a Tn21-like transposon that is nearly identical to that of R100, and a unique 9-kb sequence that bears some resemblance to sequences found in certain Klebsiella oxytoca strains. These three regions of R1 are separated by copies of the insertion sequence IS1. Overall, the structure of R1 and comparison to F and R100 suggest a fairly stable shared conjugative plasmid backbone into which a variety of mobile elements have inserted to form an "accessory" genome, containing multiple antibiotic resistance genes, transposons, remnants of phage genes, and genes whose functions remain unknown.


Assuntos
Mapeamento Cromossômico , Conjugação Genética , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/genética , Fatores R/química , Salmonella/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Elementos de DNA Transponíveis , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Fator F/química , Fator F/metabolismo , Klebsiella oxytoca/efeitos dos fármacos , Klebsiella oxytoca/genética , Klebsiella oxytoca/metabolismo , Anotação de Sequência Molecular , Fatores R/metabolismo , Salmonella/efeitos dos fármacos , Salmonella/metabolismo , Análise de Sequência de DNA
16.
Microbes Environ ; 32(1): 40-46, 2017 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28302951

RESUMO

A conjugative F plasmid induces mature biofilm formation by Escherichia coli by promoting F-pili-mediated cell-cell interactions and increasing the expression of biofilm-related genes. We herein demonstrated another function for the F plasmid in E. coli biofilms; it contributes to the emergence of genetic and phenotypic variations by spontaneous mutations. Small colony variants (SCVs) were more frequently generated in a continuous flow-cell biofilm than in the planktonic state of E. coli harboring the F plasmid. E. coli SCVs represented typical phenotypic changes such as slower growth, less biofilm formation, and greater resistance to aminoglycoside antibiotics than the parent strain. Genomic and complementation analyses indicated that the small colony phenotype was caused by the insertion of Tn1000, which was originally localized in the F plasmid, into the hemB gene. Furthermore, the Tn1000 insertion was removed from hemB in the revertant, which showed a normal colony phenotype. This study revealed that the F plasmid has the potential to increase genetic variations not only by horizontal gene transfer via F pili, but also by site-specific recombination within a single cell.


Assuntos
Biofilmes/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Fator F , Fenótipo , Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Biofilmes/efeitos dos fármacos , Elementos de DNA Transponíveis , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Variação Genética , Genoma Bacteriano , Mutagênese Insercional
17.
Neurology ; 88(17): 1642-1649, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28356461

RESUMO

OBJECTIVE: To determine the association of parental family history with risk of dementia by age at onset and sex of affected parent in a population-based cohort. METHODS: From 2000 to 2002, we assessed parental history of dementia in participants without dementia of the Rotterdam Study. We investigated associations of parental history with risk of dementia until 2015, adjusting for demographics, cardiovascular risk factors, and known genetic risk variants. Furthermore, we determined the association between parental history and markers of neurodegeneration and vascular disease on MRI. RESULTS: Of 2,087 participants (mean age 64 years, 55% female), 407 (19.6%) reported a history of dementia in either parent (mean age at diagnosis 79 years). During a mean follow-up of 12.2 years, 142 participants developed dementia. Parental history was associated with risk of dementia independently of known genetic risk factors (hazard ratio [HR] 1.67, 95% confidence interval [CI] 1.12-2.48), in particular when parents were diagnosed at younger age (<80 years: HR 2.58, 95% CI 1.61-4.15; ≥80 years: HR 1.01, 95% CI 0.58-1.77). Accordingly, age at diagnosis in probands was highly correlated with age at diagnosis in their parents <80 years (r = 0.57, p = 0.001) but not thereafter (r = 0.17, p = 0.55). Among 1,161 participants without dementia with brain MRI, parental history was related to lower cerebral perfusion and higher burden of white matter lesions and microbleeds. Dementia risk and MRI markers were similar for paternal and maternal history. CONCLUSIONS: Parental history of dementia increases risk of dementia, primarily when age at parental diagnosis is <80 years. Unexplained heredity may be attributed in part to cerebral hypoperfusion and small vessel disease. We found no evidence of preferential maternal compared to paternal transmission.


Assuntos
Demência/epidemiologia , Predisposição Genética para Doença , Idade de Início , Idoso , Apolipoproteínas E/genética , Encéfalo/diagnóstico por imagem , Doenças Cardiovasculares/epidemiologia , Estudos de Coortes , Demência/diagnóstico por imagem , Demência/genética , Fator F , Feminino , Seguimentos , Técnicas de Genotipagem , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Pais , Fatores de Risco
18.
J Virol Methods ; 239: 9-16, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27777078

RESUMO

Fecal contamination of water poses a significant risk to public health due to the potential presence of pathogens, including enteric viruses. Therefore, sensitive, reliable and easy to use methods for the concentration, detection and quantification of microorganisms associated with the safety and quality of water are needed. In this study, we performed a field evaluation of an anion exchange resin-based method to concentrate male-specific (F+) RNA coliphages (FRNA), fecal indicator organisms, from diverse environmental waters that were suspected to be contaminated with feces. In this system, FRNA coliphages are adsorbed to anion exchange resin and direct nucleic acid isolation is performed, yielding a sample amenable to real-time reverse transcriptase (RT)-PCR detection. Matrix-dependent inhibition of this method was evaluated using known quantities of spiked FRNA coliphages belonging to four genogroups (GI, GII, GII and GIV). RT-PCR-based detection was successful in 97%, 72%, 85% and 98% of the samples spiked (106 pfu/l) with GI, GII, GIII and GIV, respectively. Differential FRNA coliphage genogroup detection was linked to inhibitors that altered RT-PCR assay efficiency. No association between inhibition and the physicochemical properties of the water samples was apparent. Additionally, the anion exchange resin method facilitated detection of naturally present FRNA coliphages in 40 of 65 environmental water samples (61.5%), demonstrating the viability of this system to concentrate FRNA coliphages from water.


Assuntos
Resinas de Troca de Ânions , Colífagos/isolamento & purificação , Leviviridae/isolamento & purificação , Microbiologia da Água , Poluição da Água , Adsorção , Resinas de Troca de Ânions/economia , Colífagos/química , Colífagos/genética , Colífagos/fisiologia , Monitoramento Ambiental/métodos , Fator F , Fezes/virologia , Humanos , Leviviridae/química , Leviviridae/genética , Leviviridae/fisiologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Poluição da Água/análise
19.
PLoS One ; 11(12): e0166890, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27907029

RESUMO

Plasmid-based gene expression is a fundamental tool in the field of biotechnology. However, overexpression of genes of interest with multi-copy plasmids often causes detrimental effects on host cells. To overcome this problem, chromosomal integration of target genes has been used for decades; however, insufficient protein expression occurred with this method. In this study, we developed a novel cloning and expression system named the chromosomal vector (ChroV) system, that has features of stable and high expression of target genes on the F' plasmid in the Escherichia coli JM109(DE3) strain. We used an RMT cluster (KCTC 11994BP) containing a silent cat gene from a previous study to clone a gene into the F' plasmid. The ChroV system was applied to clone two model targets, GFPuv and carotenoids gene clusters (4 kb), and successfully used to prove the inducible tightly regulated protein expression in the F' plasmid. In addition, we verified that the expression of heterologous genes in ChroV system maintained stably in the absence of antibiotics for 1 week, indicating ChroV system is applicable to antibiotics-free production of valuable proteins. This protocol can be widely applied to recombinant protein expression for antibiotics-free, stable, and genome-based expression, providing a new platform for recombinant protein synthesis in E. coli. Overall, our approach can be widely used for the economical and industrial production of proteins in E. coli.


Assuntos
Cromossomos Bacterianos/metabolismo , Clonagem Molecular/métodos , Escherichia coli/genética , Fator F/metabolismo , Antibacterianos , Carotenoides/biossíntese , Cromossomos Bacterianos/química , Escherichia coli/metabolismo , Fator F/química , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Família Multigênica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
20.
Cell ; 166(6): 1436-1444.e10, 2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-27610568

RESUMO

Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F "sex" pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/fisiologia , Fator F/química , Fímbrias Bacterianas/química , Modelos Moleculares , Fosfolipídeos/química , Sítios de Ligação Microbiológicos/genética , Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Fator F/genética , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Lipídeos/química , Mutação , Fosfolipídeos/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sistemas de Secreção Tipo V/química , Sistemas de Secreção Tipo V/metabolismo
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