RESUMO
Bacteriophage fitness is determined by factors influencing both their replication within bacteria and their ability to maintain infectivity between infections. The latter becomes particularly crucial under adverse environmental conditions or when host density is low. In such scenarios, the damage experienced by viral particles could lead to the loss of infectivity, which might be mitigated if the virus undergoes evolutionary optimization through replication. In this study, we conducted an evolution experiment involving bacteriophage Qß, wherein it underwent 30 serial transfers, each involving a cycle of freezing and thawing followed by replication of the surviving viruses. Our findings show that Qß was capable of enhancing its resistance to this selective pressure through various adaptive pathways that did not impair the virus replicative capacity. Notably, these adaptations predominantly involved mutations located within genes encoding capsid proteins. The adapted populations exhibited higher resistance levels than individual viruses isolated from them, and the latter surpassed those observed in single mutants generated via site-directed mutagenesis. This suggests potential interactions among mutants and mutations. In conclusion, our study highlights the significant role of extracellular selective pressures in driving the evolution of phages, influencing both the genetic composition of their populations and their phenotypic properties.
Assuntos
Congelamento , Mutação , Fagos RNA/genética , Fagos RNA/fisiologia , Adaptação Fisiológica/genética , Evolução Molecular , Replicação Viral/genética , Proteínas do Capsídeo/genéticaRESUMO
Evolution of RNA bacteriophages of the family Leviviridae is governed by the high error rates of their RNA-dependent RNA polymerases. This fact, together with their large population sizes, leads to the generation of highly heterogeneous populations that adapt rapidly to most changes in the environment. Throughout adaptation, the different mutants that make up a viral population compete with each other in a non-trivial process in which their selective values change over time due to the generation of new mutations. In this work we have characterised the intra-population dynamics of a well-studied levivirus, Qß, when it is propagated at a higher-than-optimal temperature. Our results show that adapting populations experienced rapid changes that involved the ascent of particular genotypes and the loss of some beneficial mutations of early generation. Artificially reconstructed populations, containing a fraction of the diversity present in actual populations, fixed mutations more rapidly, illustrating how population bottlenecks may guide the adaptive pathways. The conclusion is that, when the availability of beneficial mutations under a particular selective condition is elevated, the final outcome of adaptation depends more on the occasional occurrence of population bottlenecks and how mutations combine in genomes than on the selective value of particular mutations.
Assuntos
Adaptação Biológica , Fagos RNA/fisiologia , Temperatura , Evolução Biológica , Evolução Molecular , Regulação Viral da Expressão Gênica , Genoma Viral , Genômica/métodos , Mutação , RNA Viral/genética , Seleção GenéticaRESUMO
Healthy human infants are typically born without high concentrations of viral particles in their intestines, but after a few weeks of life particle counts typically reach a billion per gram of stool. Where do these vast populations come from? Recent studies support the idea that colonization is stepwise. First pioneer bacteria seed the infant gut. Bacteria commonly harbor prophage sequences integrated in their genomes, which periodically induce to make particles, providing a first wave of viral particles. Later more viruses infecting human cells are detected. Analysis showed that lower accumulation of viruses that grow in human cells is associated with breastfeeding. Thus these studies emphasize the environmental influences on formation of the early life virome, and begin to point the way toward modulating viral colonization to optimize health.
Assuntos
Trato Gastrointestinal/virologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Viroma/fisiologia , Adulto , Aleitamento Materno , Vírus de DNA/fisiologia , Fezes/microbiologia , Fezes/virologia , Microbioma Gastrointestinal , Humanos , Recém-Nascido , Fagos RNA/fisiologia , VírionRESUMO
Single-stranded (ss)RNA viruses are thought to evolve rapidly due to an inherently high mutation rate. However, it remains unclear how ssRNA viruses adapt to novel environments and/or how many and what types of substitutions are needed to facilitate this evolution. In this study, we followed the adaptation of the ssRNA bacteriophage Qß using thermally adapted Escherichia coli as a host, which can efficiently grow at temperatures between 37.2 and 45.3 °C. This made it possible to evaluate Qß adaptation to the highest known temperature that supports growth, 45.3 °C. We found that Qß was capable of replication at this temperature; within 114 days (~1260 generations), we detected more than 34 novel point mutations in the genome of the thermally adapted Qß population, representing 0.8% of the total Qß genome. In addition, we returned the 45.3 °C-adapted Qß populations to 37.2 °C and passaged them for 8 days (~124 generations). We found that the reverse-adapted Qß population showed little to no decrease in fitness. These results indicate that Qß can evolve in response to increasing temperatures in a short period of time with the accumulation of point mutations.
Assuntos
Evolução Biológica , Fagos RNA/fisiologia , Adaptação Biológica , Escherichia coli/virologia , Temperatura Alta , Mutação Puntual , Fagos RNA/genética , RNA Viral/genéticaRESUMO
Noroviruses (NoV) are responsible for many shellfish outbreaks. Purification processes may be applied to oysters before marketing to decrease potential fecal pollution. This step is rapidly highly effective in reducing Escherichia coli; nevertheless, the elimination of virus genomes has been described to be much slower. It is therefore important to identify (i) the purification conditions that optimize virus removal and (ii) the mechanism involved. To this end, the effects of oyster stress, nutrients, and the presence of a potential competitor to NoV adhesion during purification were investigated using naturally contaminated oysters. Concentrations of NoV (genomes) and of the viral indicator F-specific RNA bacteriophage (FRNAPH; genomes and infectious particles) were regularly monitored. No significant differences were observed under the test conditions. The decrease kinetics of both virus genomes were similar, again showing the potential of FRNAPH as an indicator of NoV behavior during purification. The T90 (time to reduce 90% of the initial titer) values were 47.8 days for the genogroup I NoV genome, 26.7 days for the genogroup II NoV genome, and 43.9 days for the FRNAPH-II genome. Conversely, monitoring of the viral genomes could not be used to determine the behavior of infectious viruses because the T90 values were more than two times lower for infectious FRNAPH (20.6 days) compared to their genomes (43.9 days). Finally, this study highlighted that viruses are primarily inactivated in oysters rather than released in the water during purification processes.IMPORTANCE This study provides new data about the behavior of viruses in oysters under purification processes and about their elimination mechanism. First, a high correlation has been observed between F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and norovirus (NoV) in oysters impacted by fecal contamination when both are detected using molecular approaches. Second, when using reverse transcription-quantitative PCR and culture to detect FRNAPH-II genomes and infectious FRNAPH in oysters, respectively, it appears that genome detection provides limited information about the presence of infectious particles. The comparison of both genomes and infectious particles highlights that the main mechanism of virus elimination in oysters is inactivation. Finally, this study shows that none of the conditions tested modify virus removal.
Assuntos
Crassostrea/virologia , Fagos RNA/fisiologia , Inativação de Vírus , Eliminação de Partículas Virais , Animais , Ácido Cítrico/análise , Norovirus/fisiologia , Nutrientes/análise , Estresse FisiológicoRESUMO
A population's growth rate is determined by multiple 'life history traits'. To quantitatively determine which life history traits should be improved to allow a living organism to adapt to an inhibitory environment is an important issue. Previously, we conducted thermal adaptation experiments on the RNA bacteriophage Qß using three independent replicates and reported that all three end-point populations could grow at a temperature (43.6°C) that inhibited the growth of the ancestral strain. Even though the fitness values of the endpoint populations were almost the same, their genome sequence was not, indicating that the three thermally adapted populations may have different life history traits. In this study, we introduced each mutation observed in these three end-point populations into the cDNA of the Qß genome and prepared three different mutants. Quantitative analysis showed that they tended to increase their fitness by increasing the adsorption rate to their host, shortening their latent period (i.e., the duration between phage infection and progeny release), and increasing the burst size (i.e., the number of progeny phages per infected cell), but all three mutants decreased their thermal stability. However, the degree to which these traits changed differed. The mutant with the least mutations showed a smaller decrease in thermal stability, the largest adsorption rate to the host, and the shortest latent period. These results indicated that several different adaptive routes exist by which Qß can adapt to higher temperatures, even though Qß is a simple RNA bacteriophage with a small genome size, encoding only four genes.
Assuntos
Mutação , Fagos RNA/genética , Adaptação Fisiológica , Escherichia coli/virologia , Genoma Viral , Temperatura Alta , Fenótipo , Fagos RNA/química , Fagos RNA/fisiologiaRESUMO
Bacteriophages of the Leviviridae family are small viruses with short single-stranded RNA (ssRNA) genomes. Protein-RNA interactions play a key role throughout the phage life cycle, and all of the conserved phage proteins - the maturation protein, the coat protein and the replicase - are able to recognize specific structures in the RNA genome. The phage-coded replicase subunit associates with several host proteins to form a catalytically active complex. Recognition of the genomic RNA by the replicase complex is achieved in a remarkably complex manner that exploits the RNA-binding properties of host proteins and the particular three-dimensional structure of the phage genome. The coat protein recognizes a hairpin structure at the beginning of the replicase gene. The binding interaction serves to regulate the expression of the replicase gene and can be remarkably different in various ssRNA phages. The maturation protein is a minor structural component of the virion that binds to the genome, mediates attachment to the host and guides the genome into the cell. The maturation protein has two distinct RNA-binding surfaces that are in contact with different regions of the genome. The maturation and coat proteins also work together to ensure the encapsidation of the phage genome in new virus particles. In this chapter, the different ssRNA phage protein-RNA interactions, as well as some of their practical applications, are discussed in detail.
Assuntos
Genoma Viral/fisiologia , Fagos RNA , RNA Viral , RNA Polimerase Dependente de RNA , Proteínas Virais , Fagos RNA/química , Fagos RNA/fisiologia , RNA Viral/biossíntese , RNA Viral/química , RNA Viral/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Bacteriophages are the most numerous biological entities on Earth. They are on the basis of most ecosystems, regulating the diversity and abundance of bacterial populations and contributing to the nutrient and energy cycles. Bacteriophages have two well differentiated phases in their life cycle, one extracellular, in which they behave as inert particles, and other one inside their hosts, where they replicate to give rise to a progeny. In both phases they are exposed to environmental conditions that often act as selective pressures that limit both their survival in the environment and their ability to replicate, two fitness traits that frequently cannot be optimised simultaneously. In this study we have analysed the evolutionary ability of an RNA bacteriophage, the bacteriophage Qß, when it is confronted with a temperature increase that affects both the extracellular and the intracellular media. Our results show that Qß can optimise its survivability when exposed to short-term high temperature extracellular heat shocks, as well as its replicative ability at higher-than-optimal temperature. Mutations responsible for simultaneous adaptation were the same as those selected when adaptation to each condition proceeded separately, showing the absence of important trade-offs between survival and reproduction in this virus.
Assuntos
Adaptação Fisiológica/genética , Evolução Molecular , Interações Hospedeiro-Patógeno , Fagos RNA/fisiologia , Temperatura , Aclimatação/genética , Ecossistema , Escherichia coli/virologia , Aptidão Genética , Resposta ao Choque Térmico/genética , Temperatura Alta , Fenótipo , Fagos RNA/genética , Seleção GenéticaRESUMO
The association of viruses with settling particles is certainly a major process controlling the spread of viral pollution in surface water and sediment. To better understand the viral distribution in a river system, the behavior of F-specific RNA bacteriophages (FRNAPHs) was investigated in relationship with the suspended solids and sediment. The partitioning of phage particles (free or associated with solids) in surface water and the attachment capabilities of eight distinct strains of phages to sediment were studied in lab experiments. In situ observations were also performed with the genotyping of 166 individual plaques of FRNAPHs isolated from surface water and sediment. The results reported here demonstrate a variation of the status of infectious phages as a function of the hydro-climatological conditions. Phage-solid association seems to mainly occur during the peak of rainfall-runoff events but also to a certain extent during the recession phase compared to low flow conditions. The transfer of phages from the water column to sediment may occur at this time. Furthermore, the ability of FRNAPHs to interact with sediment was established for six strains out of eight, belonging to genogroups II, III and IV. A similar dynamic was observed for strains within a same genogroup despite different intensity of attachment and inactivation rates for strains of genogroups III and IV. The latter results match the in situ observations in the water and sediment compartments of the studied area. Infectious FRNAPH genogroup II was more abundant in sediment than in surface water. Its capability to sorb to sediment and its higher persistence in the environment compared to genogroups III and IV were the two main explanations. Together, lab and in situ experiments produce an overall vision of the mechanisms governing FRNAPH distribution among the water column and riverbed sediment.
Assuntos
Poluição Ambiental , Sedimentos Geológicos , Fagos RNA/fisiologia , Rios/virologia , Monitoramento Ambiental , Genótipo , Luxemburgo , Fagos RNA/genéticaRESUMO
Despite the expanding interest in bacterial viruses (bacteriophages), insights into the intracellular development of bacteriophage and its impact on bacterial physiology are still scarce. Here we investigate during lytic infection the whole-genome transcription of the giant phage vB_YecM_φR1-37 (φR1-37) and its host, the gastroenteritis causing bacterium Yersinia enterocolitica. RNA sequencing reveals that the gene expression of φR1-37 does not follow a pattern typical observed in other lytic bacteriophages, as only selected genes could be classified as typically early, middle or late genes. The majority of the genes appear to be expressed constitutively throughout infection. Additionally, our study demonstrates that transcription occurs mainly from the positive strand, while the negative strand encodes only genes with low to medium expression levels. Interestingly, we also detected the presence of antisense RNA species, as well as one non-coding intragenic RNA species. Gene expression in the phage-infected cell is characterized by the broad replacement of host transcripts with phage transcripts. However, the host response in the late phase of infection was also characterized by up-regulation of several specific bacterial gene products known to be involved in stress response and membrane stability, including the Cpx pathway regulators, ATP-binding cassette (ABC) transporters, phage- and cold-shock proteins.
Assuntos
Interações Hospedeiro-Patógeno , Fagos RNA/fisiologia , Yersinia enterocolitica/virologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Genoma Viral , RNA não Traduzido , RNA Viral , Sequências Reguladoras de Ácido Ribonucleico , Análise de Sequência de RNA , Transcriptoma , Yersinia enterocolitica/crescimento & desenvolvimentoRESUMO
Chlamydia trachomatis is the most common cause of curable bacterial sexually transmitted infections worldwide. Although the pathogen is well established, the pathogenic mechanisms remain unclear. Given the current challenges of antibiotic resistance and blocked processes of vaccine development, the use of a specific chlamydiaphage may be a new treatment solution. φCPG1 is a lytic phage specific for Chlamydia caviae, and shows over 90% nucleotide sequence identity with other chlamydiaphages. Vp1 is the major capsid protein of φCPG1. Purified Vp1 was previously confirmed to inhibit Chlamydia trachomatis growth. We here report the first attempt at exploring the relationship between Vp1-treated C. trachomatis and the protein and gene levels of the mitogen-activated/extracellular regulated protein kinase (MAPK/ERK) pathway by Western blotting and real-time PCR, respectively. Moreover, we evaluated the levels of pro-inflammatory cytokines interleukin (IL)-8 and IL-1 by enzyme-linked immunosorbent assay after Vp1 treatment. After 48 h of incubation, the p-ERK level of the Vp1-treated group decreased compared with that of the Chlamydia infection group. Accordingly, ERK1 and ERK2 mRNA expression levels of the Vp1-treated group also decreased compared with the Chlamydia infection group. IL-8 and IL-1 levels were also decreased after Vp1 treatment compared with the untreated group. Our results demonstrate that the inhibition effect of the chlamydiaphage φCPG1 capsid protein Vp1 on C. trachomatis is associated with the MAPK pathway, and inhibits production of the pro-inflammatory cytokines IL-8 and IL-1. The bacteriophages may provide insight into a new signaling transduction mechanism to influence their hosts, in addition to bacteriolysis.
Assuntos
Proteínas do Capsídeo/metabolismo , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/virologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagos RNA/fisiologia , Transdução de Sinais , Animais , Antibacterianos/farmacologia , Azitromicina/farmacologia , Linhagem Celular , Chlamydia trachomatis/efeitos dos fármacos , Humanos , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , FosforilaçãoRESUMO
Bacterial biofilms are a growing concern in a broad range of areas. In this study, a mixture of RNA bacteriophages isolated from municipal wastewater was used to control and remove biofilms. At the concentrations of 400 and 4 × 10(7) PFU/mL, the phages inhibited Pseudomonas aeruginosa biofilm formation by 45 ± 15% and 73 ± 8%, respectively. At the concentrations of 6,000 and 6 × 10(7) PFU/mL, the phages removed 45 ± 9% and 75 ± 5% of pre-existing P. aeruginosa biofilms, respectively. Chlorine reduced biofilm growth by 86 ± 3% at the concentration of 210 mg/L, but it did not remove pre-existing biofilms. However, a combination of phages (3 × 10(7) PFU/mL) and chlorine at this concentration reduced biofilm growth by 94 ± 2% and removed 88 ± 6% of existing biofilms. In a continuous flow system with continued biofilm growth, a combination of phages (a one-time treatment at the concentration of 1.9 × 10(8) PFU/mL for 1 h first) with chlorine removed 97 ± 1% of biofilms after Day 5 while phage and chlorine treatment alone removed 89 ± 1% and 40 ± 5%, respectively. For existing biofilms, a combined use of a lower phage concentration (3.8 × 10(5) PFU/mL) and chlorination with a shorter time duration (12 h) followed by continuous water flushing removed 96 ± 1% of biofilms in less than 2 days. Laser scanning confocal microscopy supplemented with electron microscopy indicated that the combination treatment resulted in biofilms with lowest cell density and viability. These results suggest that the combination treatment of phages and chlorine is a promising method to control and remove bacterial biofilms from various surfaces.
Assuntos
Biofilmes/efeitos dos fármacos , Cloro/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Fagos RNA/fisiologia , Biofilmes/crescimento & desenvolvimento , Desinfecção/métodos , Viabilidade Microbiana/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/virologia , Ensaio de Placa Viral , Águas Residuárias/virologiaRESUMO
Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation.
Assuntos
Biofilmes/crescimento & desenvolvimento , Colífagos/fisiologia , Escherichia coli/fisiologia , Plasmídeos/fisiologia , Conjugação Genética , Escherichia coli/genética , Escherichia coli/virologia , Fímbrias Bacterianas/metabolismo , Interações Microbianas , Plasmídeos/genética , Fagos RNA/fisiologiaRESUMO
Detection of specific genetic markers can rapidly identify the presence of enteric viruses in groundwater. However, comparison of stability characteristics between genetic and infectivity markers is necessary to better interpret molecular data. Human adenovirus serotype 2 (HAdV2), in conjunction with MS2 phages or GA phages, was spiked into raw groundwater microcosms. Viral stability was periodically assessed by both infectivity and real-time PCR methods. The results of this yearlong study suggest that adenoviruses have the most stable persistence profile and an ability to survive for a long time in groundwater. According to a linear regression model, infectivity reductions of HAdV2 ranged from 0.0076 log(10)/day (4°C) to 0.0279 log(10)/day (20°C) and were significantly lower than those observed for phages. No adenoviral genome degradation was observed at 4°C, and the reduction was estimated at 0.0036 log(10)/day at 20°C. Occurrence study showed that DNA of human adenoviruses could be observed in groundwater from a confined aquifer (7 of the 60 samples were positive by real-time PCR), while no fecal indicators were detected. In agreement with the persistence of genetic markers, the presence of adenoviral DNA in groundwater may be misleading in term of health risk, especially in the absence of information on the infective status.
Assuntos
Adenovírus Humanos/isolamento & purificação , Fagos RNA/isolamento & purificação , Microbiologia do Solo , Microbiologia da Água , Adenovírus Humanos/fisiologia , Humanos , Viabilidade Microbiana , Reação em Cadeia da Polimerase , Fagos RNA/fisiologia , Fatores de Tempo , Ensaio de Placa ViralRESUMO
The genetic structure of natural bacteriophage populations is poorly understood. Recent metagenomic studies suggest that phage biogeography is characterized by frequent migration. Using virus samples mostly isolated in Southern California, we recently showed that very little population structure exists in segmented RNA phage of the Cystoviridae family due to frequent segment reassortment (sexual genetic mixis) between unrelated virus individuals. Here we use a larger genetic dataset to examine the structure of Cystoviridae phage isolated from three geographic locations in Southern New England. We document extensive natural variation in the physical sizes of RNA genome segments for these viruses. In addition, consistent with earlier findings, our phylogenetic analyses and calculations of linkage disequilibrium (LD) show no evidence of within-segment recombination in wild populations. However, in contrast to the prior study, our analysis finds that reassortment of segments between individual phage plays a lesser role among cystoviruses sampled in New England, suggesting that the evolutionary importance of genetic mixis in Cystoviridae phage may vary according to geography. We discuss possible explanations for these conflicting results across the studies, such as differing local ecology and its impact on phage growth, and geographic differences in selection against hybrid phage genotypes.
Assuntos
Cystoviridae/genética , Evolução Molecular , Variação Genética , Fagos RNA/genética , California , Genética Populacional , Genótipo , Hibridização Genética , New England , Filogenia , Fagos RNA/fisiologia , Vírus Reordenados/genéticaRESUMO
The microbial quality of raw greywater was found to be much better than that of municipal wastewater, with 1.6 x 10(7)cfu ml(-1) heterotrophic plate count (HPC), and 3.8 x 10(4), 9.9 x 10(3), 3.3 x 10(3) and 4.6 x 10(0)cfu 100 ml(-1) faecal coliforms (FC), Staphylococcus aureus sp., Pseudomonas aeruginosa sp. and Clostridium perfringes sp., respectively. Further, three viral indicators monitored (somatic phage, host: Escherichia coli CN(13) and F-RNA phages, hosts: E. coli F+(amp), E. coli K12) were not present in raw greywater. The greywater was treated by an RBC followed by sedimentation. The treatment removed two orders of magnitude of all bacteria. UV disinfection kinetics, survival and regrowth of HPC, FC, P. aeruginosa sp. and S. aureus sp. were examined. At doses up to 69 mW s cm(-2) FC were found to be the most resistant bacteria, followed by HPC, P. aeruginosa sp. and S. aureus sp. (inactivation rate coefficients: 0.0687, 0.113, 0.129 and 0.201 cm2 mW(-1)s(-1), respectively). At higher doses (69-439 mW s cm(-2)) all but HPC (which exhibited a tailing curve) were completely eliminated. Microscopic examination showed that FC self-aggregate in the greywater effluent. This provides FC an advantage at low doses, since the concentration of suspended matter (that can provide shelter from UV radiation) in the effluent was very low. FC, P. aeruginosa sp. and S. aureus sp. did not exhibit regrowth up to 6h after exposure to increasing UV doses (19-439 mW s cm(-2)). HPC regrowth was proven to be statistically significant in un-disinfected effluent and after irradiation with high UV doses (147 and 439 mW s cm(-2)). At these doses regrowth resulted from growth of UV-resistant bacteria due to decreased competition with other bacteria eliminated by the irradiation.
Assuntos
Bactérias/efeitos da radiação , Colífagos/efeitos da radiação , Desinfecção/métodos , Fagos RNA/efeitos da radiação , Raios Ultravioleta , Poluentes da Água/efeitos da radiação , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Bactérias/virologia , Colífagos/isolamento & purificação , Colífagos/fisiologia , Contagem de Colônia Microbiana , Cinética , Luz , Fagos RNA/isolamento & purificação , Fagos RNA/fisiologia , Eliminação de Resíduos Líquidos/métodos , Microbiologia da Água , Poluentes da Água/isolamento & purificaçãoRESUMO
AIMS: The pressure responses of four genotypes of F-specific RNA bacteriophages, f2, GA, Qbeta and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium. METHOD AND RESULTS: The pressure responses were studied with respect to pressure magnitude (350 to 600 MPa), treatment temperature (-10 to 50 degrees C) and suspending media. Phages f2 and GA had much higher pressure resistances than Qbeta and SP. Pressure resistances of Qbeta and SP were enhanced with increase in salt concentrations in the range of 350 to 600 MPa from -10 to 50 degrees C in PBS. Qbeta and SP had greater pressure resistances when suspended in phosphate-buffered saline (PBS) with added glucose (5%, w/w), UHT whole milk and Dulbecco's Modified Eagle's Medium plus 10% fetal bovine sera than they did in PBS. Two surfactants, sucrose laurate and monolaurin, and one chelating agent, ethylenediamine tetraacetic acid (EDTA), increased the pressure resistance of Qbeta and SP, but had modest effect on either f2 or GA. CONCLUSIONS: Four representative F-specific RNA bacteriophages, f2 (serotype I), GA (serotype II), Qbeta (serotype III) and SP (serotype IV) showed different resistances to hydrostatic pressure in the range of 350-600 MPa. SIGNIFICANCE AND IMPACT OF THE STUDY: This study screened for practical surrogates of HAV for validation of commercial high hydrostatic pressure processing.
Assuntos
Pressão Hidrostática , Fagos RNA/fisiologia , Quelantes/farmacologia , Meios de Cultura , Ácido Edético/farmacologia , Genótipo , Glucose/metabolismo , Lauratos/farmacologia , Monoglicerídeos/farmacologia , Fagos RNA/efeitos dos fármacos , Fagos RNA/metabolismo , Cloreto de Sódio/metabolismo , Sacarose/análogos & derivados , Sacarose/farmacologia , Tensoativos/farmacologia , Temperatura , Inativação de Vírus/efeitos dos fármacosRESUMO
Many viruses employ molecular motors to package their genomes into preformed empty capsids (procapsids). In dsRNA bacteriophages the packaging motor is a hexameric ATPase P4, which is an integral part of the multisubunit procapsid. Structural and biochemical studies revealed a plausible RNA-translocation mechanism for the isolated hexamer. However, little is known about the structure and regulation of the hexamer within the procapsid. Here we use hydrogen-deuterium exchange and mass spectrometry to delineate the interactions of the P4 hexamer with the bacteriophage phi12 procapsid. P4 associates with the procapsid via its C-terminal face. The interactions also stabilize subunit interfaces within the hexamer. The conformation of the virus-bound hexamer is more stable than the hexamer in solution, which is prone to spontaneous ring openings. We propose that the stabilization within the viral capsid increases the packaging processivity and confers selectivity during RNA loading.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Motores Moleculares/metabolismo , Fagos RNA/enzimologia , Fagos RNA/fisiologia , RNA Polimerase Dependente de RNA/metabolismo , Montagem de Vírus/fisiologia , Adenosina Trifosfatases/química , Escherichia coli/virologia , Modelos Moleculares , Proteínas Motores Moleculares/química , Ligação Proteica , Conformação Proteica , RNA Polimerase Dependente de RNA/químicaRESUMO
Conjugative pili are extracellular filaments elaborated by Gram-negative bacteria expressing certain type IV secretion systems. They are required at the earliest stages of conjugal DNA transfer to establish specific and secure cell-cell contacts. Conjugative pili also serve as adsorption organelles for both RNA and DNA bacteriophages. Beyond these facts, the structure, formation and function of these filaments are poorly understood. This paper describes a rapid, quantitative assay for F-pili encoded by the F plasmid type IV secretion system. The assay is based on the specific lateral adsorption of icosahedral RNA bacteriophage R17 by F-pili. Bacteriophage particles conjugated with a fluorescent dye, Alexa 488, and bound to F-pili defined filaments visible by immunofluorescence microscopy. F-pili attached to F+ cells and free F-pili were both visible by this method. For quantification, cell-bound bacteriophage were separated from free bacteriophage particles by sedimentation and released by suspending cell pellets in 0.1 % SDS. Fluorescence in cell-free supernatant fractions was measured by fluorometry. The authors present a characterization of this assay and its application to F-pilus formation by cells carrying mutations in the gene for the F-pilus subunit F-pilin. Each mutation introduced a cysteine, which F-pilin normally lacks, at a different position in its primary structure. Cysteine residues in the N-terminal domain I abolished filament formation as measured by fluorescent R17 binding. This was confirmed by measurements of DNA donor activity and filamentous DNA bacteriophage infection. With one exception (G53C), cysteines elsewhere in the F-pilin primary structure did not abolish filament formation, although some mutations differentially affected F-pilus functions.