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1.
Metallomics ; 11(4): 799-809, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30869729

RESUMO

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca2+-dependent l-alanyl-d-glutamate peptidase, although the mode of Ca2+ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111-130) in Ca2+ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca2+-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca2+ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active "open" conformation. The possible mechanism of Ca2+ regulation of EndoT5 activity during bacteriophage T5's life cycle through the Ca2+ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.


Assuntos
Cálcio/metabolismo , Escherichia coli/virologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Fagos T/enzimologia , Proteínas Virais/metabolismo , Ativação Enzimática , Escherichia coli/citologia , Modelos Moleculares , Fagos T/metabolismo
2.
Science ; 362(6417): 918-922, 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30467165

RESUMO

Measurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomechanical resonators, nanomechanical mass spectrometers often suffer from prohibitive sample loss, extended analysis time, or inadequate resolution. We report on a system architecture combining nebulization of the analytes from solution, their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomechanical resonator arrays. This system determined the mass distribution of ~30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed molecular mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 picomole of sample and with an instrument resolution above 100.


Assuntos
Capsídeo/química , Capsídeo/ultraestrutura , Espectrometria de Massas/métodos , Nanotecnologia/métodos , DNA Viral/química , Campos Eletromagnéticos , Nanopartículas/química , Poliestirenos/química , Fagos T/química , Fagos T/ultraestrutura
3.
Mol Biol (Mosk) ; 52(1): 3-9, 2018.
Artigo em Russo | MEDLINE | ID: mdl-29512629

RESUMO

A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2-35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.


Assuntos
Mutação , RNA de Transferência/genética , Fagos T/genética , Sequência de Bases , DNA Viral/genética , Deleção de Sequência
4.
Food Res Int ; 103: 59-67, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29389643

RESUMO

A continuous-flow UV reactor operating at 254nm wave-length was used to investigate inactivation of microorganisms including bacteriophage in coconut water, a highly opaque liquid food. UV-C inactivation kinetics of two surrogate viruses (MS2, T1UV) and three bacteria (E. coli ATCC 25922, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes ATCC 19115) in buffer and coconut water were investigated (D10 values ranging from 2.82 to 4.54mJ·cm-2). A series of known UV-C doses were delivered to the samples. Inactivation levels of all organisms were linearly proportional to UV-C dose (r2>0.97). At the highest dose of 30mJ·cm-2, the three pathogenic organisms were inactivated by >5 log10 (p<0.05). Results clearly demonstrated that UV-C irradiation effectively inactivated bacteriophage and pathogenic microbes in coconut water. The inactivation kinetics of microorganisms were best described by log linear model with a low root mean square error (RMSE) and high coefficient of determination (r2>0.97). Models for predicting log reduction as a function of UV-C irradiation dose were found to be significant (p<0.05) with low RMSE and high r2. The irradiated coconut water showed no cytotoxic effects on normal human intestinal cells and normal mouse liver cells. Overall, these results indicated that UV-C treatment did not generate cytotoxic compounds in the coconut water. This study clearly demonstrated that high levels of inactivation of pathogens can be achieved in coconut water, and suggested potential method for UV-C treatment of other liquid foods. INDUSTRIAL RELEVANCE: This research paper provides scientific evidence of the potential benefits of UV-C irradiation in inactivating bacterial and viral surrogates at commercially relevant doses of 0-120mJ·cm-2. The irradiated coconut water showed no cytotoxic effects on normal intestinal and healthy mice liver cells. UV-C irradiation is an attractive food preservation technology and offers opportunities for horticultural and food processing industries to meet the growing demand from consumers for healthier and safe food products. This study would provide technical support for commercialization of UV-C treatment of beverages.


Assuntos
Cocos/microbiologia , Escherichia coli/efeitos da radiação , Manipulação de Alimentos/instrumentação , Microbiologia de Alimentos/instrumentação , Sucos de Frutas e Vegetais/microbiologia , Listeria monocytogenes/efeitos da radiação , Salmonella typhimurium/efeitos da radiação , Raios Ultravioleta , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cocos/toxicidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Desenho de Equipamento , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Manipulação de Alimentos/métodos , Microbiologia de Alimentos/métodos , Sucos de Frutas e Vegetais/toxicidade , Levivirus/crescimento & desenvolvimento , Levivirus/efeitos da radiação , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/virologia , Listeriose/microbiologia , Listeriose/prevenção & controle , Intoxicação Alimentar por Salmonella/microbiologia , Intoxicação Alimentar por Salmonella/prevenção & controle , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/virologia , Fagos T/crescimento & desenvolvimento , Fagos T/efeitos da radiação , Raios Ultravioleta/efeitos adversos
5.
Virology ; 515: 215-222, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29306059

RESUMO

Homing endonucleases are a group of site-specific endonucleases that initiate homing, a nonreciprocal transfer of its own gene into a new allele lacking this gene. This work describes a novel phage T4 endonuclease, SegD, which is homologous to the GIY-YIG family of homing endonucleases. Like other T4 homing endonucleases SegD recognizes an extended, 16bp long, site, cleaves it asymmetrically to form 3'-protruding ends and digests both unmodified DNA and modified T-even phage DNA with similar efficiencies. Surprisingly, we revealed that SegD cleavage site was identical in the genomes of segD- and segD+ phages. We found that segD gene was expressed during the T4 developmental cycle. Nevertheless, endonuclease SegD was not able to initiate homing of its own gene as well as genetic recombination between phages in its site inserted into the rII locus.


Assuntos
Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Endonucleases/metabolismo , Translocação Genética , Proteínas Virais/metabolismo , Bacteriófago T4/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Endonucleases/química , Endonucleases/genética , Íntrons , Família Multigênica , Fagos T/enzimologia , Fagos T/genética , Fagos T/metabolismo , Proteínas Virais/química , Proteínas Virais/genética
6.
Nucleic Acids Res ; 46(2): 873-885, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29253268

RESUMO

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA Viral/metabolismo , Fagos T/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Sequência de Bases , DNA Viral/genética , Escherichia coli/genética , Escherichia coli/virologia , Ligação Proteica , Fagos T/genética
7.
Phys Biol ; 14(5): 055004, 2017 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-28825411

RESUMO

We re-examined data from the classic Luria-Delbrück fluctuation experiment, which is often credited with establishing a Darwinian basis for evolution. We argue that, for the Lamarckian model of evolution to be ruled out by the experiment, the experiment must favor pure Darwinian evolution over both the Lamarckian model and a model that allows both Darwinian and Lamarckian mechanisms (as would happen for bacteria with CRISPR-Cas immunity). Analysis of the combined model was not performed in the original 1943 paper. The Luria-Delbrück paper also did not consider the possibility of neither model fitting the experiment. Using Bayesian model selection, we find that the Luria-Delbrück experiment, indeed, favors the Darwinian evolution over purely Lamarckian. However, our analysis does not rule out the combined model, and hence cannot rule out Lamarckian contributions to the evolutionary dynamics.


Assuntos
Evolução Biológica , Escherichia coli/genética , Modelos Genéticos , Teorema de Bayes , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Fagos T/genética , Fagos T/fisiologia
8.
Nucleic Acids Res ; 45(4): 1946-1957, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28130424

RESUMO

CRISPR-Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR-Cas systems against different viruses should vary. Here, we created a collection of Escherichia coli strains with type I-E CRISPR-Cas system targeting various positions in the genomes of bacteriophages λ, T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR-Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR-Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release.


Assuntos
Bacteriólise , Bacteriófagos/fisiologia , Sistemas CRISPR-Cas , Escherichia coli/fisiologia , Escherichia coli/virologia , Bacteriófago lambda/genética , Marcação de Genes , Variação Genética , Genoma Viral , Fagos T/genética
9.
Sci Rep ; 6: 39414, 2016 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-28009009

RESUMO

Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms.


Assuntos
Fagos T/genética , Proteínas Virais/genética , Adenosina Trifosfatases/genética , Archaea/genética , Biologia Computacional/métodos , DNA/genética , Pegada de DNA/métodos , DNA Helicases/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Nucleotídeos/genética , Especificidade por Substrato
10.
PLoS One ; 8(12): e84376, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24376806

RESUMO

We present an innovative method to couple electrophysiological measurements with fluorescence imaging of functionalized suspended bilayers. Our method combines several advantages: it is well suited to study transmembrane proteins that are difficult to incorporate in suspended bilayers, it allows single molecule resolution both in terms of electrophysiological measurements and fluorescence imaging, and it enables mechanical stimulations of the membrane. The approach comprises of two steps: first the reconstitution of membrane proteins in giant unilamellar vesicles; then the formation of a suspended bilayer spanning a 5 to 15 micron-wide aperture that can be visualized by high NA microscope objectives. We exemplified how the technique can be used to detect in real time the translocation of T5 DNA across the bilayer during its ejection from the bacteriophage capsid.


Assuntos
Membrana Celular/ultraestrutura , Fenômenos Eletrofisiológicos/fisiologia , Bicamadas Lipídicas/metabolismo , Imagem Óptica/métodos , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , DNA Viral/metabolismo , Proteínas de Escherichia coli/metabolismo , Micromanipulação , Fagos T/genética , Liberação de Vírus/fisiologia
11.
J Mol Biol ; 425(22): 4125-33, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24029071

RESUMO

The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors.


Assuntos
Replicação do DNA , Recombinação Genética , Fagos T/genética , Fagos T/metabolismo , Fatores de Transcrição/química , Transcrição Genética , Sequência de Aminoácidos , Evolução Biológica , Biologia Computacional/métodos , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Bases de Dados Genéticas , Genoma Viral , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Mapeamento Físico do Cromossomo , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Curr Opin Microbiol ; 16(4): 500-6, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23725668

RESUMO

In the reductionist era T-type coliphage research became one of the foundations for molecular biology. The technological progress in systems biology makes it now possible to study T-type phage-Escherichia coli interaction in the natural ecological niche, the gut of warm blooded animals. This development gives a second chance to phages as anti-microbial agents ('phage therapy'). Bacteria growing in biofilms are difficult to treat with antibiotics while many phages express naturally depolymerases which attack the polysaccharide matrix that enmesh bacteria in biofilms. Phages were already used successfully to reduce contamination levels with medical catheters and might likewise be of use against infections frequently forming bacterial biofilms.


Assuntos
Escherichia coli/virologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/virologia , Fagos T/fisiologia , Animais , Biofilmes , Terapia Biológica/métodos , Ecossistema , Escherichia coli/fisiologia , Infecções por Escherichia coli/terapia , Humanos , Fagos T/crescimento & desenvolvimento
13.
J Vis Exp ; (75): e3899, 2013 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-23728084

RESUMO

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation (1-3). Viruses, which are ubiquitous and the most numerous entities on our planet (4) and important in all environments (5), have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.


Assuntos
Genoma Viral , Genômica/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírion/genética , Bacteriófago lambda/genética , DNA Viral/química , DNA Viral/genética , DNA Viral/isolamento & purificação , Citometria de Fluxo/métodos , Microscopia Confocal , Fagos T/genética
14.
Biomacromolecules ; 14(5): 1257-61, 2013 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-23590700

RESUMO

These studies illustrate synthetic paths to covalently attach T1 and Φ11 bacteriophages (phages) to inert polymeric surfaces while maintaining the bacteriophage's biological activities capable of killing deadly human pathogens. The first step involved the formation of acid (COOH) groups on polyethylene (PE) and polytetrafluoroethylene (PTFE) surfaces using microwave plasma reactions in the presence of maleic anhydride, followed by covalent attachment of T1 and Φ11 species via primary amine groups. The phages effectively retain their biological activity manifested by a rapid infection with their own DNA and effective destruction of Escherichia coli and Staphylococcus aureus human pathogens. These studies show that simultaneous covalent attachment of two biologically active phages effectively destroy both bacterial colonies and eliminate biofilm formation, thus offering an opportunity for an effective combat against multibacterial colonies as well as surface detections of other pathogens.


Assuntos
Infecções Bacterianas/prevenção & controle , Materiais Revestidos Biocompatíveis/química , Escherichia coli/virologia , Fagos de Staphylococcus/química , Staphylococcus aureus/virologia , Fagos T/química , Biofilmes , Humanos , Anidridos Maleicos/química , Gases em Plasma , Polietileno/química , Politetrafluoretileno/química , Fagos de Staphylococcus/patogenicidade , Fagos de Staphylococcus/fisiologia , Fagos T/patogenicidade , Fagos T/fisiologia , Ensaio de Placa Viral
15.
Nucleic Acids Res ; 41(8): 4587-600, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23435232

RESUMO

Bacteriophage T5 has a 120 kb double-stranded linear DNA genome encoding most of the genes required for its own replication. This lytic bacteriophage has a burst size of ∼500 new phage particles per infected cell, demonstrating that it is able to turn each infected bacterium into a highly efficient DNA manufacturing machine. To begin to understand DNA replication in this prodigious bacteriophage, we have characterized a putative helicase encoded by gene D2. We show that bacteriophage T5 D2 protein is the first viral helicase to be described with bipolar DNA unwinding activities that require the same core catalytic residues for unwinding in either direction. However, unwinding of partially single- and double-stranded DNA test substrates in the 3'-5' direction is more robust and can be distinguished from the 5'-3' activity by a number of features including helicase complex stability, salt sensitivity and the length of single-stranded DNA overhang required for initiation of helicase action. The presence of D2 in an early gene cluster, the identification of a putative helix-turn-helix DNA-binding motif outside the helicase core and homology with known eukaryotic and prokaryotic replication initiators suggest an involvement for this unusual helicase in DNA replication initiation.


Assuntos
DNA Helicases/metabolismo , Fagos T/enzimologia , Proteínas Virais/metabolismo , Difosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA de Cadeia Simples/metabolismo , Cloreto de Sódio/farmacologia , Especificidade por Substrato , Proteínas Virais/química , Proteínas Virais/genética
16.
Mol Microbiol ; 87(4): 818-34, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23289425

RESUMO

We report isolation and characterization of the novel T4-like Salmonella bacteriophage vB_SenM-S16. S16 features a T-even morphology and a highly modified 160 kbp dsDNA genome with 36.9 mol % G+C, containing 269 putative coding sequences and three tRNA genes. S16 is a virulent phage, and exhibits a maximally broad host range within the genus Salmonella, but does not infect other bacteria. Synthesis of functional S16 full-length long tail fibre (LTF) in Escherichia coli was possible by coexpression of gp37 and gp38. Surface plasmon resonance analysis revealed nanomolar equilibrium affinity of the LTF to its receptor on Salmonella cells. We show that OmpC serves as primary binding ligand, and that S16 adsorption can be transferred to E. coli by substitution of ompC with the Salmonella homologue. S16 also infects 'rough' Salmonella strains which are defective in lipopolysaccharide synthesis and/or its carbohydrate substitution, indicating that this interaction does not require an intact LPS structure. Altogether, its virulent nature, broad host range and apparent lack of host DNA transduction render S16 highly suitable for biocontrol of Salmonella in foods and animal production. The S16 LTF represents a highly specific affinity reagent useful for cell decoration and labelling, as well as bacterial immobilization and separation.


Assuntos
Proteínas de Bactérias/metabolismo , Myoviridae/metabolismo , Porinas/metabolismo , Receptores Virais/metabolismo , Fagos de Salmonella/metabolismo , Salmonella enterica/virologia , Fagos T/metabolismo , Proteínas da Cauda Viral/metabolismo , Proteínas de Bactérias/genética , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Myoviridae/genética , Porinas/genética , Receptores Virais/genética , Fagos de Salmonella/genética , Salmonella enterica/genética , Salmonella enterica/metabolismo , Fagos T/genética , Proteínas da Cauda Viral/genética
17.
Ecol Lett ; 16(4): 446-53, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23331662

RESUMO

Suicide upon infection by lytic phages is known in several bacteria species and represents an effective defence strategy to limit phage spread. However, the ecological conditions favouring the evolution of such a radically altruistic behaviour are unclear. Here, we model the feedback of epidemiology on host evolution in a spatially structured environment and we generate several specific predictions on altruistic suicide evolution. We test these predictions experimentally by competing E. coli cells carrying the suicide gene Lit against non-carrier cells in the presence or in the absence of the lytic phage T6. We show that in accord with our theoretical analysis altruistic suicide is only favoured in the presence of the phage in spatially structured environments at intermediate levels of mixing. Our work provides a general explanation for the evolution of altruistic defence strategies against pathogens. We discuss the implications of these results for oncolytic virus therapy.


Assuntos
Evolução Biológica , Endopeptidases/genética , Escherichia coli K12/virologia , Proteínas de Escherichia coli/genética , Interações Hospedeiro-Patógeno/fisiologia , Proteínas de Membrana/genética , Fagos T/patogenicidade , Endopeptidases/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos
18.
Chem Commun (Camb) ; 49(9): 910-2, 2013 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-23247551

RESUMO

Functionalization of bacterial cell surfaces has the potential to introduce new activities by chemical modification. Here we show that a bacteriophage-receptor complex can be used to functionalize the surface of two Gram-negative proteobacteria, Escherichia coli and Ralstonia eutropha with CdSe/ZnS nanoparticles. This work highlights the potential for using microbe-phage interactions to generate new functions on living cells.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Cupriavidus necator/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Nanopartículas/química , Fagos T/química , Proteínas Virais/química , Compostos de Cádmio/química , Compostos de Selênio/química , Sulfetos/química , Compostos de Zinco/química
19.
Virology ; 434(2): 222-32, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23102968

RESUMO

The genomic diversity of 99 T4-like coliphages was investigated by sequencing an equimolar mixture with Illumina technology and screening them against different databases for horizontal gene transfer and undesired genes. A 9-phage cocktail was given to 15 healthy adults from Bangladesh at a dose of 3×10(9) and 3×10(7) plaque-forming units and placebo respectively. Phages were detected in 64% of the stool samples when subjects were treated with higher titer phage, compared to 30% and 28% with lower-titer phage and placebo, respectively. No Escherichia coli was present in initial stool samples, and no amplification of phage was observed. One percent of the administered oral phage was recovered from the feces. No adverse events were observed by self-report, clinical examination, or from laboratory tests for liver, kidney, and hematology function. No impact of oral phage was seen on the fecal microbiota composition with respect to bacterial 16S rRNA from stool.


Assuntos
Produtos Biológicos/administração & dosagem , Terapia Biológica/métodos , Fagos T , Administração Oral , Adulto , Bangladesh , Produtos Biológicos/efeitos adversos , Fezes/virologia , Feminino , Experimentação Humana , Humanos , Masculino , Placebos/administração & dosagem , Adulto Jovem
20.
Biochem Mol Biol Educ ; 40(4): 277-83, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22807434

RESUMO

Mutate is a program developed for teaching purposes to impart a virtual laboratory class for undergraduate students of Genetics in Biology. The program emulates the so-called fluctuation test whose aim is to distinguish between spontaneous and adaptive mutation hypotheses in bacteria. The plan is to train students in certain key multidisciplinary aspects of current genetics such as sequence databases, DNA mutations, and hypothesis testing, while introducing the fluctuation test. This seminal experiment was originally performed studying Escherichia coli resistance to the infection by bacteriophage T1. The fluctuation test initiated the modern bacterial genetics that 25 years later ushered in the era of the recombinant DNA. Nowadays we know that some deletions in fhuA, the gene responsible for E. coli membrane receptor of T1, could cause the E. coli resistance to this phage. For the sake of simplicity, we will introduce the assumption that a single mutation generates the resistance to T1. During the practical, the students use the program to download some fhuA gene sequences, manually introduce some stop codon mutations, and design a fluctuation test to obtain data for distinguishing between preadaptative (spontaneous) and induced (adaptive) mutation hypotheses. The program can be launched from a browser or, if preferred, its executable file can be downloaded from http://webs.uvigo.es/acraaj/MutateWeb/Mutate.html. It requires the Java 5.0 (or higher) Runtime Environment (freely available at http://www.java.com).


Assuntos
Biologia Computacional/métodos , Genética/educação , Mutação , Software , Materiais de Ensino , Proteínas da Membrana Bacteriana Externa/genética , Bases de Dados Genéticas , Escherichia coli/genética , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Genes Bacterianos , Internet , Fagos T/metabolismo
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