RESUMO
Spatial organization of biological processes allows for variability in molecular outcomes and coordinated development. Here, we investigate how organization underpins phage lambda development and decision-making by characterizing viral components and processes in subcellular space. We use live-cell and in situ fluorescence imaging at the single-molecule level to examine lambda DNA replication, transcription, virion assembly, and resource recruitment in single-cell infections, uniting key processes of the infection cycle into a coherent model of phage development encompassing space and time. We find that different viral DNAs establish separate subcellular compartments within cells, which sustains heterogeneous viral development in single cells. These individual phage compartments are physically separated by the E. coli nucleoid. Our results provide mechanistic details describing how separate viruses develop heterogeneously to resemble single-cell phenotypes.
Assuntos
Bacteriófago lambda/genética , Replicação do DNA/genética , Escherichia coli/virologia , Montagem de Vírus/genética , Bacteriófago lambda/crescimento & desenvolvimento , DNA Viral/biossíntese , DNA Viral/genética , Escherichia coli/genética , Lisogenia/genética , Transcrição Gênica/genéticaRESUMO
The bacterial virus lambda (λ) is a temperate bacteriophage that can lysogenize host Escherichia coli (E. coli) cells. Lysogeny requires λ repressor, the cI gene product, which shuts off transcription of the phage genome. The λ N protein, in contrast, is a transcriptional antiterminator, required for expression of the terminator-distal genes, and thus, λ N mutants are growth-defective. When E. coli is infected with a λ double mutant that is defective in both N and cI (i.e., λN-cI-), at high multiplicities of 50 or more, it forms polylysogens that contain 20-30 copies of the λN-cI- genome integrated in the E. coli chromosome. Early studies revealed that the polylysogens underwent "conversion" to long filamentous cells that form tiny colonies on agar. Here, we report a large set of altered biochemical properties associated with this conversion, documenting an overall degeneration of the bacterial envelope. These properties reverted back to those of nonlysogenic E. coli as the metastable polylysogen spontaneously lost the λN-cI- genomes, suggesting that conversion is a direct result of the multiple copies of the prophage. Preliminary attempts to identify lambda genes that may be responsible for conversion ruled out several candidates, implicating a potentially novel lambda function that awaits further studies.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , Lisogenia/fisiologia , Prófagos/crescimento & desenvolvimento , Bacteriófago lambda/efeitos dos fármacos , Bacteriófago lambda/genética , Bacteriófago lambda/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dactinomicina/farmacologia , Escherichia coli/virologia , Genes Virais , Lisogenia/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Ácido Nalidíxico/farmacologia , Peptidoglicano/metabolismo , Prófagos/efeitos dos fármacos , Prófagos/ultraestrutura , Proteínas Virais/metabolismoRESUMO
The arms race between bacteria and the phages that infect them drives the continual evolution of diverse anti-phage defences. Previously described anti-phage systems have highly varied defence mechanisms1-11; however, all mechanisms rely on protein components to mediate defence. Here we report a chemical anti-phage defence system that is widespread in Streptomyces. We show that three naturally produced molecules that insert into DNA are able to block phage replication, whereas molecules that target DNA by other mechanisms do not. Because double-stranded DNA phages are the most numerous group in the biosphere and the production of secondary metabolites by bacteria is ubiquitous12, this mechanism of anti-phage defence probably has a major evolutionary role in shaping bacterial communities.
Assuntos
Bacteriófagos/efeitos dos fármacos , Bacteriófagos/genética , Metabolismo Secundário , Streptomyces/química , Streptomyces/virologia , Replicação Viral/efeitos dos fármacos , Bacteriófago lambda/efeitos dos fármacos , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Bacteriófago lambda/fisiologia , Bacteriófagos/crescimento & desenvolvimento , Evolução Biológica , DNA Viral/biossíntese , DNA Viral/genética , Daunorrubicina/farmacologia , Escherichia coli/virologia , Pseudomonas aeruginosa/virologia , Streptomyces/metabolismoRESUMO
The exo-xis region of lambdoid bacteriophage genomes contains several established and potential genes that are evolutionarily conserved, but not essential for phage propagation under laboratory conditions. Nevertheless, deletion or overexpression of either the whole exo-xis region and important regulatory elements can significantly influence the regulation of phage development. This report defines specific roles for orf60a and orf61 in bacteriophage λ and Φ24B, a specific Shiga toxin-converting phage with clinical relevance. We observed that mutant phages bearing deletions of orf60a and orf61 impaired two central aspects of phage development: the lysis-versus-lysogenization decision and prophage induction. These effects were more pronounced for phage Φ24B than for λ. Surprisingly, adsorption of phage Φ24B on Escherichia coli host cells was less efficient in the absence of either orf60a or orf61. We conclude that these open reading frames (ORFs) play important, but not essential, roles in the regulation of lambdoid phage development. Although phages can propagate without these ORFs in nutrient media, we suggest that they may be involved in the regulatory network, ensuring optimization of phage development under various environmental conditions.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , Fases de Leitura Aberta , Proteínas Virais/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , Escherichia coli/genética , Escherichia coli/fisiologia , Escherichia coli/virologia , Regulação Viral da Expressão Gênica , Lisogenia , Proteínas Virais/genética , Ativação ViralRESUMO
The final step of lysis in phage λ infections of Escherichia coli is mediated by the spanins Rz and Rz1. These proteins form a complex that bridges the cell envelope and that has been proposed to cause fusion of the inner and outer membranes. Accordingly, mutations that block spanin function are found within coiled-coil domains and the proline-rich region, motifs essential in other fusion systems. To gain insight into spanin function, pseudorevertant alleles that restored plaque formation for lysis-defective mutants of Rz and Rz1 were selected. Most second-site suppressors clustered within a coiled-coil domain of Rz near the outer leaflet of the cytoplasmic membrane and were not allele specific. Suppressors largely encoded polar insertions within the hydrophobic core of the coiled-coil interface. Such suppressor changes resulted in decreased proteolytic stability of the Rz double mutants in vivo Unlike the wild type, in which lysis occurs while the cells retain a rod shape, revertant alleles with second-site suppressor mutations supported lysis events that were preceded by spherical cell formation. This suggests that destabilization of the membrane-proximal coiled coil restores function for defective spanin alleles by increasing the conformational freedom of the complex at the cost of its normal, all-or-nothing functionality.IMPORTANCECaudovirales encode cell envelope-spanning proteins called spanins, which are thought to fuse the inner and outer membranes during phage lysis. Recent genetic analysis identified the functional domains of the lambda spanins, which are similar to class I viral fusion proteins. While the pre- and postfusion structures of model fusion systems have been well characterized, the intermediate structure(s) formed during the fusion reaction remains elusive. Genetic analysis would be expected to identify functional connections between intermediates. Since most membrane fusion systems are not genetically tractable, only few such investigations have been reported. Here, we report a suppressor analysis of lambda spanin function. To our knowledge this is the first suppression analysis of a class I-like complex and also the first such analysis of a prokaryote membrane fusion system.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , Escherichia coli/virologia , Proteínas Mutantes/metabolismo , Supressão Genética , Proteínas Virais/metabolismo , Bacteriófago lambda/genética , Análise Mutacional de DNA , Modelos Biológicos , Proteínas Mutantes/genética , Conformação Proteica , Domínios Proteicos , Ensaio de Placa Viral , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Deciphering the way gene expression regulatory aspects are encoded in viral genomes is a challenging mission with ramifications related to all biomedical disciplines. Here, we aimed to understand how the evolution shapes the bacteriophage lambda genes by performing a high resolution analysis of ribosomal profiling data and gene expression related synonymous/silent information encoded in bacteriophage coding regions.We demonstrated evidence of selection for distinct compositions of synonymous codons in early and late viral genes related to the adaptation of translation efficiency to different bacteriophage developmental stages. Specifically, we showed that evolution of viral coding regions is driven, among others, by selection for codons with higher decoding rates; during the initial/progressive stages of infection the decoding rates in early/late genes were found to be superior to those in late/early genes, respectively. Moreover, we argued that selection for translation efficiency could be partially explained by adaptation to Escherichia coli tRNA pool and the fact that it can change during the bacteriophage life cycle.An analysis of additional aspects related to the expression of viral genes, such as mRNA folding and more complex/longer regulatory signals in the coding regions, is also reported. The reported conclusions are likely to be relevant also to additional viruses.
Assuntos
Adaptação Biológica , Bacteriófago lambda/genética , Regulação Viral da Expressão Gênica , Biossíntese de Proteínas , Bacteriófago lambda/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Evolução Molecular , Perfilação da Expressão Gênica , Genes Virais , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismoRESUMO
The infection of Escherichia coli cells by bacteriophage lambda results in bifurcated means of propagation, where the phage decides between the lytic and lysogenic pathways. Although traditionally thought to be mutually exclusive, increasing evidence suggests that this lysis-lysogeny decision is more complex than once believed, but exploring its intricacies requires an improved resolution of study. Here, with a newly developed fluorescent reporter system labeling single phage and E. coli DNAs, these two distinct pathways can be visualized by following the DNA movements in vivo. Surprisingly, we frequently observed an interesting "lyso-lysis" phenomenon in lytic cells, where phage integrates its DNA into the host, a characteristic event of the lysogenic pathway, followed by cell lysis. Furthermore, the frequency of lyso-lysis increases with the number of infecting phages, and specifically, with CII activity. Moreover, in lytic cells, the integration site attB on the E. coli genome migrates toward the polar region over time, leading to more spatial overlap with the phage DNA and frequent colocalization/collision of attB and phage DNA, possibly contributing to a higher chance for DNA integration.
Assuntos
Sítios de Ligação Microbiológicos/genética , Bacteriófago lambda/genética , Escherichia coli/virologia , Lisogenia/genética , Integração Viral/genética , Bacteriófago lambda/crescimento & desenvolvimento , DNA Viral/genética , Escherichia coli/genéticaRESUMO
Studies over more than half a century have resulted in what some consider a complete narrative for the life cycle of bacteriophage λ. However, this narrative is only complete within the limited resolution offered by the traditional genetic and biochemical approaches that were used to create it. A recent series of studies performed at the single-cell and single-phage levels has revealed a wealth of previously unknown features. By pointing to many open questions, these new studies highlight the limitations of our current understanding of λ, but they also initiate the process of forming a more detailed and quantitative narrative for the system.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , Bacteriófago lambda/genética , Escherichia coli/virologia , Lisogenia/genética , Análise de Célula Única/métodos , Escherichia coli/genética , Estágios do Ciclo de Vida/genética , Ativação Viral/genéticaRESUMO
The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (Rif(R)) cells acquiring mutations within the rpoB gene encoding the ß-subunit of RNA polymerase (RNAP), nine of which proved unique. Using fluctuation assays, we show that the intracellular expression of P evokes a mutator effect. Most of the Rif(R) mutants remained P(S) and localized to the Rif binding pocket in RNAP, but a subset acquired a P(R) phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One P(R) mutation was identical to rpo*Q148P, which alleviates the UV-sensitivity of ruv strains defective in the migration and resolution of Holliday junctions and destabilizes stalled RNAP elongation complexes. The results suggest that P-DnaB sequestration is mutagenic and supports an earlier observation that P can interact with RNAP.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , RNA Polimerases Dirigidas por DNA/metabolismo , DnaB Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/virologia , Interações Hospedeiro-Parasita , Mutação , Proteínas Virais/toxicidade , Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/genética , Ligação Proteica , Rifampina/farmacologiaRESUMO
In this work, we explore the refactoring of the circuitry of λ phage by engineering a new-to-nature regulator that responds to an ad hoc input signal that behaves orthogonal with respect to the host cell. We tailored a chimeric regulator, termed Qλ, between the CI protein of the λ phage and the BzdR repressor from Azoarcus sp. strain CIB that responds to benzoyl-CoA. When the Qλ was expressed in the appropriate Escherichia coli cells, it was able to reprogram the lytic/lysogenic λ phage decision according to the intracellular production of benzoyl-CoA. Our results are also an example of how generating new artificial regulators that respond to effectors of choice may be useful to control different cellular processes.
Assuntos
Acil Coenzima A/metabolismo , Bacteriófago lambda/crescimento & desenvolvimento , Bacteriófago lambda/genética , Regulação Bacteriana da Expressão Gênica/genética , Lisogenia/genética , Regiões Promotoras Genéticas/genética , Escherichia coli/genética , Escherichia coli/virologia , Transcrição Gênica/genéticaRESUMO
Many pathogens have evolved the ability to induce latent infections of their hosts. The bacteriophage λ is a classical model for exploring the regulation and the evolution of latency. Here, I review recent experimental studies on phage λ that identify specific conditions promoting the evolution of lysogenic life cycles. In addition, I present specific adaptations of phage λ that allow this virus to react plastically to variations in the environment and to reactivate its lytic life cycle. All of these different examples are discussed in the light of evolutionary epidemiology theory to disentangle the different evolutionary forces acting on temperate phages. Understanding phage λ adaptations yield important insights into the evolution of latency in other microbes, including several life-threatening human pathogens.
Assuntos
Bacteriófago lambda/fisiologia , Adaptação Fisiológica , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Evolução Biológica , Meio Ambiente , Humanos , Lisogenia , Latência ViralRESUMO
Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts' fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity.
Assuntos
Bacteriófago lambda/fisiologia , Escherichia coli/virologia , Trato Gastrointestinal/virologia , Interações Hospedeiro-Patógeno/fisiologia , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Bacteriófago lambda/crescimento & desenvolvimento , Bacteriófago lambda/patogenicidade , Contagem de Colônia Microbiana , Transferência Genética Horizontal , Lisogenia , Camundongos , Modelos Biológicos , Mutação/genética , VirulênciaRESUMO
One of the key determinants of the size, composition, structure, and development of a microbial community is the predation pressure by bacteriophages. Accordingly, bacteria have evolved a battery of antiphage defense strategies. Since maintaining constantly elevated defenses is costly, we hypothesize that some bacteria have additionally evolved the abilities to estimate the risk of phage infection and to adjust their strategies accordingly. One risk parameter is the density of the bacterial population. Hence, quorum sensing, i.e., the ability to regulate gene expression according to population density, may be an important determinant of phage-host interactions. This hypothesis was investigated in the model system of Escherichia coli and phage λ. We found that, indeed, quorum sensing constitutes a significant, but so far overlooked, determinant of host susceptibility to phage attack. Specifically, E. coli reduces the numbers of λ receptors on the cell surface in response to N-acyl-l-homoserine lactone (AHL) quorum-sensing signals, causing a 2-fold reduction in the phage adsorption rate. The modest reduction in phage adsorption rate leads to a dramatic increase in the frequency of uninfected survivor cells after a potent attack by virulent phages. Notably, this mechanism may apply to a broader range of phages, as AHLs also reduce the risk of χ phage infection through a different receptor. IMPORTANCE To enable the successful manipulation of bacterial populations, a comprehensive understanding of the factors that naturally shape microbial communities is required. One of the key factors in this context is the interactions between bacteria and the most abundant biological entities on Earth, namely, the bacteriophages that prey on bacteria. This proof-of-principle study shows that quorum sensing plays an important role in determining the susceptibility of E. coli to infection by bacteriophages λ and χ. On the basis of our findings in the classical Escherichia coli-λ model system, we suggest that quorum sensing may serve as a general strategy to protect bacteria specifically under conditions of high risk of infection.
Assuntos
Bacteriófago lambda/crescimento & desenvolvimento , Escherichia coli/fisiologia , Escherichia coli/virologia , Percepção de Quorum , Acil-Butirolactonas/metabolismo , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Parasita , Receptores Virais/biossínteseRESUMO
The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step.
Assuntos
Bacteriófago lambda/metabolismo , Proteínas Virais/metabolismo , Alelos , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Replicação do DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ordem dos Genes , Genes Letais , Teste de Complementação Genética , Mutação , Fenótipo , Plasmídeos/genética , Resposta SOS em Genética , Transativadores/genética , Transativadores/imunologia , Transativadores/metabolismo , Transformação Bacteriana , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação ViralRESUMO
It is generally thought that the adsorption rate of a bacteriophage correlates positively with fitness, but this view neglects that most phages rely only on exponentially growing bacteria for productive infections. Thus, phages must cope with the environmental stochasticity that is their hosts' physiological state. If lysogeny is one alternative, it is unclear how strictly lytic phages can survive the host stationary phase. Three scenarios may explain their maintenance: (1) pseudolysogeny, (2) diversified, or (3) conservative bet hedging. To better understand how a strictly lytic phage survives the stationary phase of its host, and how phage adsorption rate impacts this survival, we challenged two strictly lytic phage λ, differing in their adsorption rates, with stationary phase Escherichia coli cells. Our results showed that, pseudolysogeny was not responsible for phage survival and that, contrary to our expectation, high adsorption rate was not more detrimental during stationary phase than low adsorption rate. Interestingly, this last observation was due to the presence of the "residual fraction" (phages exhibiting extremely low adsorption rates), protecting phage populations from extinction. Whether this cryptic phenotypic variation is an adaptation (diversified bet hedging) or merely reflecting unavoidable defects during protein synthesis remains an open question.
Assuntos
Bacteriófago lambda/fisiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Adsorção , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Aptidão Genética , Lisogenia , Modelos Biológicos , Fenótipo , Processos Estocásticos , Fatores de Tempo , Ensaio de Placa Viral , Proteínas da Cauda Viral/genéticaRESUMO
As native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. In this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, eGFP. To achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the phages produced. Phage display of eGFP resulted in altered side scatter and fluorescence profile, and sub-populations could be identified within what would otherwise be considered uniform populations. Surprisingly, this study has found that side scatter may be used in the future to characterize the display of nonfluorescent proteins.
Assuntos
Bacteriófago lambda/química , Técnicas de Visualização da Superfície Celular/métodos , Gráficos por Computador , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/química , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Escherichia coli/química , Escherichia coli/virologia , Fluorescência , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Plasmídeos/química , Plasmídeos/genética , TemperaturaRESUMO
The terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. Yet, these motors are hindered at the end of the packaging process by the progressive buildup of a force-resisting packaging associated with already packaged DNA. In this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be packaged by the terminase motor of phage λ and still yield infectious virions and the conditions under which this can be efficiently performed. Using a packaging strategy developed in our laboratory of building phage λ from scratch, together with plaque assay monitoring, we have been able to show that the terminase motor of phage λ is able to produce infectious particles with up to 110% of the wild-type λ-DNA length. However, the phage production rate, and thus the infectivity, decreased exponentially with increasing DNA length and was a factor of 10(3) lower for the 110% λ-DNA phage. Interestingly, our in vitro strategy was still efficient in fully packaging phages with DNA lengths as high as 114% of the wild-type length, but these viruses were unable to infect bacterial cells efficiently. Further, we demonstrated that the phage production rate is modulated by the presence of multivalent ionic species. The biological consequences of these findings are discussed.
Assuntos
Bacteriófago lambda/enzimologia , Bacteriófago lambda/fisiologia , Empacotamento do DNA , Endodesoxirribonucleases/metabolismo , Montagem de Vírus , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Carga Viral , Ensaio de Placa ViralRESUMO
BACKGROUND: Despite identical genotypes and seemingly uniform environments, stochastic gene expression and other dynamic intracellular processes can produce considerable phenotypic diversity within clonal microbes. One trait that provides a good model to explore the molecular basis of stochastic variation is the timing of host lysis by bacteriophage (phage). RESULTS: Individual lysis events of thermally-inducible λ lysogens were observed using a temperature-controlled perfusion chamber mounted on an inverted microscope. Both mean lysis time (MLT) and its associated standard deviation (SD) were estimated. Using the SD as a measure of lysis time stochasticity, we showed that lysogenic cells in controlled environments varied widely in lysis times, and that the level of lysis time stochasticity depended on allelic variation in the holin sequence, late promoter (pR') activity, and host growth rate. In general, the MLT was positively correlated with the SD. Both lower pR' activities and lower host growth rates resulted in larger SDs. Results from premature lysis, induced by adding KCN at different time points after lysogen induction, showed a negative correlation between the timing of KCN addition and lysis time stochasticity. CONCLUSIONS: Taken together with results published by others, we conclude that a large fraction of λ lysis time stochasticity is the result of random events following the expression and diffusion of the holin protein. Consequently, factors influencing the timing of reaching critical holin concentrations in the cell membrane, such as holin production rate, strongly influence the mean lysis time and the lysis time stochasticity.
Assuntos
Bacteriólise , Bacteriófago lambda/crescimento & desenvolvimento , Bacteriófago lambda/fisiologia , Escherichia coli/fisiologia , Escherichia coli/virologia , Modelos Estatísticos , Regiões Promotoras Genéticas , Temperatura , Fatores de Tempo , Proteínas Virais/genéticaRESUMO
Phenoloxidase (PO) and its activation system are implicated in several defense responses of insects. Upon wounding or infection, inactive prophenoloxidase (proPO) is converted to active PO through a cascade of serine proteases and their homologs. PO generates reactive compounds such as 5,6-dihydroxyindole (DHI), which have a broad-spectrum antibacterial and antifungal activity. Here we report that DHI and its spontaneous oxidation products are also active against viruses and parasitic wasps. Preincubation of a baculovirus stock with 1.25 mM DHI for 3 h near fully disabled recombinant protein production. The LC50 for lambda bacteriophage and eggs of the wasp Microplitis demolitor were 5.6 ± 2.2 and 111.0 ± 1.6 µM, respectively. The toxicity of DHI and related compounds also extended to cells derived from insects that serve as hosts for several of the aforementioned pathogens. Pretreatment of Sf9 cells with 1.0 mM DHI for 4 h resulted in 97% mortality, and LC50 values of 20.3 ± 1.2 µM in buffer and 131.8 ± 1.1 µM in a culture medium. Symptoms of DHI toxicity in Sf9 cells included DNA polymerization, protein crosslinking, and lysis. Taken together, these data showed that proPO activation and DHI production is strongly toxic against various pathogens but can also damage host tissues and cells if not properly controlled.
Assuntos
Indóis/imunologia , Proteínas de Insetos/imunologia , Monofenol Mono-Oxigenase/imunologia , Spodoptera/enzimologia , Animais , Bacteriófago lambda/crescimento & desenvolvimento , Linhagem Celular , Citotoxicidade Imunológica , DNA/imunologia , DNA/metabolismo , Ativação Enzimática , Indóis/metabolismo , Proteínas de Insetos/metabolismo , Cinética , Monofenol Mono-Oxigenase/metabolismo , Nucleopoliedrovírus/crescimento & desenvolvimento , Oxirredução , Polimerização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Spodoptera/imunologia , Spodoptera/microbiologia , Especificidade por Substrato , Vespas/crescimento & desenvolvimentoRESUMO
We have identified a holin-like gene from a goat skin surface metagenome. The ORF designated tmp1 coding for 34 amino acids shared sequence similarity with putative holin-like toxin genes. To analyze the antibacterial activity of tmp1 encoded protein, this ORF was cloned and expressed in Escherichia coli BL21(DE3). The expressed gene product Tmp1 exhibited antibacterial activity against Gram-positive bacteria but not to Gram-negative bacteria. A single transmembrane domain (TMD) was identified within Tmp1 and deletion analysis of the N-terminal region and TMD indicated TMD to be responsible for antibacterial activity. The TMD-dependent antibacterial activity was validated using a synthetic peptide with the amino acid sequence of TMD. Besides antibacterial activity, Tmp1 also complemented the function of holin in a lysis-defective bacteriophage lambda. To broaden the spectrum of antibacterial activity, a mutant library of tmp1 was generated by random mutagenesis. Four mutants with amino acid substitutions at the N-terminus of Tmp1 exhibited increased antibacterial activity against Gram-positive and Gram-negative bacteria and were not hemolytic. An improved activity of these mutant proteins is attributed to their increased hydrophobicity.